U.S. patent application number 09/978600 was filed with the patent office on 2003-05-08 for diagnostic and therapeutic compositions for alzheimer's disease.
This patent application is currently assigned to MITOKOR. Invention is credited to Ghosh, Soumitra S., Herrnstadt, Corinna.
Application Number | 20030087858 09/978600 |
Document ID | / |
Family ID | 34272319 |
Filed Date | 2003-05-08 |
United States Patent
Application |
20030087858 |
Kind Code |
A1 |
Herrnstadt, Corinna ; et
al. |
May 8, 2003 |
Diagnostic and therapeutic compositions for alzheimer's disease
Abstract
The present invention relates to genetic mutations in
mitochondrial cytochrome c oxidase genes that segregate with
Alzheimer's disease (AD). The invention provides methods for
detecting such mutations, as a diagnostic for Alzheimer's Disease,
either before or after the onset of clinical symptoms. The
invention further provides treatment of cytochrome c oxidase
dysfunction.
Inventors: |
Herrnstadt, Corinna; (San
Diego, CA) ; Ghosh, Soumitra S.; (San Diego,
CA) |
Correspondence
Address: |
SEED INTELLECTUAL PROPERTY LAW GROUP PLLC
701 FIFTH AVE
SUITE 6300
SEATTLE
WA
98104-7092
US
|
Assignee: |
MITOKOR
11494 Sorrento Valley Road
San Diego
CA
92121
|
Family ID: |
34272319 |
Appl. No.: |
09/978600 |
Filed: |
October 15, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09978600 |
Oct 15, 2001 |
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09448312 |
Nov 23, 1999 |
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09448312 |
Nov 23, 1999 |
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08413740 |
Mar 30, 1995 |
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6171859 |
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08413740 |
Mar 30, 1995 |
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08219842 |
Mar 30, 1994 |
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5565323 |
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Current U.S.
Class: |
514/44A ;
435/6.14; 536/24.3 |
Current CPC
Class: |
C12N 9/0053 20130101;
C12Q 1/6806 20130101; C07K 14/4711 20130101; A61K 38/00 20130101;
A61K 47/545 20170801; C12Q 1/6806 20130101; C07D 219/08 20130101;
C12Q 2527/101 20130101; A61K 47/541 20170801; C07K 14/4713
20130101 |
Class at
Publication: |
514/44 ; 435/6;
536/24.3 |
International
Class: |
A61K 048/00; C12Q
001/68; C07H 021/04 |
Claims
We claim:
1. A method for inhibiting the transcription or translation of
mutant cytochrome c oxidase encoding genes, comprising the steps
of: a) contacting said genes with antisense sequences which are
specific to said mutant sequences; and b) allowing hybridization
between said target mutant cytochrome c oxidase gene and said
antisense sequences under conditions under which said antisense
sequences bind to and inhibit transcription or translation of said
target mutant cytochrome c oxidase genes without preventing
transcription or translation of wild-type cytochrome c oxidase
genes.
2. The method of claim 1 wherein Alzheimer's disease is treated and
wherein said cytochrome c oxidase genes contain mutations at one or
more codons selected from the group of: (a) codon 155, codon 167,
codon 178, codon 193, codon 194, and codon 415 of the cytochrome c
oxidase I gene; and (b) codon 20, codon 22, codon 68, codon 71,
codon 74, codon 90, codon 95, codon 110, and codon 146 of the
cytochrome c oxidase II gene.
3. A probe for detection of a disease state associated with one or
more mutations in mitochondrial cytochrome c oxidase genes
comprising a nucleotide sequence complementary to either of the
sense and anti-sense strands of said one or more mutations in said
mitochondrial cytochrome c oxidase genes.
4. The probe of claim 3 wherein said probe includes a region
complementary to the sense and anti-sense strands of one or more
codons selected from the group of: (a) codon 155, codon 167, codon
178, codon 193, codon 194, and codon 415 of the cytochrome c
oxidase I gene; and (b) codon 20, codon 22, codon 68, codon 71,
codon 74, codon 90, codon 95, codon 110, and codon 146 of the
cytochrome c oxidase II gene.
5. A kit comprising a probe for detection of an Alzheimer's disease
genotype, said probe comprising a nucleotide sequence complementary
to either of the sense and anti-sense strands of a mitochondrial
cytochrome c oxidase gene.
6. The kit of claim 5, wherein said probe includes a region
complementary to the sense and anti-sense strands of one or more
codons selected from the group of: (a) codon 155, codon 167, codon
178, codon 193, codon 194, and codon 415 of the cytochrome c
oxidase I gene; and (b) codon 20, codon 22, codon 68, codon 71,
codon 74, codon 90, codon 95, codon 110, and codon 146 of the
cytochrome c oxidase II gene.
7. A therapeutic composition comprising antisense sequences which
are specific to mutant cytochrome c oxidase genes or mutant
messenger RNA transcribed therefrom, said antisense sequences
adapted to bind to and inhibit transcription or translation of said
target mutant cytochrome c oxidase genes without preventing
transcription or translation of wild-type cytochrome c oxidase
genes.
8. The therapeutic composition of claim 7, wherein Alzheimer's
disease is treated and wherein said cytochrome c oxidase genes
contain mutations at one or more codons selected from the group of:
(a) codon 155, codon 167, codon 178, codon 193, codon 194, and
codon 415 of the cytochrome c oxidase I gene; and (b) codon 20,
codon 22, codon 68, codon 71, codon 74, codon 90, codon 95, codon
110, and codon 146 of the cytochrome c oxidase II gene.
9. A method for detecting the presence of Alzheimer's disease in a
subject, comprising the steps of: a) obtaining a biological sample
containing mitochondria from said subject; and b) interrogating at
least one variant polypeptide, arising from one or more mutations
in one or more subunits of mitochondrial cytochrome c oxidase
genes, which correlates with the presence of Alzheimer's
disease.
10. The method of claim 9, wherein said mutation is interrogated
using monoclonal antibodies or polyclonal antibodies.
11. A ribozyme adapted to hybridize to and cleave mitochondrial
mRNA molecules that encode for mutant cytochrome c oxidase
subunits.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a continuation of co-pending U.S. patent
application Ser. No. 09/448,312, filed Nov. 23, 1999, now allowed;
which is a continuation of U.S. patent application Ser. No.
08/413,740, filed Mar. 30, 1995 and issued as U.S. Pat. No.
6,171,859 on Jan. 9, 2001; which is a continuation-in-part of U.S.
patent application Ser. No. 08/219,842 filed Mar. 30, 1994, and
issued as U.S. Pat. No. 5,565,323 on Oct. 15, 1996; the disclosures
of which are herein incorporated by reference in their
entirety.
FIELD OF THE INVENTION
[0002] The present invention relates generally to the diagnosis and
treatment of Alzheimer's disease. More specifically, the invention
relates to detecting genetic mutations in mitochondrial cytochrome
c oxidase genes as a means for diagnosing Alzheimer's disease and
suppressing these same mutations or the effects of these mutations
in the treatment of Alzheimer's disease.
BACKGROUND OF THE INVENTION
[0003] Alzheimer's disease (AD) is a progressive neurodegenerative
disorder characterized by loss and/or atrophy of neurons in
discrete regions of the brain, accompanied by extracellular
deposits of .beta.-amyloid and the intracellular accumulation of
neurofibrillary tangles. It is a uniquely human disease, affecting
over 13 million people worldwide. It is also a uniquely tragic
disease. Many individuals who have lived normal, productive lives
are slowly stricken with AD as they grow older, and the disease
gradually robs them of their memory and other mental faculties.
Eventually, they even cease to recognize family and loved ones, and
they often require continuous care until their eventual death.
[0004] Alzheimer's disease is incurable and untreatable, except
symptomatically. Persons suffering from Alzheimer's disease may
have one of two forms of this disease: "familial" AD or "sporadic"
AD.
[0005] Familial Alzheimer's disease accounts for only about 5 to
10% of all Alzheimer's cases and has an unusually early-onset,
generally before the age of fifty. Familial AD is inherited and
follows conventional patterns of mendelian inheritance: This form
of AD has been linked to nuclear chromosomal abnormalities.
[0006] In contrast, the second form of Alzheimer's disease,
sporadic AD, is a late-onset disease which is neither inherited nor
caused by nuclear chromosomal abnormalities. This late onset form
of the disease is the more common type of Alzheimer's disease and
is believed to account for approximately 90 to 95% of all
Alzheimer's cases.
[0007] It has been recognized that some degenerative diseases such
as Leber's hereditary optic neuropathy, myoclonus, epilepsy, lactic
acidosis and stroke (MELAS), and myoclonic epilepsy ragged red
fiber syndrome, are transmitted through mitochondrial DNA
mutations. Mitochondrial DNA mutations have also been implicated in
explaining the apparently "sporadic" (nonmendelian) occurrence of
some degenerative neurologic disorders, such as Parkinson's and
Alzheimer's disease. Proteins encoded by the mitochondrial genome
are components of the electron transport chain, and deficits in
electron transport function have been reported in Parkinson's and
Alzheimer's disease. In particular, it has been reported that
defects in cytochrome c oxidase, an important terminal component of
the electron transport chain located in the mitochondria of
eukaryotic cells, may be involved in Alzheimer's disease.
[0008] One report suggesting a relation between AD and cytochrome c
oxidase is Parker et al., Neurology 40:1302 (1990), which finds
that patients with Alzheimer's disease have reduced cytochrome c
oxidase activity. It has also been shown by Bennett et al., J.
Geriatric Psychiatry and Neurology 5:93-101 (1992), that when
sodium azide, a specific inhibitor of cytochrome c oxidase (COX)
was infused into rats, the rats suffered impaired memory and
learning (a form of dementia). The rats mimicked the effect of
Alzheimer's disease in humans. In addition, the sodium azide-tested
rats failed to display long term potentiation, demonstrating loss
of neuronal plasticity. It has been hypothesized that the reduced
cytochrome c oxidase activity leads to increased intracellular
levels of oxygen free radicals, and that the cumulative effects of
free radical-mediated lipid oxidation ultimately cause the
degenerative neurological changes that are characteristic of AD.
Wallace, D. C., Science, 256:628-632 (1992).
[0009] Despite these findings, prior to the present invention, the
exact mechanism producing the electron transport dysfunctions was
not known for Alzheimer's disease, nor had a genetic or structural
basis for these dysfunctions been identified. Without knowing what
causes these electron transport dysfunctions and in particular the
genetic or structural basis, it is difficult to diagnose or treat
Alzheimer's disease, especially the predominant form, sporadic
AD.
[0010] To date, the diagnosis of probable Alzheimer's disease is
only by clinical observation and is a diagnosis of exclusion.
Unfortunately, definitive diagnosis can be accomplished only by
pathological examination at autopsy. While attempts have been made
to diagnose Alzheimer's disease by identifying differences in
certain biological markers, including protease nexin II and
apolipoprotein E alleles, this approach has not been successful.
Incomplete penetrance in AD patients or crossover into normal or
other disease populations makes identification of biological
markers an unreliable method of diagnosis. Clearly, a reliable
diagnosis of Alzheimer's at its earliest stages is critical for
efficient and effective intercession and treatment of this
debilitating disease. Thus, there exists a definite need for an
effective diagnostic of Alzheimer's disease, and especially for the
more prevalent form, sporadic AD. There also exists a need for a
non-invasive diagnostic that is reliable at or before the earliest
manifestations of AD symptoms.
[0011] Not only does the Alzheimer's field currently lack a
reliable, early means of detection, there is at present no
effective therapy for AD, other than certain palliative treatments.
Current therapies in clinical evaluation are designed to treat the
symptoms of the disease and not impact the underlying pathology of
AD. These therapies include Cognex, E2020, and other similar agents
known in the field. However, since the primary etiologic events in
AD are not yet known in the art, rational therapies have not been
designed. As a result, there exists a need for effective therapies,
particularly those that address the primary cause of AD.
[0012] The present invention satisfies these needs for a useful
diagnostic and effective treatment of Alzheimer's disease and
provides related advantages as well.
SUMMARY OF THE INVENTION
[0013] The present invention relates to the identification of
genetic mutations in mitochondrial cytochrome c oxidase genes which
segregate with Alzheimer's disease. The invention provides methods
for detecting such mutations as a diagnostic for Alzheimer's
disease, either before or after the onset of clinical symptoms.
[0014] According to an embodiment of the present invention for
detecting the presence of Alzheimer's disease, a biological sample
containing mitochondria from a subject is obtained and one or more
mutations in the sequence of a mitochondrial cytochrome c oxidase
gene which correlates with the presence of Alzheimer's disease is
interrogated. Such interrogated mutations are preferably positioned
between codon 155 and codon 415 of the cytochrome c oxidase I gene
and/or between codon 20 and codon 150 of the cytochrome c oxidase
II gene. More preferably, the mutations are interrogated at one or
more of the following positions: codon 155, codon 167, codon 178,
codon 193, codon 194, and codon 415 of the cytochrome c oxidase I
gene; and codon 20, codon 22, codon 68, codon 71, codon 74, codon
95, codon 110, and codon 146 of the cytochrome c oxidase II gene.
If desired, the codon of interest can be amplified prior to
interrogation.
[0015] Preferred methods for interrogating the above mutations
include: (a) hybridization with oligonucleotide probes, (b) methods
based on the ligation of oligonucleotide sequences that anneal
adjacent to one another on target nucleic acids, such as the ligase
chain reaction, (c) the polymerase chain reaction or variants
thereof which depend on using sets of primers, and (d) single
nucleotide primer-guided extension assays.
[0016] The present invention also encompasses nucleic acid
sequences which are useful in the above mentioned diagnostics,
namely those which correspond, or are complementary, to portions of
mitochondrial cytochrome c oxidase gene that contain gene mutations
which correlate with the presence of Alzheimer's disease. According
to one embodiment, the nucleic acid sequences are labelled with
detectable agents. Preferred detectable agents include
radioisotopes (such as .sup.32P), haptens (such as digoxigenin),
biotin, enzymes (such as alkaline phosphatase or horseradish
peroxidase), fluorophores (such as fluorescein or Texas Red), or
chemilumiphores (such as acridine).
[0017] According to another embodiment for detecting the presence
of Alzheimer's disease, a biological sample is interrogated for the
presence of protein products. In particular, protein products of
mitochondria with one or more cytochrome c oxidase mutations that
correlate with the presence of Alzheimer's disease are
interrogated. Preferred agents for the interrogation of such
proteins include monoclonal antibodies.
[0018] According to another embodiment of the present invention,
genetic mutations which cause Alzheimer's disease are detected by
determining the sequence of mitochondrial cytochrome c oxidase
genes from subjects known to have Alzheimer's disease, and
comparing the sequence to that of known wild-type mitochondrial
cytochrome c oxidase genes.
[0019] Other embodiments of the present invention pertain to
suppression of the undesired biological activity of the mutations.
This affords a therapeutic treatment for Alzheimer's disease. More
specifically, one embodiment of the invention pertains to methods
of inhibiting the transcription or translation of mutant cytochrome
c oxidase encoding genes by contacting the genes with antisense
sequences which are specific for mutant sequences and which
hybridize to a target mutant cytochrome c oxidase gene or messenger
RNA transcribed therefrom.
[0020] Another embodiment of the invention concerns the selective
introduction of a conjugate molecule into mitochondria with
defective cytochrome c oxidase genes. The conjugate comprises a
targeting molecule conjugated to a toxin or to an imaging ligand
using a linker. The targeting molecule can be, for example, a
lipophilic cation such as an acridine orange derivative, a
rhodamine 123 derivative, or a JC-1
(5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidiazolo-carbocyanine
iodide) derivative. The linker can include, for example, an ester,
ether, thioether, phosphorodiester, thiophosphorodiester,
carbonate, carbamate, hydrazone, oxime, amino or amide
functionality. The imaging ligand can be, for example, a
radioisotope, hapten, biotin, enzyme, fluorophore or
chemilumiphore. And the toxin can be, for example, phosphate,
thiophosphate, dinitrophenol, maleimide and antisense oligonucleic
acids.
[0021] The appended claims are hereby incorporated by reference as
a further enumeration of preferred embodiments.
[0022] It is an object of the present invention to identify the
structural and genetic basis for the electron transport
dysfunctions that are known to accompany Alzheimer's disease.
[0023] It is another object of the present invention to provide
reliable and efficient means for the diagnosis of Alzheimer's
disease.
[0024] It is another object of the present invention to provide
effective therapies for the treatment of Alzheimer's disease.
[0025] One advantage of the present invention is that it provides
an effective diagnostic of Alzheimer's disease, particularly for
the more prevalent form, sporadic AD.
[0026] Another advantage of the present invention is that it
affords a non-invasive diagnostic that is reliable at or before the
earliest manifestations of AD symptoms.
[0027] Still another object of the present invention is that it
provides an effective therapy that addresses the primary cause of
AD, by suppressing the undesired biological activity of mutations
that segregate with Alzheimer's disease or by selectively
destroying defective mitochondria.
[0028] Other objects and advantages of the invention and
alternative embodiments will readily become apparent to those
skilled in the art, particularly after reading the detailed
description, and examples set forth below.
BRIEF DESCRIPTION OF THE FIGURES
[0029] FIGS. 1A and 1B list the 5' end upstream non-coding region,
the complete nucleic acid sequence encoding mitochondrial
cytochrome c oxidase subunit I and the 3' end downstream non-coding
region. (SEQ. ID. NO. 1).
[0030] FIG. 2 lists the 5' end non-coding region, the complete
nucleic acid sequence of the mitochondrial cytochrome c oxidase
subunit II coding region and the 3' end downstream non-coding
region. (SEQ. ID. NO. 2).
[0031] FIG. 3 lists the 5' end non-coding region, the complete
nucleic acid sequence of the mitochondrial cytochrome c oxidase
subunit III coding region and the 3' end downstream non-coding
region. (SEQ. ID. NO. 3).
[0032] FIG. 4 illustrates a reaction scheme for the preparation of
several acridine orange derivatives useful for the detection and
selective destruction of defective mitochondria.
[0033] FIGS. 5-8 illustrate reaction schemes for the preparation of
several JC-1 derivatives useful for the detection and selective
destruction of defective mitochondria.
DETAILED DESCRIPTION OF THE INVENTION
[0034] The present invention relates to genetic mutations in
mitochondrial cytochrome c oxidase genes which segregate with
Alzheimer's disease. The invention provides methods for detecting
such mutations, as a diagnostic for Alzheimer's disease, either
before or after the onset of clinical symptoms. Moreover, the
invention also pertains to suppression of the undesired biological
activity of the mutations and thus affords a therapeutic treatment
for Alzheimer's disease. Not only does this invention provide the
first effective diagnostic of Alzheimer's disease which is reliable
at or before the earliest manifestations of AD symptoms, it also
provides the first effective therapy for this debilitating
disease.
[0035] In order to facilitate a full and complete understanding of
the present invention, it is important to note that all terms used
herein are intended to have the same meaning as generally ascribed
to those terms by those skilled in the art of molecular genetics,
unless defined to the contrary. The references cited herein are
incorporated by reference in their entireties.
[0036] In using the terms "nucleic acid", RNA, DNA, etc., we do not
mean to limit the chemical structures that can be used in
particular steps. For example, it is well known to those skilled in
the art that RNA can generally be substituted for DNA, and as such,
the use of the term "DNA" should be read by those skilled in the
art to include this substitution. In addition, it is known that a
variety of nucleic acid analogues and derivatives can be made and
will hybridize to one another and to DNA and RNA, and the use of
such analogues and derivatives is also within the scope of the
present invention. Segregation of Cytochrome C Oxidase Mutations
with Alzheimer's Disease Cytochrome c oxidase (COX) is an important
terminal component of the electron transport chain located in the
mitochondria of eukaryotic cells. Cytochrome c oxidase, also known
as complex IV of the electron transport chain, is composed of at
least thirteen subunits. At least ten of these subunits are encoded
by nuclear genes; the remaining three subunits (I, II, and III) are
encoded by mitochondrial genes. Mitochondrial DNA (mtDNA) is a
small circular DNA molecule that is approximately 17 kB long in
humans. The mtDNA encodes for two ribosomal RNAs (rRNA), a complete
set of transfer RNAs (tRNA), and thirteen proteins, including three
cytochrome c oxidase subunits COX I, COX II, and COX III.
[0037] Most of the mtDNA present in an individual is derived from
the mtDNA contained within the ovum at the time of the individual's
conception. Mutations in mtDNA sequence which affect all copies of
mtDNA in an individual are known as homoplasmic. Mutations which
affect only some copies of mtDNA are known as heteroplasmic and
will vary between different mitochondria in the same individual. It
should also be noted that most mitochondrially encoded proteins and
all mitochondrially encoded COX proteins are transcribed from the
heavy strand of mtDNA. The other strand is called the "light
strand," because mtDNA can be separated into heavy and light single
strands on the basis of their density.
[0038] In the present invention, mtDNA from both normal individuals
and known Alzheimer's patients are isolated, cloned and sequenced.
As expected, a few nondeleterious and apparently random mutations
in each gene including some normal genes, are observed. However, in
the AD patients, a small number of homoplasmic or heteroplasmic
mutations at common sites are noted. For the three mitochondrial
COX subunits, the mutations occurred in one or more of the subunit
clones for each individual. Such mutations are especially observed
in the expressed regions of COX subunits I and II of the mtDNA.
[0039] According to the present invention, such mutations in COX
genes segregate with, and are apparently sufficient for,
Alzheimer's disease. Sporadic AD, which accounts for at least 90%
of all AD patients, is segregated with heteroplasmic mutation(s) in
the mtDNA-encoded COX subunits. Detection of these mutations,
therefore, is both predictive and diagnostic of Alzheimer's
disease.
[0040] Blood and brain samples are harvested and DNA isolated from
a number of clinically-classified or autopsy confirmed AD patients,
from a number of documented age-matched `normals` (elderly
individuals with no history of AD or any sign of clinical symptoms
of AD) and from age-matched neurodegenerative disease controls
(patients with Huntington's disease, parasupranuclear palsy, and so
forth). After cloning of cytochrome c oxidase (COX) gene fragments,
the sequences of multiple clones from each patient are obtained.
Compilation of the sequences are made, aligned, and compared with
published Cambridge and Genbank sequences (Anderson et al., Nature
290:457-465 (1981)) for known normal human COX genes. The published
Cambridge coding sequences are numbered as follows: COX I is
nucleotides 5964 to 7505, COX II is nucleotides 7646 to 8329, and
COX III is nucleotides 9267 to 10052. The corresponding sequences
are numbered as follows according to Anderson's scheme: COX I is
nucleotides 5904 to 7445, COX II is nucleotides 7586 to 8269, and
COX III is nucleotides 9207 to 9992. Id. All reference hereinbelow
is made only to the published Cambridge sequences, though it will
be appreciated by those of skill in the art that the corresponding
sequences, following a different numbering scheme, including
Anderson's, could be used in the invention.
[0041] Any variation (mutation, insertion, or deletion) from
published sequences is verified by replication and by complementary
strand sequencing. Analysis of the variations in known AD patients
indicated a significant number of mutations. Some of the mutations
observed are `silent` mutations resulting in no amino acid changes
in the expressed protein. However, a number of mutations present
result in amino acid changes in the corresponding protein. In many
instances the corresponding amino acid change may also lead to
conformational changes to the COX enzyme.
[0042] In cytochrome c oxidase subunit II, for example, the
sequence in AD patients varies from the normal sequence in at least
one base per gene. The data is summarized in Table 2 hereinbelow.
Several of the recurrent mutations observed are believed to result
in conformational alterations of the COX enzyme. For example,
mutation of the normal ACC observed at codon 22 to ATC results in a
change from the normal hydrophilic threonine (Thr) to a hydrophobic
isoleucine (Ile). Changes of this type in nucleic acid structure,
particularly when occurring in highly conserved areas, are known to
disrupt or modify enzymatic activity.
[0043] As described more fully hereinbelow, each of the COX genes
sequenced shows significant variation from the normal sequence at a
number of specific sites, or mutational "hot spots." Moreover,
these hot spots generally fall within particular regions of the COX
genes. In the first 1,530 bases (510 codons) of COX I, and in
particular between codons 155 and 415, codons 155, 167, 178, 193,
194 and 415 have a high degree of mutational similarity in the AD
sequences (see Table 1). In COX II, hot spots occur especially in
the region between codon 20 and codon 150 and in particular at
codons 20, 22, 68, 71, 74, 90, 95, 110 and 146 (see Table 2). In
COX III, codons 64, 76, 92, 121, 131, 148, 241 and 247 appear to be
highly variable hot spots.
[0044] Mutations Observed in COX I Gene of Alzheimer's Patients
[0045] Table 1 below is an example of several mutations and the
number of times a given mutation is observed in ten clones of
mitochondrial cytochrome c oxidase subunit I (COX I) gene for each
of 44 Alzheimer's patients. The, mutations listed for the AD
patients are relative to the published Cambridge sequences for
normal human COX I. The codon number indicated is determined in a
conventional manner from the open reading frame at the 5'-end of
the gene.
1 Codon # 26 52 52 66 66 84 88 103 109 111 136 155 155 Normal AA
Ala His His Ile Ile Pro Gly Trp Leu Leu Tyr Val Val Normal DNA GCT
CAC CAC ATC ATC CCC GGT TGA CTC CTC TAC GTC GTC Observed Thr Tyr
Leu Val Thr Leu Asp Arg Pro Pro His Ile Ala Mutation AD Patient ACT
CCC CTC GTC ACC CTC GAT CGA CCC CCC CAC ATC GCC #1 3A6_KE 1 2 #2
3B1_RI 5 #3 3B2_DA #4 3B3_WO #5 3B4_PI #6 3B5_TR #7 3B6_CR #8
3B7_LF #9 3B8_OB #10 3C1_GU 1 #11 3E3_GE 1 #12 3E4_MI #13 3E5_BE 1
#14 3E6_RE 1 #15 3F6_BJ #16 3G6_BL #17 3G7_SD 1 1 #18 3H1_JY 1 #19
3H2_ML #20 3H3_HA #21 3H5_AS 1 1 #22 3H6_AI #23 3I1_AA 1 #24 3I2_NU
#25 3I6_BC #26 3I7_DN 1 #27 3I8_CO #29 3J1_GR #30 3J3_HW 1 #31
3K2_DM #32 3K8_ZI 1 #33 3D3_LW #34 3D4_AL #35 3A5_YA 2 1 1 #36
8A6_BR 1 #37 8A7_SA 1 #38 8A8_BA #39 8B2_SP #40 8D2_MD 1 #41 8D3_LC
#42 8D4_WI #43 8D5_JE 1 #44 8D6_DE Codon # 167 170 178 193 193 194
200 200 216 221 261 276 330 Normal AA Thr Asn Gln Val Val Leu Pro
Pro Asn Asp Tyr Ala Ser Normal DNA ACA AAT CAA GTC GTC CTA CCA CCA
AAC GAC TAC GCT AGC Observed Ala Ser Leu Ala Ile Phe Leu Ser Asp
Asn Cys Thr Gly Mutation AD Patient GCA AGT CTA GCC ATC TTA CTA TCA
GAC AAC TGC ACT GGC #1 3A6_KE 1 #2 3B1_RI 4 1 #3 3B2_DA 1 1 #4
3B3_WO 1 #5 3B4_PI 1 #6 3B5_TR 1 #7 3B6_CR #8 3B7_LF 1 1 #9 3B8_OB
1 #10 3C1_GU #11 3E3_GE 1 1 #12 3E4_MI 1 #13 3E5_BE #14 3E6_RE 1
#15 3F6_BJ #16 3G6_BL #17 3G7_SD #18 3H1_JY #19 3H2_ML #20 3H3_HA
#21 3H5_AS 1 #22 3H6_AI 1 1 #23 3I1_AA #24 3I2_NU #25 3I6_BC #26
3I7_DN #27 3I8_CO 1 #29 3J1_GR #30 3J3_HW #31 3K2_DM #32 3K8_ZI #33
3D3_LW 1 #34 3D4_AL #35 3A5_YA 1 #36 8A6_BR 7 1 #37 8A7_SA #38
8A8_BA 1 1 #39 8B2_SP 1 #40 8D2_MD #41 8D3_LC #42 8D4_WI 1 #43
8D5_JE 2 #44 8D6_DE 1 Codon # 357 369 415 415 416 456 456 466 468
474 504 Normal AA Val Asp Thr Thr Ile Val Val Met Met Glu Thr
Normal DNA GTA GAC ACT ACT ATC GTA GTA ATA ATA GAA ACA Observed Ala
Gly Ala Ile Thr Ala Mel Thr Val Gly Ala Mutation AD Patient GCA GGC
GCT ATT ACC GCA ATA ACA GTA GGA GCA #1 3A6_KE 2 #2 3B1_RI 5 #3
3B2_DA 1 #4 3B3_WO 2 #5 3B4_PI #6 3B5_TR #7 3B6_CR 1 #8 3B7_LF #9
3B8_OB #10 3C1_GU #11 3E3_GE 1 #12 3E4_MI #13 3E5_BE #14 3E6_RE #15
3E6_BJ 1 #16 3G6_BL 10 #17 3G7_SD 10 1 #18 3H1_JY 1 1 #19 3H2_ML 1
#20 3H3_HA #21 3H5_AS #22 3H6_AI 1 1 #23 3I1_AA 1 1 1 #24 3I2_NU
#25 3I6_BC #26 3I7_DN 1 1 #27 3I8_CO 1 1 #29 3J1_GR #30 3J3_HW #31
3K2_DM #32 3K8_ZI #33 3D3_LW #34 3D4_AL #35 3A5_YA #36 8A6_BR #37
8A7_SA #38 8A8_BA #39 8B2_SP 1 #40 8D2_MD #41 8D3_LC #42 8D4_WI #43
8D5_JE 2 1 #44 8D6_DE
[0046] As evidenced by Table 1, mutational hot spots of COX I in AD
patients are codons 155, 167, 178, 193, 194 and 415.
[0047] Mutations Observed in COX II Gene of Alzheimer's
Patients
[0048] Table 2 below is an example of several mutations and the
number of times a given mutation is observed in ten clones of
mitochondrial cytochrome c oxidase subunit II (COX II) gene for
each of the 44 Alzheimer's patients. The mutations listed for the
AD patients are relative to the published Cambridge sequences for
normal human COX II. The codon number indicated is determined in a
conventional manner from the open reading frame at the 5'-end of
the gene.
2 Codon # 20 21 22 25 26 26 61 68 68 70 71 74 74 76 89 Normal AA
Leu Ile Thr Asp His His Met Leu Leu Ala Ile Val Val Ile Glu Normal
DNA CTT ATC ACC GAT CAC CAC ATA CTG CTG GCC ATC GTC GTC ATC GAG
Observed Pro Thr Ile Asn Tyr Arg Val Pro Phe Thr Thr Ala Ile Val
Gly Mutation ADPatient CCT ACC ATC AAT TAC CGC GTA CCG TTG ACC ACC
GCC ATC GTC GGG #1 3A6_KE 3 2 1 #2 3B1_RI 3 #3 3B2_DA 1 1 #4 3B3_WO
#5 3B4_PI 1 1 #6 3B5_TR #7 3B6_CR 1 1 #8 3B7_LF #9 3B8_OB 1 #10
3C1_GU #11 3E3_GE #12 3E4_MI 1 1 #13 3E5_BE 1 #14 3E6_RE #15 3F6_BJ
#16 3G6_BL #17 3G7_SD 1 #18 3H1_JY 1 1 #19 3H2_ML 1 #20 3H3_HA 1
#21 3H5_AS #22 3H6_AI 1 #23 3I1_AA #24 3I2_NU #25 3I6_BC #26 3I7_DN
1 1 1 #27 3I8_CO 1 #29 3J1_GR 1 1 #30 3J3_HW #31 3K2_DM 1 #32
3K8_ZI #33 3D3_LW #34 3D4_AL 10 #35 8A5_YA #36 8A6_BR #37 8A7_SA 1
10 #38 8A8_BA 1 #39 8B2_SP #40 8D2_MD 10 #41 8D3_LC #42 8D4_WI 1
#43 8D5_JE #44 8D6_DE Codon # 90 95 95 95 110 110 126 146 146 152
157 205 207 224 228 Normal AA Val Leu Leu Leu Tyr Tyr Phe Ile Ile
Met Gln Ser Met Val Ter Normal DNA GTC CTT CTT CTT TAC TAC TTA ATT
ATT ATA CAA AGT ATG GTA TAG Observed Ile Phe Pro Ile Cys His Leu
Val Thr Val Ter Gly Val Met Trp Mutation ADPatient ATC TTT CCT ATT
TGC CAC CTA GTT ACT GTA TAA GGT GTG ATA TGG #1 3A6_KE 8 3 2 #2
3B1_RI 2 3 #3 3B2_DA #4 3B3_WO #5 3B4_PI #6 3B5_TR 1 #7 3B6_CR 1 #8
3B7_LF #9 3B8_OB #10 3C1_GU #11 3E3_GE 10 #12 3E4_MI 1 #13 3E5_BE 1
#14 3E6_RE #15 3F6_BJ 1 1 1 #16 3G6_BL #17 3G7_SD 1 #18 3H1_JY 1
#19 3H2_ML #20 3H3_HA 1 #21 3H5_AS #22 3H6_AI #23 3I1_AA #24 3I2_NU
1 #25 3I6_BC #26 3I7_DN #27 3I8_CO #29 3J1_GR #30 3J3_HW #31 3K2_DM
#32 3K8_ZI #33 3D3_LW #34 3D4_AL 1 1 #35 8A5_YA 1 #36 8A6_BR 1 1
#37 8A7_SA #38 8A8_BA #39 8B2_SP #40 8D2_MD 1 1 #41 8D3_LC 1 #42
8D4_WI #43 8D5_JE 1 #44 8D6_DE 10
[0049] As evidenced by Table 2, the mutational hot spots of COX II
in AD patients are codons 20, 22, 68, 71, 74, 90, 95, 110 and
146.
[0050] At each mutational hot spot, the specific variations noted
in AD patients appear universally. For example, at codon 415 in COX
I, the normal codon is threonine; each of nine AD mutations
observed in codon 415 in COX I codes for alanine. At position 194
in COX I, the aromatic phenylalanine codon replaces the normally
hydrophobic leucine. These specific mutations do not occur randomly
and are not observed in normal or neurological patients which do
not have Alzheimer's disease.
[0051] Table 3 below demonstrates the use of the above mutational
hot spots in the diagnosis of Alzheimer's disease. For each patient
in Table 3, the presence of a mutation at each of codons 155, 167,
178, 193, 194 and 415 of COX I, and each of codons 20, 22, 68, 71,
74, 95, 110 and 146 of COX II is indicated by a shaded box.
[0052] Blood samples are obtained and DNA isolated from a number of
living subjects that are either clinically-classified AD patients
("Blood/AD") or documented age-matched `normals` (elderly
individuals with no family history of AD or any sign of clinical
symptoms of AD) ("Blood/Control"). Of the clinically-classified AD
patients ("Blood/AD"), 61% (22 out of 36) have mutations at one or
more of the above hot spots. 36% (13 out of 36) contain no
mutations. However, as noted above, the diagnosis of probable
Alzheimer's disease is presently limited to clinical observation,
with definitive analysis accomplished only by pathological
examination at autopsy. Moreover, of living patients presently
diagnosed as having AD by clinical observation only about 70 to 80%
are confirmed to have AD upon autopsy. Tierney, M. C. et al.,
Neurology 38:359-364 (1988). The remaining 20 to 30% are
incorrectly diagnosed as having AD, while they actually have
another condition such as senile dementia of the Lewy body variety,
Pick's Disease, parasupranuclear palsy, and so forth. Thus, it is
expected that a significant percentage of the blood samples taken
from living clinically-classified AD patients will not test
positive for AD. Indeed, a contrary result is cause for
concern.
[0053] Of the living documented age-matched normals (Blood/Control)
only 1 out of 14 (7%) had a single hot spot mutation. Moreover, it
is noted that this individual is 65 years old and may yet develop
symptoms of AD.
[0054] Brain samples are also harvested and DNA isolated from a
number of deceased patients that are confirmed to have AD upon
pathological examination at autopsy ("Brain/AD") or deceased
documented age matched `normals` (elderly individuals with no
family history of AD, no sign of clinical symptoms of AD during
life, and no sign of AD upon pathological examination at autopsy)
("Brain/Control"). Brain samples are also harvested and DNA
isolated from a number of deceased patients that are diagnosed upon
autopsy to have other degenerative neurologic disorders selected
from Huntington's disease ("Brain/HD"), non-specific degenerative
disease ("Brain/NSD"), parasupranuclear palsy ("Brain/PSP"), Pick's
disease (Brain/Picks"), Hallervorden Spatz ("Brain/HSP"), diffuse
Lewy body disease ("Brain/DLBD"), atypical tangles ("Brain/AT"),
argyrophyllic grains ("Brain/AG"), senile dementia of the Lewy body
variety ("Brain/LBV").
[0055] Results from the DNA isolated from brain samples clearly
illustrate the specificity of the diagnostic technique of the
present invention. Of the brain samples taken from individuals with
pathologically confirmed AD, 83% (10 or 12) contained one or more
hot spot mutations. Of the two remaining individuals (BA and DE),
BA demonstrated mutations at COX I codons 170 and 276 and COX II
codon 26, while DE demonstrated mutations at COX I codon 221 and
COX II codon 90. Accordingly, it may be desirable to extend to
above list of hot spots. In contrast, none of the age matched
`normals` are found to contain such mutations.
[0056] In addition, of the individuals having other neurologic
disorders, only 2 of 18 (11%) contained a single mutation. This
illustrates that the diagnosis of the present invention is specific
to AD. Moreover, pathologists involved with the autopsy of one of
the two individuals (SC) are unable to definitively clearly
differentiate the dementia with argyrophyllic grains from AD.
Finally, one cannot rule out the possibility that the other
individual (KI) would have manifested symptoms of AD if the
individual had not succumbed to Para-Supranuclear Palsy.
[0057] The invention also includes the isolated nucleotide
sequences which correspond to or are complementary to portions of
mitochondrial cytochrome c oxidase genes which contain gene
mutations that correlate with the presence of Alzheimer's disease.
The isolated nucleotide sequences which contain gene mutations
include COX I nucleotides 5964 to 7505, COX II nucleotides 7646 to
8329 and COX III nucleotides 9267 to 10052.
[0058] Diagnostic Detection of Alzheimer's Disease-Associated
Mutations:
[0059] According to the present invention, base changes in the
mitochondrial COX genes can be detected and used as a diagnostic
for Alzheimer's disease. A variety of techniques are available for
isolating DNA and RNA and for detecting mutations in the isolated
mitochondrial COX genes.
[0060] A number of sample preparation methods are available for
isolating DNA and RNA from patient blood samples. For example, the
DNA from a blood sample is obtained by cell lysis following alkali
treatment. Often, there are multiple copies of RNA message per DNA.
Accordingly, it is useful from the standpoint of detection
sensitivity to have a sample preparation protocol which isolates
both forms of nucleic acid. Total nucleic acid may be isolated by
guanidium isothiocyanate/phenol-chloroform extraction, or by
proteinase K/phenol-chloroform treatment. Commercially available
sample preparation methods such as those from Qiagen Inc.
(Chatsworth, Calif.) can also be utilized.
[0061] As discussed more fully hereinbelow, hybridization with one
or more labelled probes containing complements of the variant
sequences enables detection of the AD mutations. Since each AD
patient can be heteroplasmic (possessing both the AD mutation and
the normal sequence) a quantitative or semi-quantitative measure
(depending on the detection method) of such heteroplasmy can be
obtained by comparing the amount of signal from the AD probe to the
amount from the AD.sup.- (normal or wild-type) probe.
[0062] A variety of techniques, as discussed more fully
hereinbelow, are available for detecting the specific mutations in
the mitochondrial COX genes. The detection methods include, for
example, cloning and sequencing, ligation of oligonucleotides, use
of the polymerase chain reaction and variations thereof, use of
single nucleotide primer-guided extension assays, hybridization
techniques using target-specific oligonucleotides and sandwich
hybridization methods.
[0063] Cloning and sequencing of the COX genes can serve to detect
AD mutations in patient samples. Sequencing can be carried out with
commercially available automated sequencers utilizing fluorescently
labelled primers. An alternate sequencing strategy is the
"sequencing by hybridization" method using high density
oligonucleotide arrays on silicon chips (Fodor et al., Nature
364:555-556 (1993); Pease et al., Proc. Natl. Acad. Sci. USA,
91:5022-5026 (1994). For example, fluorescently-labelled target
nucleic acid generated, for example from PCR amplification of the
target genes using fluorescently labelled primers, are hybridized
with a chip containing a set of short oligonucleotides which probe
regions of complementarity with the target sequence. The resulting
hybridization patterns are useful for reassembling the original
target DNA sequence.
[0064] Mutational analysis can also be carried out by methods based
on ligation of oligonucleotide sequences which anneal immediately
adjacent to each other on a target DNA or RNA molecule (Wu and
Wallace, Genomics 4:560-569 (1989); Landren et al., Science
241:1077-1080 (1988); Nickerson et al., Proc. Natl. Acad. Sci.
87:8923-8927 (1990); Barany, F., Proc. Natl. Acad. Sci. 88:189-193
(1991)). Ligase-mediated covalent attachment occurs only when the
oligonucleotides are correctly base-paired. The Ligase Chain
Reaction (LCR), which utilizes the thermostable Taq ligase for
target amplification, is particularly useful for interrogating AD
mutation loci. The elevated reaction temperatures permits the
ligation reaction to be conducted with high stringency (Barany, F.,
PCR Methods and Applications 1:5-16 (1991)).
[0065] Analysis of point mutations in DNA can also be carried out
by using the polymerase chain reaction (PCR) and variations
thereof. Mismatches can be detected by competitive oligonucleotide
priming under hybridization conditions where binding of the
perfectly matched primer is favored (Gibbs et al., Nucl. Acids.
Res. 17:2437-2448 (1989)). In the amplification refractory mutation
system technique (ARMS), primers are designed to have perfect
matches or mismatches with target sequences either internal or at
the 3' residue (Newton et al., Nucl. Acids. Res. 17:2503-2516
(1989)). Under appropriate conditions, only the perfectly annealed
oligonucleotide functions as a primer for the PCR reaction, thus
providing a method of discrimination between normal and mutant (AD)
sequences.
[0066] Genotyping analysis of the COX genes can also be carried out
using single nucleotide primer-guided extension assays, where the
specific incorporation of the correct base is provided by the high
fidelity of the DNA polymerase (Syvanen et al., Genomics 8:684-692
(1990); Kuppuswamy et al., Proc. Natl. Acad. Sci. USA. 88:1143-1147
(1991)). Another primer extension assay, which allows for the
quantification of heteroplasmy by simultaneously interrogating both
wild-type and mutant nucleotides, is disclosed in a co-pending U.S.
patent application entitled, "Multiplexed Primer Extension
Methods", naming Eoin Fahy and Soumitra Ghosh as inventors, filed
on Mar. 24, 1995, Ser. No. 08/410,658, the disclosure of which is
incorporated by reference.
[0067] Detection of single base mutations in target nucleic acids
can be conveniently accomplished by differential hybridization
techniques using target-specific oligonucleotides (Suggs et al.,
Proc. Natl. Acad. Sci. 78:6613-6617 (1981); Conner et al., Proc.
Natl. Acad. Sci. 80:278-282 (1983); Saiki et al., Proc. Natl. Acad.
Sci. 86:6230-6234 (1989)). For example, mutations are diagnosed on
the basis of the higher thermal stability of the perfectly matched
probes as compared to the mismatched probes. The hybridization
reactions may be carried out in a filter-based format, in which the
target nucleic acids are immobilized on nitrocellulose or nylon
membranes and probed with oligonucleotide probes. Any of the known
hybridization formats may be used, including Southern blots, slot
blots, "reverse" dot blots, solution hybridization, solid support
based sandwich hybridization, bead-based, silicon chip-based and
microtiter well-based hybridization formats.
[0068] An alternative strategy involves detection of the COX genes
by sandwich hybridization methods. In this strategy, the mutant and
wild-type (normal) target nucleic acids are separated from
non-homologous DNA/RNA using a common capture oligonucleotide
immobilized on a solid support and detected by specific
oligonucleotide probes tagged with reporter labels. The capture
oligonucleotides can be immobilized on microtitre plate wells or on
beads (Gingeras et al., J. Infect. Dis. 164:1066-1074 (1991);
Richman et al., Proc. Natl. Acad. Sci. 88:11241-11245 (1991)).
[0069] While radio-isotopic labeled detection oligonucleotide
probes are highly sensitive, non-isotopic labels are preferred due
to concerns about handling and disposal of radioactivity. A number
of strategies are available for detecting target nucleic acids by
non-isotopic means (Matthews et al., Anal. Biochem., 169:1-25
(1988)). The non-isotopic detection method may be direct or
indirect.
[0070] The indirect detection process is generally where the
oligonucleotide probe is covalently labelled with a hapten or
ligand such as digoxigenin (DIG) or biotin. Following the
hybridization step, the target-probe duplex is detected by an
antibody- or streptavidin-enzyme complex. Enzymes commonly used in
DNA diagnostics are horseradish peroxidase and alkaline
phosphatase. One particular indirect method, the Genius.TM.
detection system (Boehringer Mannheim) is especially useful for
mutational analysis of the mitochondrial COX genes. This indirect
method uses digoxigenin as the tag for the oligonucleotide probe
and is detected by an anti-digoxigenin-antibody-alkaline
phosphatase conjugate.
[0071] Direct detection methods include the use of
fluorophor-labeled oligonucleotides, lanthanide chelate-labeled
oligonucleotides or oligonucleotide-enzyme conjugates. Examples of
fluorophor labels are fluorescein, rhodamine and phthalocyanine
dyes. Examples of lanthanide chelates include complexes of
Eu.sup.3+ and Tb.sup.3+. Directly labeled oligonucleotide-enzyme
conjugates are preferred for detecting point mutations when using
target-specific oligonucleotides as they provide very high
sensitivities of detection.
[0072] Oligonucleotide-enzyme conjugates can be prepared by a
number of methods (Jablonski et al., Nucl. Acids Res., 14:6115-6128
(1986); Li et al., Nucl. Acids Res. 15:5275-5287 (1987); Ghosh et
al., Bioconjugate Chem. 1:71-76 (1990)), and alkaline phosphatase
is the enzyme of choice for obtaining high sensitivities of
detection. The detection of target nucleic acids using these
conjugates can be carried out by filter hybridization methods or by
bead-based sandwich hybridization (Ishii et al., Bioconjugate
Chemistry 4:34-41 (1993)).
[0073] Detection of the probe label may be accomplished by the
following approaches. For radioisotopes, detection is by
autoradiography, scintillation counting or phosphor imaging. For
hapten or biotin labels, detection is with antibody or streptavidin
bound to a reporter enzyme such as horseradish peroxidase or
alkaline phosphatase, which is then detected by enzymatic means.
For fluorophor or lanthanide-chelate labels, fluorescent signals
may be measured with spectrofluorimeters with or without
time-resolved mode or using automated microtitre plate readers.
With enzyme labels, detection is by color or dye deposition
(p-nitrophenyl phosphate or 5-bromo-4-chloro-3-indolyl
phosphate/nitroblue tetrazolium for alkaline phosphatase and
3,3'-diaminobenzidine-NiCl.sub.2 for horseradish peroxidase),
fluorescence (e.g., 4-methyl umbelliferyl phosphate for alkaline
phosphatase) or chemiluminescence (the alkaline phosphatase
dioxetane substrates LumiPhos 530 from Lumigen Inc., Detroit Mich.
or AMPPD and CSPD from Tropix, Inc.). Chemiluminescent detection
may be carried out with X-ray or polaroid film or by using single
photon counting luminometers. This is the preferred detection
format for alkaline phosphatase labelled probes.
[0074] The oligonucleotide probes for detection preferably range in
size between 10 and 100 bases, more preferably between 15 and 30
bases in length. Examples of such nucleotide probes are found below
in Tables 4 and 5. Tables 4 and 5 provide representative sequences
of probes for detecting mutations in COX genes and representative
antisense sequences. In order to obtain the required target
discrimination using the detection oligonucleotide probes, the
hybridization reactions are preferably run between 20.degree. C.
and 60.degree. C., and more preferably between 30.degree. C. and
55.degree. C. As known to those skilled in the art, optimal
discrimination between perfect and mismatched duplexes can be
obtained by manipulating the temperature and/or salt concentrations
or inclusion of formamide in the stringency washes.
3TABLE 4 SENSE PROBES-DNA DETECTION OF ANTISENSE STRAND SEQ SEQ AA
LENGTH ID ID GENE NO. (WT) % GC WILD-TYPE NO. MUTANT NO. CCXI 155
23 52.2 5'-ACCTAGCAGGTGTCTCCTCTATC-3' 4
5-ACCTAGCAGGTATCTCCTCTATCT-3' 18 CCXI 167 27 22.2
5'-CAATTTCATCACAACAATTATCAATAT-3' 5
5'-CAATTTCATCACAGCAATTATCAATAT-3' 19 CCXI 178 21 47.6
5'-GCCATAACCCAATACCAAACG-3' 6 5'-GCCATAACCCTATACCAAACG-3' 20 CCXI
193 23 47.8 5-40 -AATCACAGCAGTCCTACTTCTCC-3' 7
5'-AATCACAGCAGCCTACTTCTCC-3' 21 5'-AATCACAGCAATCCTACTTCTCC-3' 22
CCXI 194 25 50.0 5'-TCACAGCAGTCCTACTTCTCCTATC-3' 8
5'-TCACACAGCAGTCTTACTTCTCCTATC-3' 23 CCXI 415 26 26.9
5'-CAAAATCCATTTCACTATCATATTCA-3' 9 5'-AAAATCCATTTCGCTATCATATTCA-3'
24 CCXII 20 25 37.5 5'-TCATAGAAGAGCTTATCACCTTTCA-3' 10
5'-TCATAGGAAGAGCCTATCACCTTTCA-3' 25 CCXII 22 25 37.5
5'-AGAGCTTATCACCCTTTCATGATCA-3' 11 5'-AGAGCTTATCATCTTTCATGATCA-3'
26 CCXII 68 18 61.1 5'-TGCCCGCCATCATCCTAG-3' 12
5'-TGAACTATCTTGCCCGCC-3' 27 CCXII 71 18 61.1
5'-TGCCCGCCATCATCCTAG-3' 13 5'-TGCCCGCCACCATCCTAG-3' 28 CCXII 74 21
52.4 5'-ATCATCCTAGTCCTCATCGCC-3' 14 5'-ATCATCCTAATCCTCATCGCC-3' 29
CCXII 95 21 47.6 5'-GATCCCTCCCTTACCATCAAA-3' 15
5'-GATCCCTCCTTTACCATCAAAT-3' 30 5'-GATCCCTCCCCTACCATCAAA-3' 31
CCXII 110 23 52.2 5'-AACCTACGAGACACCGACTACG-3' 16
5'-AACCTACGAGCACACCGACTAC-3' 32 5'-AACCTACGAGTGCACCGACTAC-3' 33
CCXII 146 20 55.0 5'-AGTACTCCCGATTGAAGCCC-3' 17
5'-AGTACCCGGTTGAAGCCC-3' 34
[0075]
4TABLE 5 ANTISENSE PROBES-DNA AND RNA DETECTION OF SENSE SEQUENCE
SEQ SEQ AA LENGTH ID ID GENE NO. (WT) % GC WILD-TYPE NO. MUTANT NO.
COXI 155 23 52.2 5'-GATAGAGGAGACACCTGCTAGGT-3' 35
5'-AGATAGAGGAGATACCTGCTAGGT-3' 49 CDXI 167 27 22.2
5'-ATATTGATAATTGTTGTAGATGAAATTG-3' 36
5'-ATATTGATAATTGCTGTGATGAAATTG-3' 50 CDXI 178 21 47.6
5'-CGTTTGGTATTGGGTTATGGC-3' 37 5'-CGTTTGGTATAGGGTTATGGC-3' 51 CDXI
193 23 47.8 5'-GGAGAAGTAGGACTGCTGTGATT-3' 38
5'-GGAGAAGTAGGGCTGCTGTGATT-3' 52 5'-GGAGAAGTAGGATTGCTGTGATT-- 3' 53
CDXI 194 25 50.0 5'-GATAGGAGAAGTAGGACTGCTGTGA-3' 39
5'-GATAGGAGAAGTAAGACTGCTGTGA-3' 54 CDXI 415 26 26.9 5'-
TGAATATGATAGTGAAATGGATTTTG-3' 40 5'-TGAATATGATAGCGAAATGGATTTT-3' 55
CDXII 20 25 37.5 5'-TGAAAGGTGATAAGCTCTTCTATGA-3' 41
5'-TGAAAGGTGATAGGCTCTTCTATGA-3' 56 COXII 22 24 37.5
5'-TGATCATGAAAGGTGATAAGCTCTT-3' 42 5'-TGATCATGAAAGATGATAAGCTCT-3'
57 CDXII 68 18 61.1 5'-GGCGGGCAGGATAGTTCA-3' 43
5'-GGCGGGCAAGATAGTTCA-3' 58 CDXII 71 18 61.1
5'-CIAGGATGATGGCGGGCA-3' 44 5'-GGCGGGCAAGATAGTTCA-3' 59 CDXII 74 21
52.4 5'-GGCGATGACCACTAGGATGAT-3' 45 5'-GGCGATGAGGATTAGGATGAT-3' 60
CDXII 95 21 47.6 5'-TTTGATGGTAAGGGAGGGATC-3' 46
5'-ATTTGATGGTAAAGGAGGGATC-3' 61 5'-TTTGATGGTAGGGGGAGGGATC-3' 62
CDXII 110 23 52.2 5'-CGTAGTCGGTGTACTCGTAGGTT-3' 47
5'-GTAGTCGGTCTGCTCGTAGGTT-3' 63 CDXII 110 23 52.2
5'-GTAGTCGGTGCACTCGTAGGTT-3' 64 CCXII 146 20 55.0
5'-GGGCTTCAATCGGGAGTACT-3' 48 5'-GGGCTCAACCGGGAAGTACT-3' 65
[0076] As an alternative to detection of mutations in the nucleic
acids associated with the COX genes, it is also possible to analyze
the protein products of the COX genes. In particular, point
mutations in cytochrome c oxidase subunits 1 and 2 are expected to
alter the structure of the proteins for which these gene encode.
These altered proteins (variant polypeptides) can be isolated and
used to prepare antisera and monoclonal antibodies that
specifically detect the products of the mutated genes and not those
of non-mutated or wild-type genes. Mutated gene products also can
be used to immunize animals for the production of polyclonal
antibodies. Recombinantly produced peptides can also be used to
generate polyclonal antibodies. These peptides may represent small
fragments of gene products produced by expressing regions of the
mitochondrial genome containing point mutations.
[0077] More particularly, as discussed, for example, in
PCT/US93/10072, variant polypeptides from point mutations in
cytochrome c oxidase subunits 1 and 2 can be used to immunize an
animal for the production of polyclonal antiserum. For example, a
recombinantly produced fragment of a variant polypeptide can be
injected into a mouse along with an adjuvant so as to generate an
immune response. Murine immunoglobulins which bind the recombinant
fragment with a binding affinity of at least 1.times.10.sup.7
M.sup.-1 can be harvested from the immunized mouse as an antiserum,
and may be further purified by affinity chromatography or other
means. Additionally, spleen cells are harvested from the mouse and
fused to myeloma cells to produce a bank of antibody-secreting
hybridoma cells. The bank of hybridomas can be screened for clones
that secrete immunoglobulins which bind the recombinantly produced
fragment with an affinity of at least 1.times.10.sup.6 M.sup.-1.
More specifically, immunoglobulins that selectively bind to the
variant polypeptides but poorly or not at all to wild-type
polypeptides are selected, either by pre-absorption with wild-type
proteins or by screening of hybridoma cell lines for specific
idiotypes that bind the variant, but not wild-type,
polypeptides.
[0078] Nucleic acid sequences capable of ultimately expressing the
desired variant polypeptides can be formed from a variety of
different polynucleotides (genomic or cDNA, RNA, synthetic
oligonucleotides, etc.) as well as by a variety of different
techniques.
[0079] The DNA sequences can be expressed in hosts after the
sequences have been operably linked to (i.e., positioned to ensure
the functioning of) an expression control sequence. These
expression vectors are typically replicable in the host organisms
either as episomes or as an integral part of the host chromosomal
DNA. Commonly, expression vectors can contain selection markers
(e.g., markers based on tetracyclinic resistance or hygromycin
resistance) to permit detection and/or selection of those cells
transformed with the desired DNA sequences. Further details can be
found in U.S. Pat. No. 4,704,362.
[0080] Polynucleotides encoding a variant polypeptide may include
sequences that facilitate transcription (expression sequences) and
translation of the coding sequences such that the encoded
polypeptide product is produced. Construction of such
polynucleotides is well known in the art. For example, such
polynucleotides can include a promoter, a transcription termination
site (polyadenylation site in eukaryotic expression hosts), a
ribosome binding site, and, optionally, an enhancer for use in
eukaryotic expression hosts, and, optionally, sequences necessary
for replication of a vector.
[0081] E. coli is one prokaryotic host useful particularly for
cloning DNA sequences of the present invention. Other microbial
hosts suitable for use include bacilli, such as Bacillus subtilus,
and other enterobacteriaceae, such as Salmonella, Serratia, and
various Pseudomonas species. In these prokaryotic hosts one can
also make expression vectors, which will typically contain
expression control sequences compatible with the host cell (e.g.,
an origin of replication). In addition, any number of a variety of
well-known promoters will be present, such as the lactose promoter
system, a tryptophan (Trp) promoter system, a beta-lactamase
promoter system, or a promoter system from phage lambda. The
promoters will typically control expression, optionally with an
operator sequence, and have ribosome binding site sequences, for
example, for initiating and completing transcription and
translation.
[0082] Other microbes, such as yeast, may also be used for
expression. Saccharomyces can be a suitable host, with suitable
vectors having expression control sequences, such as promoters,
including 3-phosphoglycerate kinase or other glycolytic enzymes,
and an origin of replication, termination sequences, etc. as
desired.
[0083] In addition to microorganisms, mammalian tissue cell culture
may also be used to express and produce the polypeptides of the
present invention. Eukaryotic cells are actually preferred, because
a number of suitable host cell lines capable of secreting intact
human proteins have been developed in the art, and include the CHO
cell lines, various COS cell lines, HeLa cells, myeloma cell lines,
Jurkat cells, and so forth. Expression vectors for these cells can
include expression control sequences, such as an origin of
replication, a promoter, an enhancer, and necessary information
processing sites, such as ribosome binding sites, RNA splice sites,
polyadenylation sites, and transcriptional terminator sequences.
Preferred expression control sequences are promoters derived from
immunoglobulin genes, SV40, Adenovirus, Bovine Papilloma Virus, and
so forth. The vectors containing the DNA segments of interest
(e.g., polypeptides encoding a variant polypeptide) can be
transferred into the host cell by well-known methods, which vary
depending on the type of cellular host. For example, calcium
chloride transfection is commonly utilized for prokaryotic cells,
whereas calcium phosphate treatment or electroporation may be used
for other cellular hosts.
[0084] The method lends itself readily to the formulation of test
kits which can be utilized in diagnosis. Such a kit would comprise
a carrier compartmentalized to receive in close confinement one or
more containers wherein a first container may contain suitably
labeled DNA probes. Other containers may contain reagents useful in
the localization of the labeled probes, such as enzyme substrates.
Still other containers may contain restriction enzymes, buffers
etc., together with instructions for use. Therapeutic treatment of
Alzheimer's Disease:
[0085] Suppressing the effects of the mutations through antisense
technology provides an effective therapy for AD. Much is known
about `antisense` therapies targeting messenger RNA (mRNA) or
nuclear DNA. Hlen et al., Biochem. Biophys. Acta 1049:99-125
(1990). The diagnostic test of the present invention is useful for
determining which of the specific AD mutations exist in a
particular AD patient; this allows for "custom" treatment of the
patient with antisense oligonucleotides only for the detected
mutations. This patient-specific antisense therapy is also novel,
and minimizes the exposure of the patient to any unnecessary
antisense therapeutic treatment. As used herein, an "antisense"
oligonucleotide is one that base pairs with single stranded DNA or
RNA by Watson-Crick base pairing and with duplex target DNA via
Hoogsteen hydrogen bonds.
[0086] Without wishing to be held to any particular theory, it has
been postulated that the destructive effects of mutations in the
cytochrome c oxidase gene arise from the production of the radicals
due to faults in the election transport chain. The effects of such
free radicals is expected to be cumulative, especially in view of
the lack of mechanisms for suppressing mutations in
mitochondria.
[0087] The destructive effect of the AD mutations in cytochrome c
oxidase genes is preferably reduced or eliminated using antisense
oligonucleotide agents. Such antisense agents target mitochondrial
DNA, by triplex formation with double-stranded DNA, by duplex
formation with single-stranded DNA during transcription, or both.
In a preferred embodiment, antisense agents target messenger RNA
coding for the mutated cytochrome c oxidase gene(s). Since the
sequences of both the DNA and the mRNA are the same, it is not
necessary to determine accurately the precise target to account for
the desired effect. Procedures for inhibiting gene expression in
cell culture and in vivo can be found, for example, in C. F.
Bennett, et al. J. Liposome Res., 3:85 (1993) and C. Wahlestedt, et
al. Nature, 363:260 (1993).
[0088] Antisense oligonucleotide therapeutic agents demonstrate a
high degree of pharmaceutical specificity. This allows the
combination of two or more antisense therapeutics at the same time,
without increased cytotoxic effects. Thus, when a patient is
diagnosed as having two or more AD mutations in COX genes, the
therapy is preferably tailored to treat the multiple mutations
simultaneously. When combined with the present diagnostic test,
this approach to "patient-specific therapy" results in treatment
restricted to the specific mutations detected in a patient. This
patient-specific therapy circumvents the need for `broad spectrum`
antisense treatment using all possible mutations. The end result is
less costly treatment, with less chance for toxic side effects.
[0089] One method to inhibit the synthesis of proteins is through
the use of antisense or triplex oligonucleotides, analogues or
expression constructs. These methods entail introducing into the
cell a nucleic acid sufficiently complementary in sequence so as to
specifically hybridize to the target gene or to mRNA. In the event
that the gene is targeted, these methods can be extremely efficient
since only a few copies per cell are required to achieve complete
inhibition. Antisense methodology inhibits the normal processing,
translation or half-life of the target message. Such methods are
well known to one skilled in the art.
[0090] Antisense and triplex methods generally involve the
treatment of cells or tissues with a relatively short
oligonucleotide, although longer sequences can be used to achieve
inhibition. The oligonucleotide can be either deoxyribo- or
ribonucleic acid and must be of sufficient length to form a stable
duplex or triplex with the target RNA or DNA at physiological
temperatures and salt concentrations. It should also be
sufficiently complementary or sequence specific to specifically
hybridize to the target nucleic acid. Oligonucleotide lengths
sufficient to achieve this specificity are preferably about 10 to
60 nucleotides long, more preferably about 10 to 20 nucleotides
long. However, hybridization specificity is not only influenced by
length and physiological conditions but may also be influenced by
such factors as GC content and the primary sequence of the
oligonucleotide. Such principles are well known in the art and can
be routinely determined by one who is skilled in the art.
[0091] As an example, many of the oligonucleotide sequences used in
connection with probes in Tables 4 and 5 can also be used as
antisense agents, directed to either the mitochondrial DNA or
resultant messenger RNA.
[0092] A great range of antisense sequences can be designed for a
given mutation. For example, oligonucleotide sequences can be
selected from the following list to function as RNA and DNA
antisense sequences for the mutant mitochondrial gene COX1, Codon
193.
[0093] As can be seen, permutations can be generated for a selected
mutant antigene by truncating the 5' end, truncating the 3' end,
extending the 5' end, or extending the 3' end. Both light chain and
heavy chain mtDNA can be targeted. Other variations such as
truncating the 5' end and truncating the 3' end, extending the 5'
end and extending the 3' end, and truncating the 5' end and
extending the 3' end, extending the 5' end and truncating the 3'
end, and so forth are possible.
5 Antigens to heavy chain mtDNA, wild-type sequence: 5'-AAT CAC AGC
AGT CCT ACT TCT CC SEQ ID NO:7 Antigens to heavy chain mtDNA,
mutant sequence: 5'-AAT CAC AGC AGC CCT ACT TCT CC SEQ ID NO:21 3'
truncation: 5'-AAT CAC AGG AGC CCT ACT TCT C SEQ ID NO:66 5'-AAT
CAC AGC AGC CCT ACT TCT SEQ ID NO:67 5'-AAT CAC AGC AGG CCT ACT TC
SEQ ID NO:68 5'-AAT CAC AGC AGC CCT ACT T SEQ ID NO:69 5'-AAT CAC
AGC AGG CCT ACT SEQ ID NO:70 5'-AAT CAC AGC AGC CCT AC SEQ ID NO:71
5'-AAT CAC AGC AGC CCT A SEQ ID NO:72 5' truncation: 5'-AT CAC AGC
AGC CCT ACT TCT CC SEQ ID NO:73 5'-T CAC AGC AGC CCT ACT TCT CC SEQ
ID NO:74 5'-CAC AGC AGC CCT ACT TCT CC SEQ ID NO:75 5'-AC AGC AGC
CCT ACT TCT CC SEQ ID NO:76 5'-C AGC AGC CCT ACT TCT CC SEQ ID
NO:77 5'-AGC AGC CCT ACT TCT CC SEQ ID NO:78 3' and 5' truncation:
5'-AT CAC AGC AGC CCT ACT TCT C SEQ ID NO:79 5'-T CAC AGC AGC CCT
ACT TCT SEQ ID NO:80 5'-CAC AGC AGC CCT ACT TC SEQ ID NO:81 5'-AC
AGC AGC CCT ACT T SEQ ID NO:82 5' and 3' extension 5'-C CGT CCT AAT
CAC AGC AGC CCT ACT TCT CCT ATC TCT SEQ ID NO:83 5'-CGT CCT AAT CAC
AGC AGC CCT ACT TCT CCT ATC TCT SEQ ID NO:84 5'-GT CCT AAT CAC AGC
AGC CCT ACT TCT CCT ATC TCT SEQ ID NO:85 5'-T CCT AAT CAC AGC AGC
CCT ACT TCT CCT ATC TCT SEQ ID NO:86 5'-CCT AAT CAC AGC AGC CCT ACT
TCT CCT ATC TCT SEQ ID NO:87 5'-CT AAT CAC AGC AGC CCT ACT TCT CCT
ATC TCT SEQ ID NO:88 5'-T AAT CAC AGC AGC CCT ACT TCT CCT ATC TCT
SEQ ID NO:89 5' extension, 3' extension, or both, keeping length
constant: 5'-C CGT CCT AAT CAC AGC AGC CCT ACT TCT CC SEQ ID NO:90
5'-CGT CCT AAT CAC AGC AGC CCT ACT TCT CCT SEQ ID NO:91 5'-GT CCT
AAT CAC AGC AGC CCT ACT TCT CCT A SEQ ID NO:92 5'-T CCT AAT CAC AGC
AGC CCT ACT TCT CCT AT SEQ ID NO:93 5'-CCT AAT CAC AGC AGC CCT ACT
TCT CCT ATC SEQ ID NO:94 5'-CT AAT CAC AGC AGC CCT ACT TCT CCT ATC
T SEQ ID NO:95 5'-T AAT CAC AGC AGC CCT ACT TCT CCT ATC TO SEQ ID
NO:96 5'-AAT CAC AGC AGC CCT ACT TCT CCT ATC TCT SEQ ID NO:97
Antigens to light chain mtDNA, wild-type sequence: 3'-TTA GTG TCG
TCA GGA TGA AGA GG SEQ ID NO:98 Antigens to light chain mtDNA,
mutant sequence: 3'-TTA GTG TCG TCC GGA TGA AGA GG SEQ ID NO:99 5'
truncation: 3'-TTA GTG TCG TCC GGA TGA AGA G SEQ ID NO:100 3'-TTA
GTG TCG TCC GGA TGA AGA SEQ ID NO:101 3'-TTA GTG TCG TCC GGA TGA AG
SEQ ID NO:102 3'-TTA GTG TCG TCC GGA TGA A SEQ ID NO:103 3'-TTA GTG
TCG TCC GGA TGA SEQ ID NO:104 3'-TTA GTG TCG TCC GGA TG SEQ ID
NO:105 3'-TTA GTG TCG TCC GGA T SEQ ID NO:106 3' truncation: 3'-TA
GTG TCG TCC GGA TGA AGA GG SEQ ID NO:107 3'-A GTG TCG TCC GGA TGA
AGA GG SEQ ID NO:108 3'-GTG TCG TCC GGA TGA AGA GG SEQ ID NO:109
3'-TG TCG TCC GGA TGA AGA GG SEQ ID NO:110 3'-G TCG TCC GGA TGA AGA
GG SEQ ID NO:111 3'-TCG TCC GGA TGA AGA GG SEQ ID NO:112 3' and 5'
truncation: 3'-TA GTG TCG TCC GGA TGA AGA G SEQ ID NO:113 3'-A GTG
TCG TCC GGA TGA AGA SEQ ID NO:114 3'-GTG TCG TCC GGA TGA AG SEQ ID
NO:115 3'-TG TCG TCC GGA TGA A SEQ ID NO:116 3' and 5' extension:
3'-G GCA GGA TTA GTG TCG TCC GGA TGA AGA GGA TAG AGA SEQ ID NO:117
3'-GCA GGA TTA GTG TCG TCC GGA TGA AGA GGA TAG AGA SEQ ID NO:118
3'-OA GGA TTA GTG TCG TCC GGA TGA AGA GGA TAG AGA SEQ ID NO:119
3'-A GGA TTA GTG TCG TCC GGA TGA AGA GGA TAG AGA SEQ ID NO:120
3'-GGA TTA GTG TCG TCC GGA TGA AGA GGA TAG AGA SEQ ID NO:121 3'-GA
TTA GTG TCG TCC GGA TGA AGA GGA TAG AGA SEQ ID NO:122 3'-A TTA GTG
TCG TCC GGA TGA AGA GGA TAG AGA SEQ ID NO:123 3' extension, 5'
extension, or both, keeping length constant: 3'-G GCA GGA TTA GTG
TCG TCC GGA TGA AGA GG SEQ ID NO:124 3'-GCA GGA TTA GTG TCG TCC GGA
TGA AGA GGA SEQ ID NO:125 3'-CA GGA TTA GTG TCG TCC GGA TGA AGA GGA
T SEQ ID NO:126 3'-A GGA TTA GTG TCG TCC GGA TGA AGA GGA TA SEQ ID
NO:127 3'-GGA TTA GTG TCG TCC GGA TGA AGA GGA TAG SEQ ID NO:128
3'-GA TTA GTG TCG TCC GGA TGA AGA GGA TAG A SEQ ID NO:129 3'-A TTA
GTG TCG TCC GGA TGA AGA GGA TAG AG SEQ ID NO:130 3'-TTA GTG TCG TCC
GGA TGA AGA GGA TAG AGA SEQ ID NO:131
[0094] The composition of the antisense or triplex oligonucleotides
can also influence the efficiency of inhibition. For example, it is
preferable to use oligonucleotides that are resistant to
degradation by the action of endogenous nucleases. Nuclease
resistance will confer a longer in vivo half-life to the
oligonucleotide thus increasing its efficacy and reducing the
required dose. Greater efficacy may also be obtained by modifying
the oligonucleotide so that it is more permeable to cell membranes.
Such modifications are well known in the art and include the
alteration of the negatively charged phosphate backbone bases, or
modification of the sequences at the 5' or 3' terminus with agents
such as intercalators and crosslinking molecules. Specific examples
of such modifications include oligonucleotide analogs that contain
methylphosphonate (Miller, P. S., Biotechnology, 2:358-362 (1991)),
phosphorothioate (Stein, Science 261:1004-1011 (1993)) and
phosphorodithioate linkages (Brill, W. K-D., J. Am. Chem. Soc.,
111:2322 (1989)). Other types of linkages and modifications exist
as well, such as a polyamide backbone in peptide nucleic acids
(Nielson et al., Science 254:1497 (1991)), formacetal (Matteucci,
M., Tetrahedron Lett. 31:2385-2388 (1990)) carbamate and morpholine
linkages as well as others known to those skilled in the art. In
addition to the specificity afforded by the antisense agents, the
target RNA or genes can be irreversibly modified by incorporating
reactive functional groups in these molecules which covalently link
the target sequences e.g. by alkylation.
[0095] Recombinant methods known in the art can also be used to
achieve the antisense or triplex inhibition of a target nucleic
acid. For example, vectors containing antisense nucleic acids can
be employed to express protein or antisense message to reduce the
expression of the target nucleic acid and therefore its activity.
Such vectors are known or can be constructed by those skilled in
the art and should contain all expression elements necessary to
achieve the desired transcription of the antisense or triplex
sequences. Other beneficial characteristics can also be contained
within the vectors such as mechanisms for recovery of the nucleic
acids in a different form. Phagemids are a specific example of such
beneficial vectors because they can be used either as plasmids or
as bacteriophage vectors. Examples of other vectors include
viruses, such as bacteriophages, baculoviruses and retroviruses,
cosmids, plasmids, liposomes and other recombination vectors. The
vectors can also contain elements for use in either procaryotic or
eukaryotic host systems. One of ordinary skill in the art will know
which host systems are compatible with a particular vector.
[0096] The vectors can be introduced into cells or tissues by any
one of a variety of known methods within the art. Such methods are
described for example in Sambrook et al., Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory, New York (1992),
which is hereby incorporated by reference, and in Ausubel et al.,
Current Protocols in Molecular Biology, John Wiley and Sons,
Baltimore, Md. (1989), which is also hereby incorporated by
reference. The methods include, for example, stable or transient
transfection, lipofection, electroporation and infection with
recombinant viral vectors. Introduction of nucleic acids by
infection offers several advantages over the other listed methods
which includes their use in both in vitro and in vivo settings.
Higher efficiency can also be obtained due to their infectious
nature. Moreover, viruses are very specialized and typically infect
and propagate in specific cell types. Thus, their natural
specificity can be used to target the antisense vectors to specific
cell types in vivo or within a tissue or mixed culture of cells.
Viral vectors can also be modified with specific receptors or
ligands to alter target specificity through receptor mediated
events.
[0097] A specific example of a viral vector for introducing and
expressing antisense nucleic acids is the adenovirus derived vector
Adenop53TX. This vector expresses a herpes virus thymidine kinase
(TX) gene for either positive or negative selection and an
expression cassette for desired recombinant sequences such as
antisense sequences. This vector can be used to infect cells
including most cancers of epithelial origin, glial cells and other
cell types. This vector as well as others that exhibit similar
desired functions can be used to treat a mixed population of cells
to selectively express the antisense sequence of interest. A mixed
population of cells can include, for example, in vitro or ex vivo
culture of cells, a tissue or a human subject.
[0098] Additional features may be added to the vector to ensure its
safety and/or enhance its therapeutic efficacy. Such features
include, for example, markers that can be used to negatively select
against cells infected with the recombinant virus. An example of
such a negative selection marker is the TK gene described above
that confers sensitivity to the antibiotic gancyclovir. Negative
selection is therefor a means by which infection can be controlled
because it provides inducible suicide through the addition of
antibiotics. Such protection ensures that if, for example,
mutations arise that produce mutant forms of the viral vector or
antisense sequence, cellular transformation will not occur.
Moreover, features that limit expression to particular cell types
can also be included. Such features include, for example, promoter
and expression elements that are specific for the desired cell
type.
[0099] The present invention also provides methods for the
selective destruction of defective mitochondria. Since the
mitochondrial genome is heteroplasmic (i.e., it contains mutated
and normal DNA), this will leave intact mitochondria carrying
normal or wild-type DNA and these normal mitochondria will
repopulate the targeted tissue, normalizing mitochondrial function.
This can be accomplished by identifying unique characteristics of
mitochondria carrying mutated DNA, designing a small molecule that
is directed at one or more of these unique characteristics, and
conjugating a mitochondrial toxin to this small molecule. Thus, a
"targeting molecule" is any molecule that selectively accumulates
in mitochondria having defective cytochrome c oxidase activity, and
includes acridine orange derivatives and JC-1 derivatives as
discussed hereinbelow. "Mitochondrial toxins" are molecules that
destroy or disable the selected mitochondria, and include
phosphate, thiophosphate, dinitrophenol, maleimide and antisense
oligonucleotides such as those discussed above. The toxin will be
concentrated within the defective mitochondria by the targeting
molecule and will disable or destroy selectively the defective
mitochondria. The molecule may be an active mitochondrial toxin in
its conjugated form. However, it is preferred to design the
molecule such that it is inactive in its conjugated form. The
chemical linkage between the targeting molecule and the toxin may
be a substrate for a mitochondria-specific enzyme or sensitive to
redox cleavage. Choice of the linkage depends upon the chemical
nature of the targeting molecule and toxin and the requirements of
the cleavage process. Once the conjugate is concentrated in the
defective mitochondria, the toxin is cleaved from the targeting
molecule, activating the toxin.
[0100] Mitochondria with defective cytochrome c oxidase activity
exhibit impaired electron transport, leading to decreased synthesis
of adenosine triphosphate and general bioenergetic failure. As a
consequence, mitochondria carrying mutated DNA will become enlarged
and the intramitochondrial membrane potential increases.
[0101] Enlarged mitochondria have increased levels of cardiolipin
and other negatively charged phospholipids. The acridine orange
derivative 10N-nonylacridine orange (NAO) binds relatively
specifically to cardiolipin and accumulates in dysfunctional
mitochondria. The accumulation of NAO and other chemical
derivatives of acridine orange, including but not limited to those
with aliphatic chains of variable length attached to the ring
nitrogen of acridine orange ([3,6-bis (dimethyl-amino) acridine]),
such as 10N-pentylacridine orange, 10N-octylacridine orange, and
dodecylacridine orange, is independent of the mitochondrial
transmembrane potential. Maftah et al., Biochemical and Biophysical
Research Communications 164(1):185-190 (1989)). At concentrations
up to 1 .mu.M, NAO and its derivatives can be used to target other
molecules to the inner mitochondrial matrix. If the NAO is
chemically linked to a mitochondrial toxin such as phosphate,
thiophosphate, dinitrophenol, maleimide and antisense
oligonucleotides, then mitochondria accumulating the
NAO-mitochondrial toxin conjugate can be selectively disabled or
destroyed. Alternately, at high concentrations (3-10 .mu.M) NAO and
its derivatives inhibit electron transport, ATP hydrolysis and
P.sub.1-transport and disrupt respiration. (Maftah et al., FEBS
Letters 260(2):236-240 (1990). At these concentrations, NAO is
mitochondrial toxin.
[0102] According to an embodiment of the present invention, the
terminus of any aliphatic or other type of chain (such as
polyethylene glycol) attached to the ring nitrogen of acridine
orange is chemically derivatized with carboxylic acid, hydroxyl,
sulfhydryl, amino or similar groups to accept any mitochondrial
toxin. In other embodiments, additional sites of attachment of the
mitochondrial toxin to acridine orange and acridine orange
derivatives are selected. For example, the
10-N-(10-hydroxy-1-decyl)-3,6-bis(dimethylamino)acridine bromide
salt may be prepared and further derivatized to
10-N-(10-phosphoryl-1-decyl)-3,6-b- is(dimethylamino) acridine
chloride salt or 10-N-(10-thiophosphoryl-1-decy-
l)-3,6-bis(dimethylamino) acridine chloride salt. Alternately,
10-N-(11-undecanoic acid)-3,6-bis(dimethylamino) acridine bromide
salt may be prepared and further derivatized to
10-N-(11-undecan-1-oic acid 2,4-dinitrophenyl
ester)-3,6-bis(dimethylamino) acridine bromide salt. Upon cleavage,
the phosphate, thiphospate or dinitrophenol levels selectively
increase within defective mitochondria and destroy them. The
functionalization and covalent attachment of the toxin does not
need to depend on subsequent release of the toxin by cleavage of
the NAO from the toxin, if the attachment point on the toxin is
non-interfering with the function of the toxin within the
mitochondria.
[0103] Several examples of the preparation of acridine orange
derivatives are summarized in FIG. 4 and in Examples IX(a)-IX(f)
hereinbelow. Other modifications are permitted as known to those
skilled in the art.
[0104] Still other embodiments of the present invention target
changes in the intramitochondrial membrane potential due to
defective cytochrome c oxidase activity. Delocalized lipophilic
cations have been used to monitor mitochondrial membrane potential.
The uptake of these cations is related to the presence of the
negative sink inside the mitochondria created by the proton pump.
As mitochondria increase in size due to cytochrome c oxidase
defects, the transmembrane potential will increase and these
defective mitochondria will accumulate lipophilic cations.
According to an embodiment of the present invention, these
lipophilic cations are conjugated to mitochondrial toxins and used
to destroy defective mitochondria that possess increased
transmembrane potentials. Rhodamine-123 the hydrated form of which
is as follows: 1
[0105] has been used extensively to monitor mitochondrial membrane
potential and can conjugate to mitochondrial toxins to concentrate
toxins within the mitochondria. The compound
5,5',6,6'-tetrachloro-1,1',3,3'-tet-
raethylbenzimidiazolo-carbocyanine iodide (JC-1) also accumulates
in mitochondria dependent upon the transmembrane potential. When
JC-1 exceeds a critical concentration, J-aggregates form in the
mitochondrial matrix, and their size causes these JC-1 J-aggregates
to diffuse slowly out of the mitochondria (Reers et al.,
Biochemistry, 30(18):4480-4486 (1991)). JC-1 may be chemically
conjugated to a mitochondrial toxin, producing a long-lived toxic
compound to mitochondria displaying increased transmembrane
potential relative to normal mitochondria.
[0106] As with NAO, by adding a functional group to the JC-1
structure one can covalently attach another chemical entity to the
JC-1 subunit. Delivery to the cells then causes the dual agent to
be preferentially transported into the mitochondria, where the dual
agent may be cleaved at the covalent attachment to release a toxin
within the mitochondria where it exerts the desired effect.
Alternatively, the functionalization and covalent attachment of the
toxin does not need to depend on subsequent release of the toxin by
cleavage of the JC-1 from the active agent, if the attachment point
on the active species is non-interfering with the function of the
toxin within the mitochondria.
[0107] FIGS. 5, 6 and 7 outline the functionalization of JC-1 by
several different methods. Examples IX(g)-IX(f) hereinbelow
illustrate an oxygen functionality, but the same can be
accomplished with a nitrogen, sulfur or carboxylic acid
functionality.
[0108] By utilizing the quasi-symmetrical nature of JC-1, a new
chemical entity may be synthesized that is "half" JC-1 and contains
a functional group capable of being used as a point for covalent
attachment of another chemical entity to the JC-1 subunit. The
existence of the JC-1 subunit facilitates selective transport of
the whole molecule to the mitochondria where, if desired, enzymes
effect cleavage of the JC-1 subunit from the-toxin, allowing it to
exert the desired effect. Alternatively, the functionalization and
covalent attachment of the toxin does not need to depend on
subsequent release of the toxin by cleavage of the JC-1 subunit
from the toxin, if the attachment point on the toxin is
non-interfering with the function of the active agent within the
mitochondria.
[0109] FIG. 8 outlines the synthesis of a functionalized "half"
JC-1 subunit by several different methods. The attachment of the
active chemical species is via the heteroatom incorporated in the
JC-1 or "half" JC-1 structure. This attachment may be accomplished
by any number of linking strategies such as by taking advantage of
a functionality on the active molecule (such as a carboxylic acid
to form an ester with the oxygen of the altered JC-1) or by using a
linker to space between the JC-1 and the toxin. These strategies
are well known to those skilled in the chemistry of preparing
diagnostic or labelling molecules with reporter functions for
biological studies and include ester, amide, urethane, urea,
sulfonamide, and sulfonate ester (S. T. Smiley et al., Proc. Nat'l,
Acad. Sci. USA, 88:3671-3675 (1991)).
[0110] As noted hereinabove, mitochondria carrying mutated
cytochrome c oxidase genes have increased levels of cardiolipin and
other negatively charged phospholipids as well as increased
mitochondrial membrane potential. As a result, the mitochondria
selectively accumulate targeting molecules, including acridine
orange derivatives and lipophilic cations such as rhodamine-123 and
JC-1 derivatives. In addition to selectively introducing toxins
into the mitochondria, such targeting molecules can also
selectively introduce imaging ligands, which can form the basis of
effective in vivo and in vitro diagnostic strategies. Such
strategies include magnetic resonance imaging (MRI), single photon
emission computed topography (SPELT), and positron emission
tomography (PET). Preferred imaging ligands for the practice of the
present invention include radioisotopes (such as .sup.123I,
.sup.125I, .sup.18F, .sup.13N, .sup.15O, .sup.11C, .sup.99mTc,
.sup.67Ga and so forth), haptens (such as digoxigenin), biotin,
enzymes (such as alkaline phosphatase or horseradish peroxidase),
fluorophores (such as fluorescein lanthanide chelates, or Texas
Red.RTM.), and gadolinium chelates for MRI applications. Saha et
al., Seminars in Nuclear Medicine, 4:324-349 (1994).
[0111] As an example of an in vitro diagnosis, a targeting
molecule, such as an acridine orange or JC-1 derivative, is
labelled with fluorescein as an imaging ligand. The labelled
targeting molecule is introduced into a human tissue cell culture
such as a primary fibroblast culture. After a period of several
hours, cells having mitochondria with defective cytochrome c
oxidase genes selectively absorb the labelled targeting molecule in
amounts greater than cells without such mitochondria. The cells are
then washed and sorted in a fluorescence activated cell sorter
(FACS) such as that sold by Becton Dickinson. Threshold limits can
be established for the FACS using cells with wild-type
mitochondria. Similarly, in an in vivo diagnosis, a targeting
molecule such as an acridine orange or JC-1 derivative is labelled
with .sup.99mTc, .sup.18F or .sup.123I as an imaging ligand. This
labelled targeting molecule is introduced into the bloodstream of a
patient. After a period of several hours, the labelled targeting
molecule accumulates in those tissues having mitochondria with
cytochrome-oxidase-defective genes. Such tissues can be directly
imaged using positron-sensitive imaging equipment.
[0112] Selective destruction of defective mitochondria is also
achieved by using ribozymes. Ribozymes are a class of RNA molecules
that catalyze strand scission of RNA molecules independent of
cellular proteins. Specifically, ribozymes may be directed to
hybridize and cleave target mitochondrial mRNA molecules. The
cleaved target RNA cannot be translated, thereby preventing
synthesis of essential proteins which are critical for
mitochondrial function. The therapeutic application thus involves
designing a ribozyme which incorporates the catalytic center
nucleotides necessary for function and targeting it to mRNA
molecules which encode for dysfunctional COX subunits. The
ribozymes may be chemically synthesized and delivered to cells or
they can be expressed from an expression vector following either
permanent or transient transfection. Therapy is thus provided by
the selective removal of mutant mRNAs in defective
mitochondria.
[0113] The foregoing and following description of the invention and
the various embodiments is not intended to be limiting of the
invention but rather is illustrative thereof. Those skilled in the
art of molecular genetics can formulate further embodiments
encompassed within the scope of the present invention.
EXAMPLES
[0114] Definitions of Abbreviations:
[0115] 1.times. SSC=150 mM sodium chloride, 15 mM sodium citrate,
pH 6.5-8
[0116] SDS=sodium dodecyl sulfate
[0117] BSA=bovine serum albumin, fraction IV
[0118] probe=a labelled nucleic acid, generally a single-stranded
oligonucleotide, which is complementary to the DNA target
immobilized on the membrane. The probe may be labelled with
radioisotopes (such as .sup.32P), haptens (such as digoxigenin),
biotin, enzymes (such as alkaline phosphatase or horseradish
peroxidase), fluorophores (such as fluorescein or Texas Red), or
chemilumiphores (such as acridine).
[0119] PCR=polymerase chain reaction, as described by Erlich et
al., Nature 331:461-462 (1988) hereby incorporated by
reference.
Example I
isolation and Cloning of Cytochrome C Oxidase Genes
[0120] DNA is obtained from AD patients and from non-Alzheimer's
(normal) individuals. Age-matched normal individuals and AD
patients classified as probable AD by NINCDS criteria (McKann et
al., Neurology 34:939-944 (1984)) are used.
[0121] For blood samples, 6 ml samples are drawn, added to 18 ml of
dextrane solution (3% dextrane, average MW =250,000 kiloDaltons
(kDa), 0.9% sodium chloride, 1 mM ethylenedinitrilo tetraacetate,
mixed and maintained at room temperature for 40 minutes without
agitation to allow erythrocytes to sediment.
[0122] The plasma and leukocyte fraction is transferred to a
centrifuge tube and leukocytes are collected by centrifugation at
14,000.times. g for 5 minutes. The leukocyte pellet is resuspended
in 3.8 ml of water and vortexed for 10 seconds to lyse remaining
erythrocytes. 1.2 ml of 0.6 M sodium chloride is added and the
sample is again centrifuged at 14,000.times. g for 5 minutes to
collect the leukocytes. The leukocyte pellet is resuspended in 0.4
ml of a solution containing 0.9% sodium chloride/1 mM
ethylenedinitrilo tetraacetate and stored at -80.degree. C.
[0123] Total cellular DNA is isolated from 0.2 ml of the frozen
leukocyte sample. The frozen leukocytes are thawed, then collected
by centrifugation at 14,000.times. g in a microcentrifuge for 5
minutes. The cell pellet is washed three times with 0.8 ml of
Dulbecco's Phosphate Buffered Saline (PBS; Gibco Laboratories, Life
Technologies, Inc., Grand Island, N.Y.; catalog # 310-4040AJ) and
resuspended in 0.3 ml water. The leukocytes are lysed by adding
0.06 ml of 10% sodium dodecyl sulfate to the cell suspension, then
incubating the samples for 10 minutes in a boiling water bath.
After the samples come to room temperature, cellular debris is
pelleted by centrifugation at 14,000.times. g for 5 minutes. The
supernatant is transferred to a clean microcentrifuge tube and
extracted twice with 0.5 ml of phenol:chloroform (1:1) and twice
with chloroform. DNA is precipitated by addition of 0.03 ml of SM
sodium chloride and 0.7 ml of 100% ethanol to the sample. Following
incubation at -80.degree. C., the precipitated DNA is collected by
centrifugation at 14,000.times. g for 15 minutes. The DNA pellet is
washed with 0.8 ml of 80% ethanol, briefly dried, then resuspended
in 0.2-0.4 ml of TE buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA). The
DNA concentration is determined by UV absorption at 260 nm.
[0124] As an alternative method for isolation of DNA from blood, 5
ml blood samples are drawn and added to Accuspin.TM. Tubes (12 ml
or 50 ml capacity, Sigma Diagnostics, St. Louis, Mo.), prepared
according to the manufacturer's instructions and containing
Histopaque.TM. separation medium. The tubes are centrifuged at
1,000.times. g for 10 minutes. The plasma and leukocyte fraction is
transferred to a centrifuge tube containing 1 ml of TE buffer, and
leukocytes are collected by centrifugation at 2,500 rpm for 10
minutes. The leukocyte pellet is resuspended in 5 ml TE buffer and
0.2 ml of 20% SDS and 0.1 ml of Proteinase K at 20 mg/ml are added.
After incubation at 37.degree. C. for four hours while shaking the
lysate is extracted twice with phenol and twice with
chloroform:isoamyl alcohol (24:1). DNA is precipitated by addition
of {fraction (1/10)} volume 3.0 M sodium acetate (pH 5.0) and 2
volumes of ethanol. Following incubation at -20.degree. C.
overnight, the precipitated DNA is collected by centrifugation,
washed with 70% ethanol, briefly dried, and resuspended in 0.1-0.2
ml of TE buffer. The DNA concentration is determined by UV
absorption at 260 nm.
[0125] For brain samples, total cellular DNA is isolated from
0.1-0.2 grams of frozen brain tissue. The frozen brain tissue is
placed into a glass dounce homogenizer (Pyrex, VWR catalog #7726-5)
containing 3 ml of lysis buffer (50 mM Tris-HCl, pH 7.9, 100 mM
EDTA, 0.1 M NaCl, 0.03 M dithiothreitol, 1% sodium dodecyl sulfate,
1 mg/ml proteinase K) and homogenized with a few strokes of the
glass rod. The brain homogenate is transferred to an incubation
tube and placed at 45-50.degree. C. for 30-60 minutes. After the
addition of 5 ml of sterile water, the homogenate is extracted with
phenol/chloroform two to three times, then twice with chloroform.
DNA is precipitated by mixing the extracted sample with {fraction
(1/20)}.times. volume of 5 M NaCl and 2.5.times. volumes of 200
proof ethanol and placed at -20.degree. C. DNA is pelleted by
centrifugation at 6,000.times. g for 15 minutes. The DNA pellet is
washed with 10 ml of 80% ethanol, briefly dried, and resuspended in
200-400 .mu.l of TE buffer. The DNA concentration is determined by
UV absorption at 260 nm.
[0126] The target cytochrome c oxidase gene sequences are amplified
by Polymerase Chain Reaction (PCR) (Erlich et al., Nature
331:461-462 (1988)). Primers are designed using the published
Cambridge sequences for normal human COX genes. Primers are
specific for COX gene sequences located approximately 100
nucleotides upstream and downstream of the mitochondrial COX genes
encoding subunits I, II, and III. Primers have the following
sequences: COX I-forward primer (5'-CAATATGAAAATCACCTCGGAGC- -3')
(SEQ. ID. NO. 132), COX I-reverse primer
(5'-TTAGCCTATAATTTAACTTTGAC-- 3') (SEQ. ID. NO. 133), COX
II-forward primer (5'-CAAGCCAACCCCATGGCCTCC-3'- ) (SEQ. ID. NO.
134), COX II-reverse primer (5'-AGTATTTAGTTGGGGCATTTCAC-3'- ) (SEQ.
ID. NO. 135), COX III-forward primer (5'-ACAATTCTAATTCTACTGACTATCC-
-3') (SEQ. ID. NO. 136), COX III-reverse primer
(5'-TTAGTAGTAAGGCTAGGAGGGT- G-3') (SEQ. ID. NO. 137).
[0127] Primers are chemically synthesized using a Cyclone Plus DNA
Synthesizer (Millipore Corporation, Marlborough, Mass.) or a Gene
assembler DNA Synthesizer (Pharmacia) utilizing
beta-cyanoethylphosphoram- idite chemistry. Newly synthesized
primers are deprotected using ammonium hydroxide, lyophilized and
purified by NAP-10 column chromatography (Pharmacia LKB
Biotechnology Inc., Piscataway, N.J.; catalog #17-0854-01). DNA
concentration is determined by UV absorption at 260 nm.
[0128] Alternatively, primers are chemically synthesized using an
ABI 394 DNA/RNA Synthesizer (Applied Biosystems, Inc., Foster City,
Calif.) using standard beta-cyanoethylphosphoramidite chemistry.
Without cleavage of the trityl group, the primers are deprotected
with ammonium hydroxide and purified using Oligonucleotide
Purification Cartridges (Applied Biosystems, Inc., Foster City,
Calif.). The DNA concentration is determined by UV absorption at
260 nm.
[0129] Amplification is performed using 0.5-1.0 .mu.g DNA in a
reaction volume of 50-100 .mu.l containing 10 mM Tris-HCl pH
8.3-9.5, 50 mM potassium chloride, 1-4 mM magnesium chloride, 200
.mu.M each of dATP, dCTP, dGTP, and dTTP ("amplification
cocktail"), 200 ng each of the appropriate COX forward and reverse
primers and 5 units of AmpliTaq Polymerase (Perkin-Elmer
Corporation; catalog #N801-0060).
[0130] Amplification using the GeneAmp PCR System 9600 (Perkin
Elmer Corporation) is allowed to proceed for one cycle at
95.degree. C. for 10 seconds, 25 cycles at 95.degree. C. for 1
minute, 60.degree. C. for 1 minute, 72.degree. C. for 1 minute, one
cycle at 72.degree. C. for 4 minutes, after which the samples are
cooled to 4.degree. C. Five separate amplification reactions are
performed for each patient and each cytochrome c oxidase subunit.
After the reactions are complete, the samples for each patient and
subunit are combined and the amplified product is precipitated at
-80.degree. C. by the addition {fraction (1/10)} volume of 5 M
sodium chloride and 2 volumes of 100% ethanol.
[0131] The PCR amplification product is pelleted by centrifugation,
dried briefly, resuspended in 40 .mu.l of TE buffer and purified by
agarose gel electrophoresis (Sambrook et al., "Molecular Cloning: A
Laboratory Manual," Cold Spring Harbor Laboratory, 1988). DNA is
stained with ethidium bromide and visualized under long wavelength
UV light. Bands of the expected lengths (approximately 1,700 bp for
COX I, 900 bp for COX II and 1,000 bp for COX III) are excised from
the gel. The gel containing the DNA is minced into small pieces and
placed into a microcentrifuge tube. 0.3 ml of 1 M sodium chloride
is added to the gel fragments and the sample is frozen at
-80.degree. C., then thawed and incubated at 50.degree. C. for
15-20 minutes. Agarose is sedimented by centrifugation at
14,000.times. g for 5 minutes, the supernatant containing the DNA
is transferred to a new vial and the DNA fragments are collected by
ethanol precipitation.
[0132] The amplified DNA fragments are cloned into the plasmid
pCRII (Invitrogen Corp., San Diego, Calif.) using the TA-Cloning
Kit (Invitrogen Corp., San Diego, Calif.; catalog #K2000-01).
Ligations are performed in a reaction volume of 11 .mu.l containing
1-5 .mu.l of PCR amplification product, 2 .mu.l of plasmid (50 ng),
1 .mu.l of 10.times. ligation buffer and 1 .mu.l of T4 DNA Ligase
(4 units). Ligation reactions are incubated at 10-12.degree. C. for
15-16 hours.
[0133] Vector-ligated PCR fragments are transformed into competent
E. coli cells of the strains XL1-Blue MRF', XL2-Blue MRF' and SURE
(Stratagene, San Diego, Calif.). Transformed cells are spread onto
LB-agar plates containing ampicillin (50 .mu.g/ml), kanamycin (50
.mu.g/ml), IPTG (isopropyl-3-D-thiogalactopyranoside, 20 .mu.g/ml)
and X-Gal (100 .mu.g/ml). The blue/white color selection mechanism
provided by the cloning vector in combination with the E. coli
cells allows for easy detection of recombinant clones, which are
white.
[0134] Multiple white colonies are selected for each patient and
COX subunit and screened by PCR for the presence of a correct
insert using nested primers derived from the published Cambridge
sequences. The primers are specific for sequences located
approximately 40-60 nucleotides upstream and downstream of COX
genes encoding subunits I, II and III. The sequences of the primers
are as follows: COX I-forward primer (5'-AGGCCTAACCCCTGTC-3') (SEQ.
ID. NO. 138), COX I-reverse primer (5'-GGCCATGGGGTTGGC-3') (SEQ.
ID. NO. 139), COX II-forward primer (5'-AGGTATTAGAAAAACCA-3') (SEQ.
ID. NO. 140), COX II-reverse primer (5'ATCTTTAACTTAAAAGG) (SEQ. ID.
NO. 141), COX III-forward primer (5'-GCCTTAATCCAAGCC-3') (SEQ. ID.
NO. 142), COX III reverse primer (5'-GAATGTTGTCAAAACTAG-3') (SEQ.
ID. NO. 143).
[0135] DNA samples from lysed cell supernatants are used as
templates for PCR amplification. Individual colonies are selected
and incubated overnight at 37.degree. C. with shaking (225 rpm) in
LB-broth containing ampicillin and kanamycin. 100-200 .mu.l of each
culture is centrifuged at 14,000.times. g for 2 minutes. The cell
pellet is resuspended in 5-10 .mu.l of water, then lysed by
incubation in a boiling water bath for 5 minutes. Cellular debris
is removed by centrifugation at 14,000.times. g for 2 minutes.
[0136] Amplification of the cloned DNA samples is performed in a
reaction volume of 10 .mu.l containing amplification cocktail, 40
ng each of the appropriate COX-S forward and reverse primers and
0.25 units of AmpliTaq Polymerase. Amplification is performed for
one cycle at 95.degree. C. for 10 seconds, 25 cycles at 95.degree.
C. for 1 minute, 44.degree. C. for 1 minute, 72.degree. C. for 1
minute, and cooled to 4.degree. C., using the GeneAmP PCR System
9600. PCR products are analyzed by horizontal agarose gel
electrophoresis.
Example II
Sequencing of Cytochrome C Oxidase (COX) Genes
[0137] Plasmid DNA containing the COX gene inserts is obtained as
described in Example I is isolated using the Plasmid Quik.TM.
Plasmid Purification Kit (Stratagene, San Diego, Calif.) or the
Plasmid Kit (Qiagen, Chatsworth, Calif., Catalog #12145). Plasmid
DNA is purified from 50 ml bacterial cultures. For the Stratagene
protocol "Procedure for Midi Columns," steps 10-12 of the kit
protocol are replaced with a precipitation step using 2 volumes of
100% ethanol at -20.degree. C., centrifugation at 6,000.times. g
for 15 minutes, a wash step using 80% ethanol and resuspension of
the DNA sample in 100 ul TE buffer. DNA concentration is determined
by horizontal agarose gel electrophoresis, or by UV absorption at
260 nm.
[0138] Sequencing reactions using double-stranded plasmid DNA are
performed using the Sequenase Kit (United States Biochemical Corp.,
Cleveland, Ohio.; catalog #70770), the BaseStation T7 Kit
(Millipore Corp.; catalog #MBBLSEQ01), the Vent Sequencing Kit
(Millipore Corp; catalog #MBBLVEN01), the AmpliTaq Cycle Sequencing
Kit (Perkin Elmer Corp.; catalog #N808-0110) and the Taq DNA
Sequencing Kit (Boehringer Mannheim). The DNA sequences are
detected by fluorescence using the BaseStation Automated DNA
Sequencer (Millipore Corp.). For gene walking experiments,
fluorescent oligonucleotide primers are synthesized on the Cyclone
Plus DNA Synthesizer (Millipore Corp.) or the GeneAssembler DNA
Synthesizer (Pharmacia LKB Biotechnology, Inc.) utilizing
beta-cyanoethylphosphoramidite chemistry. The following primer
sequences are prepared from the published Cambridge sequences of
the COX genes for subunits I, II, and III, with fluorescein (F;
FluoreDite fluorescein amidite, Millipore Corp.; or FluorePrime
fluorescein amidite, Pharmacia LKB Biotechnology, Inc.) being
introduced in the last step of automated DNA synthesis: COX I
primer1 (5'-AGGCCTAACCCCTGTC-3') (SEQ. ID. NO. 144); COX I primer2
(5'-GTCACAGCCCATG-3') (SEQ. ID. NO. 145); COX I primer3
(5'-CCTGGAGCCTCCGTAG-3') (SEQ. ID. NO. 146); COX I primer4
(5'-CTTCTTCGACCCCG-3') (SEQ. ID. NO. 147); COX I primer5
(5'-CATATTTCACCTCCG-3') (SEQ. ID. NO. 148); COX I primer6
(5'-CCTATCAATAGGAGC-3') (SEQ. ID. NO. 149); COX I primer7
(5'-CATCCTATCATCTGTAGG-3') (SEQ. ID. NO. 150); COX II primer1
(5'-AGGTATTAGAAAAACCA-3') (SEQ. ID. NO. 151); COX II primer2
(5'-TAACTAATACTAACATCT-3') (SEQ. ID. NO. 152); COX II primer3
(5'-TGCGACTCCTTGAC-3') (SEQ. ID. NO. 153); COX III primer1
(5'-GCCTTAATCCAAGCC-3') (SEQ. ID. NO. 154); COX III primer2
(5'-CAATGATGGCGCGATG-3') (SEQ. ID. NO. 155); COX III primer3
(5'-CCGTATTACTCGCATCAGG-3') (SEQ. ID. NO. 156); COX III primer4
(5'-CCGACGGCATCTACGGC-3') (SEQ. ID. NO. 157). Primers are
deprotected and purified as described above. DNA concentration is
determined by UV absorption at 260 nm.
[0139] Sequencing reactions are performed according to
manufacturer's instructions except for the following modification:
1) the reactions are terminated and reduced in volume by heating
the samples without capping to 94.degree. C. for 5 minutes, after
which 4 .mu.l of stop dye (3 mg/ml dextran blue, 95%-99% formamide;
as formulated by Millipore Corp.) are added; 2) the temperature
cycles performed for the AmpliTaq Cycle Sequencing Kit reactions,
the Vent Sequencing kit reactions, and the Taq Sequence Kit consist
of one cycle at 95.degree. C. for 10 seconds, 30 cycles at
95.degree. C. for 20 seconds, at 44.degree. C. for 20 seconds and
at 72.degree. C. for 20 seconds followed by a reduction in volume
by heating without capping to 94.degree. C. for 5 minutes before
adding 4 .mu.l of stop dye.
[0140] Electrophoresis and gel analysis are performed using the
BioImage and BaseStation Software provided by the manufacturer for
the BaseStation Automated DNA Sequencer (Millipore Corp.).
Sequencing gels are prepared according to the manufacturer's
specifications. An average of ten different clones from each
individual is sequenced. The resulting COX sequences are aligned
and compared with published Cambridge sequences. Mutations in the
derived sequence are noted and confirmed by resequencing the
variant region.
[0141] As an alternative procedure for sequencing the COX genes,
plasmid DNA containing the COX gene inserts obtained as described
in Example I is isolated using the Plasmid Quik.TM. Plasmid
Purification Kit with Midi Columns (Qiagen, Chatsworth, Calif.)
Plasmid DNA is purified from 35 ml bacterial cultures. The isolated
DNA is resuspended in 100 .mu.l TE buffer. DNA concentrations are
determined by OD (260) absorption.
[0142] As an alternative method, sequencing reactions using double
stranded plasmid DNA are performed using the Prism.TM. Ready
Reaction DyeDeoxy.TM. Terminator Cycle Sequencing Kit (Applied
Biosystems, Inc., Foster City, Calif.). The DNA sequences are
detected by fluorescence using the ABI 373A Automated DNA Sequencer
(Applied Biosystems, Inc., Foster City, Calif.). For gene walking
experiments, oligonucleotide primers are synthesized on the ABI 394
DNA/RNA Synthesizer (Applied Biosystems, Inc., Foster City, Calif.)
using standard beta-cyanoethylphosphoramidite chemistry. The
following primer sequences are prepared from the published
Cambridge sequences of the COX genes for subunits I, II, and
III:
6 COX1 primer11 (5'-TGCTTCACTCAGCC-3'); (SEQ. ID. NO.158) COX1
primer1SF (5'-AGGCCTAACCCCTGTA-3'); (SEQ. ID. NO.159) COX1
primer11X (5'-AGTCCAATGCTTCACTCA-3'); (SEQ. ID. NO.160) COX1
primer12 (5'-GCTATAGTGGAGGC-3'); (SEQ. ID. NO.161) COX1 primer12A
(5'-CTCCTACTCCTGCTCGCA-3'); (SEQ. ID. NO.162) COX1 primer12X
(5'-TCCTGCTCGCATCTGCTA-3'); (SEQ. ID. NO.163) COX1 primer12XX
(5'-CTCCTACTCCTGCTCGCA-3'); (SEQ. ID. NO.164) COX1 primer13
(5'-CCTACCAGGATTCG-3'); (SEQ. ID. NO.165) COX1 primer13A
(5'-CCTACCAGCCTTCGGAA-3'); (SEQ. ID. NO.166) COX1 primer13X
(5'-TCCTACCAGGCTTCGGAA-3'); (SEQ. ID. NO.167) COX1 primer14
(5'-CCTATCAATAGGAGC-3'); (SEQ. ID. NO.168) COX1 primer14XX
(5'-GTCCTATCAATAGGAGCTGTA-3'); (SEQ. ID. NO.169) COX1 primer11C
(5'-GTAGAGTGTGCAACC-3'); (SEQ. ID. NO.170) COX1 primer11CN
(5'-GTCTACGGAGGCTCC-3'); (SEQ. ID. NO.171) COX1 primer11CX
(5'-AGGTCTACGGAGGCTCCA-3'); (SEQ. ID. NO.172) COX1 primer11CXX
(5'-AGGAGACACCTGCTAGGTGTA-3'); (SEQ. ID. NO.173) COX1 primer12C
(5'-CCATACCTATGTATCC-3'); (SEQ. ID. NO.174) COX1 primer12CA
(5'-TCACACGATAAACCCTAGGAA-3'); (SEQ. ID. NO.175) COX1 primer12CX
(5'-GACCATACCTATGTATCcAA-3'); (SEQ. ID. NO.176) COX1 primer13C
(5'-CCTCCTATGATGGC-3'); (SEQ. ID. NO.177) COX1 primer13CN
(5'-GTGTAGCCTGAGAATAGG-3'); (SEQ. ID. NO.178) COX1 primer13CXX
(5'-GTCTAGGGTGTAGCCTGAGAA-3'); (SEQ. ID. NO.179) COX1 primer14C
(5'-GGGTTCGATTCCTTCC-3'); (SEQ. ID. NO.180) COX1 primer14CN
(5'-TGGATTGAAACCAGC-3'); (SEQ. ID. NO.181) COX1 primer14CX
(5'-GTTGGCTTGAAACCAGCTT-3'); (SEQ. ID. NO.182) COX1 primer21
(5'-TCATAACTTTGTCGTC-3'); (SEQ. ID. NO.183) COX2 primer21N
(5'-CATTTCATAACTTTGTCGTC-3'); (SEQ. ID. NO.184) COX2 primer21NA
(5'-AGGTATTAGAAAAACCA-3'); (SEQ. ID. NO.185) COX2 primer21NB
(5'-AAGGTATTAGAAAAACC-3'); (SEQ. ID. NO.186) COX2 primer21X
(5'-TTCATAACTTTGTCGTCAA-3'); (SEQ. ID. NO.187) COX2 primer2FSF
(5'-AAGGTATTAGAAAAACC-3'); (SEQ. ID. NO.188) COX2 primer2SFA
(5'-CCATGGCCTCCATGACTT-3')- ; (SEQ. ID. NO.189) COX2 primer22
(5'-TGGTACTGAACCTACG-3'); (SEQ. ID. NO.190) COX2 primer22A
(5'-ACAGACGAGGTCAACGAT-3'); (SEQ. ID. NO.191) COX2 primer22X
(5'-CATAACAGACGAGGTCAA-3'); (SEQ. ID. NO.192) COX2 primer21C
(5'-AGTTGAAGATTAGTCC-3'); (SEQ. ID. NO.193) CCX2 primer21CN
(5'-TAGGAGTTGAAGATTAGTCC-3'); (SEQ. ID. NO.194) CCX2 primer21CX
(5'-TGAAGATAAGTCCGCCGTA-3'); (SEQ. ID. NO.195) CCX2 primer22C
(5'-GTTAATGCTAAGTTAGC-3'); (SEQ. ID. NO.196) COX2 primer22CXX
(5'-AAGGTTAATGCTAAGTTAGCT- T-3'); (SEQ. ID. NO.197) COX3 primer31
(5'-AAGCCTCTACCTGC-3'); (SEQ. ID. NO.198) COX3 primer31N
(5'-CTTAATCCAAGCCTACG-3') (SEQ. ID. NO.199) COX3 primer32
(5'-AACAGGCATCACCC-3'); (SEQ. ID. NO.200) COX3 primer32A
(5'-CATCCGTATTACTCGCATCA-3') (SEQ. ID. NO.201) COX3 primer31C
(5'-GATGCGAGTAATACG-3'); (SEQ. ID. NO.202) COX3 primer31CX
(5'-GATGCGAGTAATACGGAT-3')- ; (SEQ. ID. NO.203) COX3 primer32C
(5'-AATTGGAAGTTAACGG-3'); (SEQ. ID. NO.204) COX3 primer32CX
(5'-AATTGGAAGTTAACGGTA-3'); (SEQ. ID. NO.205) COX3 primer32CXX
(5'-GTCAAAACTAGTTAATTGGAA-3'); (SEQ. ID. NO.206)
[0143] Sequencing reactions are performed according to the
manufacturer's instructions. Electrophoresis and sequence analysis
are performed using the ABI 373A Data Collection and Analysis
Software and the Sequence Navigator Software (ABI, Foster City,
Calif.). Sequencing gels are prepared according to the
manufacturer's specifications. An average of ten different clones
from each individual is sequenced. The resulting COX sequences are
aligned and compared with the published Cambridge sequence.
Mutations in the derived sequence are noted and confirmed by
sequence of the complementary DNA strand.
[0144] Mutations in each COX gene for each individual are compiled.
Comparisons of mutations between normal and AD patients are made
and summarized in Tables I and II.
Example III
Detection of COX Mutations by Hybridization Without Prior
Amplification
[0145] This example illustrates taking test sample blood, blotting
the DNA, and detecting by oligonucleotide hybridization in a dot
blot format. This example uses two probes to determine the presence
of the abnormal mutation at codon 74 of the COX II gene (see Table
1) in mitochondrial DNA of Alzheimer's patients. This example
utilizes a dot-blot format for hybridization, however, other known
hybridization formats, such as Southern blots, slot blots,
"reverse" dot blots, solution hybridization, solid support based
sandwich hybridization, bead-based, silicon chip-based and
microtiter well-based hybridization formats can also be used.
[0146] Sample Preparation Extracts and Blotting of DNA onto
Membranes:
[0147] Whole blood is taken from the patient. The blood is mixed
with an equal volume of 0.5-1 N NaOH, and is incubated at ambient
temperature for ten to twenty minutes to lyse cells, degrade
proteins, and denature any DNA. The mixture is then blotted
directly onto prewashed nylon membranes, in multiple aliquots. The
membranes are rinsed in 10.times. SSC (1.5 M NaCl, 0.15 M Sodium
Citrate, pH 7.0) for five minutes to neutralize the membrane, then
rinsed for five minutes in 1.times. SSC. For storage, if any,
membranes are air-dried and sealed. In preparation for
hybridization, membranes are rinsed in 1.times. SSC, 1% SDS.
[0148] Alternatively, 1-10 mls of whole blood is fractionated by
standard methods, and the white cell layer ("buffy coat") is
separated. The white cells are lysed, digested, and the DNA
extracted by conventional methods (organic extraction, non-organic
extraction, or solid phase). The DNA is quantitated by UV
absorption or fluorescent dye techniques. Standardized amounts of
DNA (0.1-5 .mu.g) are denatured in base, and blotted onto
membranes. The membranes are then rinsed.
[0149] Alternative methods of preparing cellular or mitochondrial
DNA, such as isolation of mitochondria by mild cellular lysis and
centrifugation, may also be used.
[0150] Hybridization and Detection:
[0151] For examples of synthesis, labelling, use, and detection of
oligonucleotide probes, see "Oligonucleotides and Analogues: A
Practical Approach", F. Eckstein, ed., Oxford University Press
(1992); and "Synthetic Chemistry of Oligonucleotides and Analogs",
S. Agrawal, ed., Humana Press (1993), which are incorporated herein
by reference.
[0152] In this example two COX II codon 74 probes having the
following sequences are used: ATC ATC CTA GTC CTC ATC GCC (SEQ. ID.
NO. 14) (wild-type) and ATC ATC CTA ATC CTC ATC GCC (SEQ. ID. NO.
29) (mutant).
[0153] For detection and quantitation of the abnormal mutation,
membranes containing duplicate samples of DNA are hybridized in
parallel; one membrane is hybridized with the wild-type probe, the
other with the AD probe. Alternatively, the same membrane can be
hybridized sequentially with both probes and the results
compared.
[0154] For example, the membranes with immobilized DNA are hydrated
briefly (10-60 minutes) in 1.times. SSC, 1% SDS, then prehybridized
and blocked in 5.times. SSC, 1% SDS, 0.5% casein, for 30-60 minutes
at hybridization temperature (35-60.degree. C., depending on which
probe is used). Fresh hybridization solution containing probe
(0.1-10 nM, ideally 2-3 nM) is added to the membrane, followed by
hybridization at appropriate temperature for 15-60 minutes. The
membrane is washed in 1.times. SSC, 11 SDS, 1-3 times at
45-60.degree. C. for 5-10 minutes each (depending on probe used),
then 1-2 times in 1.times. SSC at ambient temperature. The
hybridized probe is then detected by appropriate means.
[0155] The average proportion of AD COX gene to wild-type gene in
the same patient can be determined by the ratio of the signal of
the AD probe to the normal probe. This is a semiquantitative
measure of % heteroplasmy in the AD patient and can be correlated
to the severity of the disease.
[0156] The above and other probes for alteration and quantitation
of wild-type and mutant DNA samples are listed in Tables 4 and 5
hereinabove.
Example IV
Detection of COX Mutations by Hybridization (Without Prior
Amplification)
[0157] A. Slot-Blot Detection of RNA/DNA with .sup.32P Probes
[0158] This example illustrates detection of COX mutations by
slot-blot detection of DNA with .sup.32p probes. The reagents are
prepared as follows: 4.times. BP: 2% (w/v) Bovine serum albumin
(BSA), 2% (w/v) polyvinylpyrrolidone (PVP, Mol. Wt.: 40,000) is
dissolved in sterile H.sub.2O and filtered through 0.22-.mu.
cellulose acetate membranes (Coming) and stored at -20.degree. C.
in 50-ml conical tubes.
[0159] DNA is denatured by adding TE to the sample for a final
volume of 90 .mu.l. 10 .mu.l of 2 N NaOH is then added and the
sample vortexed, incubated at 65.degree. C. for 30 minutes, and
then put on ice. The sample is neutralized with 100 .mu.l of 2 M
ammonium acetate.
[0160] A wet piece of nitrocellulose or nylon is cut to fit the
slot-blot apparatus according to the manufacturer's directions, and
the denatured samples are loaded. The nucleic acids are fixed to
the filter by baking at 80.degree. C. under vacuum for 1 hr or
exposing to UV light (254 nm). The filter is prehybridized for
10-30 minutes in 5 mls of 1.times. BP, 5.times. SSPE, 1% SDS at the
temperature to be used for the hybridization incubation. For
15-30-base probes, the range of hybridization temperatures is
between 35-60.degree. C. For shorter probes or probes with low G-C
content, a lower temperature is used. At least 2.times.10.sup.6 cpm
of detection oligonucleotide per ml of hybridization solution is
added. The filter is double sealed in Scotchpak.TM. heat sealable
pouches (Kapak Corporation) and incubated for 90 min. The filter is
washed 3 times at room temperature with 5-minute washes of
20.times. SSPE: 3M NaCl, 0.02M EDTA, 0.2 Sodium Phospate, pH 7.4,
1% SDS on a platform shaker. For higher stringency, the filter can
be washed once at the hybridization temperature in 1.times. SSPE,
1% SDS for 1 minute. Visualization is by autoradiography on Kodak
XAR film at -70.degree. C. with an intensifying screen. To estimate
the amount of target, compare the amount of target detected by
visual comparison with hybridization standards of known
concentration.
[0161] B. Detection of RNA/DNA by Slot-Blot Analysis with Alkaline
Phosphatase-Oligonucleotide Conjugate Probes
[0162] This example illustrates detection of COX mutations by
slot-blot detection of DNA with alkaline
phosphatase-oligonucleotide conjugate probes, using either a color
reagent or a chemiluminescent reagent. The reagents are prepared as
follows:
[0163] Color Reagent:
[0164] For the color reagent, the following are mixed together,
fresh 0.16 mg/ml 5-bromo-4-chloro-3-indolyl phosphate (BCIP), 0.17
mg/ml nitroblue tetrazolium (NBT) in 100 mM NaCl, 100 mM Tris. HCl,
5 mM MgCl.sub.2 and 0.1 mM ZnCl.sub.2, pH 9.5.
[0165] Chemiluminescent Reagent:
[0166] For the chemiluminescent reagent, the following are mixed
together, 250 .mu.M 3-adamantyl 4-methoxy 4-(2-phospho)phenyl
dioxetane (AMPPD), (Tropix Inc., Bedford, Mass.) in 100 mM
diethanolamine-HCl, 1 mM MgCl.sub.2 pH 9.5, or preformulated
dioxetane substrate Lumiphos.TM. 530 (Lumigen, Inc., Southfield,
Mich.).
[0167] DNA target (0.01-50 fmol) is immobilized on a nylon membrane
as described above. The nylon membrane is incubated in blocking
buffer (0.2% I-Block (Tropix, Inc.), 0.5.times. SSC, 0.1% Tween 20)
for 30 min. at room temperature with shaking. The filter is then
prehybridized in hybridization solution (5.times. SSC, 0.5% BSA, 1%
SDS) for 30 minutes at the hybridization temperature (37-60.degree.
C.) in a sealable bag using 50-100 .mu.l of hybridization solution
per cm of membrane. The solution is removed and briefly washed in
warm hybridization buffer. The conjugate probe is then added to
give a final concentration of 2-5 nM in fresh hybridization
solution and final volume of 50-100 .mu.l/cm.sup.2 of membrane.
After incubating for 30 minutes at the hybridization temperature
with agitation, the membrane is transferred to a wash tray
containing 1.5 ml of preheated wash-1 solution (1.times. SSC, 0.1%
SDS)/cm.sup.2 of membrane and agitated at the wash temperature
(usually optimum hybridization temperature minus 10.degree. C.) for
10 minutes. Wash-1 solution is removed and this step is repeated
once more. Then wash-2 solution (1.times. SSC) added and then
agitated at the wash temperature for 10 minutes. Wash-2 solution is
removed and immediate detection is done by color.
[0168] Detection by color is done by immersing the membrane fully
in color reagent, and incubating at 20-37.degree. C. until color
development is adequate. When color development is adequate, the
development is quenched by washing in water.
[0169] For chemiluminescent detection, the following wash steps are
performed after the hybridization step (see above). Thus, the
membrane is washed for 10 min. with wash-i solution at room
temperature, followed by two 3-5 min. washes at 50-60.degree. C.
with wash-3 solution (0.5' SSC, 0.1% SDS). The membrane is then
washed once with wash-4 solution (1.times. SSC, 1% Triton X 100) at
room temperature for 10 min., followed by a 10 min. wash at room
temperature with wash-2 solution. The membrane is then rinsed
briefly (.about.1 min.) with wash-5 solution (50 mM NaHCO.sub.3/1
mM MgCl.sub.2, pH 9.5).
[0170] Detection by chemiluminescence is done by immersing the
membrane in luminescent reagent, using 25-50 .mu.l
solution/cm.sup.2 of membrane. Kodak XAR-5 film (or equivalent;
emission maximum is at 477 .mu.m) is exposed in a light-tight
cassette for 1-24 hours, and the film developed.
Example V
Detection of COX Mutations by Amplification and Hybridization
[0171] This example illustrates taking a test sample of blood,
preparing DNA, amplifying a section of a specific COX gene by
polymerase chain reaction (PCR), and detecting the mutation by
oligonucleotide hybridization in a dot blot format.
[0172] Sample Preparation and Preparing of DNA:
[0173] Whole blood is taken from the patient. The blood is lysed,
and the DNA prepared for PCR by using procedures described in
Example 1.
[0174] Amplification of Target COX Genes by Polymerase Chain
Reaction, and Blotting onto Membranes:
[0175] The treated DNA from the test sample is amplified using
procedures described in Example 1. After amplification, the DNA is
denatured, and blotted directly onto prewashed nylon membranes, in
multiple aliquots. The membranes are rinsed in 10.times. SSC for
five minutes to neutralize the membrane, then rinsed for five
minutes in 1.times. SSC. For storage, if any, membranes are
air-dried and sealed. In preparation for hybridization, membranes
are rinsed in 1.times. SSC, 1% SDS.
[0176] Hybridization and Detection:
[0177] Hybridization and detection of the amplified genes are
accomplished as detailed in Example III.
[0178] Although the invention has been described with reference to
the disclosed embodiments, those skilled in the art will readily
appreciate that the specific examples provided herein are only
illustrative of the invention and not limitative thereof. It should
be understood that various modifications can be made without
departing from the scope of the invention.
Example VI
Synthesis of Antisense Oligonucleotides
[0179] Standard manufacturer protocols for solid phase
phosphoramidite-based DNA or RNA synthesis using an ABI DNA
synthesizer are employed to prepare antisense oligomers.
Phosphoroamidite reagent monomers (T, C, A, G, and U) are used as
received from the supplier. Applied Biosystems Division/Perkin
Elmer, Foster City, Calif. For routine oligomer synthesis, 1
.mu.mole scale syntheses reactions are carried out utilizing
THF/I.sub.2/lutidine for oxidation of the phosphoramidite and
Beaucage reagent for preparation of the phosphorothioate oligomers.
Cleavage from the solid support and deprotection are carried out
using ammonium hydroxide under standard conditions. Purification is
carried out via reverse phase HPLC and quantification and
identification is performed by UV absorption measurements at 260
nm, and mass spectrometry.
Example VII
Inhibition of Mutant Mitochondria in Cell Culture
[0180] Antisense phosphorothioate oligomer complementary to the COX
gene mutant at codon 193 and thus non-complementary to wild-type
COX gene mutant RNA is added to fresh medium containing
Lipofectin.RTM. Gibco BRL (Gaithersburg, Md.) at a concentration of
10 .mu.g/ml to make final concentrations of 0.1, 0.33, 1, 3.3, and
10 .mu.M. These are incubated for 15 minutes then applied to the
cell culture. The culture is allowed to incubate for 24 hours and
the cells are harvested and the DNA isolated and sequenced as in
previous examples. Quantitative analysis results shows a decrease
in mutant COX DNA to a level of less than 1% of total COX.
[0181] The antisense phosphorothioate oligomer non-complementary to
the COX gene mutant at codon 193 and non-complementary to wild-type
COX is added to fresh medium containing lipofectin at a
concentration of 10 .mu.g/mL to make final concentrations of 0. 1,
0.33, 1, 3.3, and 10 .mu.M. These are incubated for 15 minutes then
applied to the cell culture. The culture is allowed to incubate for
24 hours and the cells are harvested and the DNA isolated and
sequenced as in previous examples. Quantitative analysis results
showed no decrease in mutant COX DNA.
Example VIII
Inhibition of Mutant Mitochondria in vivo
[0182] Mice are divided into six groups of 10 animals per group.
The animals are housed and fed as per standard protocols. To groups
1 to 4 is administered ICV, antisense phosphorothioate
oligonucleotide, prepared as described in Example VI, complementary
to mutant COX gene RNA, respectively 0.1, 0.33, 1.0 and 3.3 nmol
each in 5 .mu.L. To group 5 is administered ICV 1.0 nmol in 5 .mu.L
of phosphorothioate oligonucleotide non-complementary to mutant COX
gene RNA and non-complementary to wild-type COX gene RNA. To group
6 is administered ICV vehicle only. Dosing is performed once a day
for ten days. The animals are sacrificed and samples of brain
tissue collected. This tissue is treated as previously described
and the DNA isolated and quantitatively analyzed as in previous
examples. Results show a decrease in mutant COX DNA to a level of
less than 1% of total COX for the antisense treated group and no
decrease for the control group.
Example IX
Agents for the Detection and Selective Destruction of Defective
Mitochondria
[0183] a. Preparation of
10-N-(10-Hydroxy-1-decyl)-3,6-bis(dimethylamino)a- cridine Bromide
Salt
[0184] 3,6-bis(dimethylamino)acridine (1.0 millimole) is dissolved
in DMF (100 mL) containing 1.1 equivalent of tertiary amine base.
To this is added 10-hydroxy-1-bromo decane (1.1 millimole), and the
mixture is heated to reflux. When monitoring by TLC shows no
remaining 3,6-bis(dimethylamino)acridine, the reaction is cooled
and the 10-N-(10-hydroxy-1-decyl)-3,6-bis(dimethylamino)acridine is
isolated (0.75 millimoles).
[0185] b. Preparation of
10-N-(10-phosphoryl-1-decyl)-3,6-bis(dimethylamin- o)acridine
Chloride Salt
[0186] 10-N-(10-Hydroxy-1-decyl)-3,6-bis(dimethylamino)acridine
(1.0 millimole) is dissolved in pyridine (100 mL). To this is added
2-(N,N-dimethylamino)-4-nitrophenyl phosphate (1.1 millimole)
according to the procedure of Taguchi (Chem. Pharm. Bull., 23:1586
(1975)), and the mixture is stirred under a nitrogen atmosphere.
When monitoring by TLC showed no remaining
10-N-(10-hydroxy-1-decyl)-3,6-bis(dimethylamino)acrid- ine, the
reaction is worked up according to Taguchi and the
10-N-(10-phosphoryl-1-decyl)-3,6-bis(dimethylamino)acridine is
isolated (0.75 millimoles).
[0187] c. Preparation of
10-N-(10-thiophosphoryl-1-decyl)-3,6-bis(dimethyl- amino)acridine
Chloride Salt
[0188] 10-N-(10-hydroxy-1-decyl)-3,6-bis(dimethylamino)acridine
(1.0 millimole) is dissolved in DMF (100 mL). To this is added
triimidazolyl-1-phosphine sulfide (1.1 millimole) according to the
procedure of Eckstein (Journal of the American Chemical Society,
92:4718, (1970)) and the mixture stirred under a nitrogen
atmosphere. When monitoring by TLC shows no remaining
10-N-(10-Hydroxy-1-decyl)-3,6-bis(di- methylamino)acridine, the
reaction is worked up according to Eckstein and the
10-N-(10-thiophosphoryl-1-decyl)-3,6-bis(dimethylamino)acridine is
isolated (0.75 millimoles).
[0189] d. Preparation of 10-N-(11-undecanoic
Acid)-3,6-bis(dimethylamino)a- cridine Bromide Salt
[0190] 3,6-Bis(dimethylamino)acridine (1.0 millimole) is dissolved
in DMF (100 mL). To this is added 11-bromo undecanoic acid (1.1
millimole) and the mixture is heated to reflux. When monitoring by
TLC shows no remaining 3,6-bis(dimethylamino)acridine, the reaction
is cooled and the 10-N-(11-undecanoic
acid)-3,6-bis(dimethylamino)acridine is isolated (0.75
millimoles).
[0191] e. Preparation of 10-N-(11-undecyl-2,4-dinitrophenyl
Urethane)-3,6-bis(dimethylamino)acridine Bromide Salt
[0192] 10-N-(11-Undecanoic acid)-3,6-bis(dimethylamino)acridine
(1.0 millimole) is dissolved in THF (100 mL). To this is added
2,4-dinitrophenol (1.1 millimole) and diphenylphosphoryl azide (1.1
millimole), and the mixture is stirred while heating to 70.degree.
C. When monitoring by TLC shows no remaining 10-N-(11-undecanoic
acid)-3,6-bis(dimethylamino)-acridine, the reaction is cooled and
the 10-N-(11-undecyl-2,4-dinitrophenyl
urethane)-3,6-bis(dimethylamino)acridi- ne is isolated (0.75
millimoles).
[0193] f. Preparation of 10-N-(11-undecan-1-oic Acid
2,4-dinitrophenyl Ester)-3,6-bis(dimethylamino)acridine Bromide
Salt
[0194] 10-N-(11-undecanoic acid)-3,6-bis(dimethylamino)acridine
(1.0 millimole) is dissolved in DMF (100 mL). To this is added
2,4-dinitrophenol (1.1 millimole), dicyclohexylcarbodimide (1.1
millimole) and hydroxybenztriazole (1.1 millimole), and the mixture
is stirred. When monitoring by TLC shows no remaining
10-N-(11-undecanoic acid)-3,6-bis(dimethylamino)-acridine, the
reaction is cooled and the 10-N-(11-undecan-1-oic acid
2,4-dintrophenyl ester)-3,6-bis(dimethylamino- )acridine is
isolated (0.75 millimoles).
[0195] g. Preparation of N'-(2-hydroxyethyl)-JC-1
[0196] According to the procedure of Yamamoto et al. (Bulletin of
the Chemical Society of Japan, 46:1509-11 (1973)),
2-methyl-5,6-dichloro-N-et- hyl-N'-(2-hydroxyethyl) benzimidazole
is heated with aniline and ethyl orthoformate at 100.degree. C. To
this is added acetic anhydride and potassium acetate and heating is
continued at 160.degree. C. The reaction is worked up as described
in Yamamoto et al. and the product isolated.
[0197] h. Preparation of Bis N'-(2-phosphoryl-1-ethyl)-JC-1
[0198] N'-(2-hydroxyethyl)-JC-1 (1.0 millimole) is dissolved in
pyridine (100 mL). To this is added
2-(N,N-dimethylamino)-4-nitrophenyl phosphate (1.1 millimole)
according to the procedure of Taguchi (Chem. Pharm. Bull, 23, 1586
(1975)), and the mixture is stirred under a nitrogen atmosphere.
When monitoring by TLC shows no remaining
10-N-(10-hydroxy-1-decyl)-3,6-b- is(dimethylamino)acridine, the
reaction is worked up according to Taguchi and bis
N'-(2-phosphoryl-1-ethyl) JC-1 was isolated (0.75 millimoles).
Sequence CWU 1
1
* * * * *