Link:  Pharm/Biotech Resources

Title:  Method of targeting conjugate molecules to mitochondria

United States Patent:  6,867,197

Issued:  March 15, 2005

Inventors:  Davis; Robert E. (San Diego, CA); Ghosh; Soumitra S. (San Diego, CA); Kiely; John S. (San Diego, CA)

Assignee:  Mitokor (San Diego, CA)

Appl. No.:  448312

Filed:  November 23, 1999

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.

Description of the Invention

FIELD OF THE INVENTION

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

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.

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.

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.

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.

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.

One report suggesting a relation between AD and cytochrome c oxidase is Parker et al., Neurology 40: 13021303 (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. Geriatic 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).

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.

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.

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.

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

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.

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, 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.

Preferred methods for interrogating the above mutations include: (a) hybridization with oligonucleotide probes, (b) methods based on the ligation of oligonucleotide sequences that annual 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.

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 ³² P), 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).

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.

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.

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.

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 JC-1 (5,5',6, 6'-tetrachloro-1,1',3,3'-tetraethylbenzimidiazolo-carbocyanine iodide) derivatives. 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.

The appended claims are hereby incorporated by reference as a further enumeration of preferred embodiments.

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.

It is another object of the present invention to provide reliable and efficient means for the diagnosis of Alzheimer's disease.

It is another object of the present invention to provide effective therapies for the treatment of Alzheimer's disease.

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.

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.

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 selecting destroying defective mitochondria.

DETAILED DESCRIPTION OF THE INVENTION

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.

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.

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.

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. Mutation 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.

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.

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.

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 sequence 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.

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 presence 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.

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.

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.

Mutations observed in COX I gene of Alzheimer's patients

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.

As evidenced by Table 1, mutational hot spots of COX I in AD patients are codons 155, 167, 178, 193, 194 and 415.

Mutations observed in COX II gene of Alzheimer's patients

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.

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.

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.

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.

Blood samples are obtained in 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.

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.

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").

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.

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.

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 nucleotide 9267 to 10052.

Diagnostic Detection of Alzheimer's Disease-Associated Mutations:

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.

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.

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^- (normal or wild-type) probe.

A variety of technique, 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.

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 sequences 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, fluoroescently-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 complementarily with the target sequence. The resulting hybridization patterns are useful for reassembling the original target DNA sequence.

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)).

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 amplication 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.

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. U.S.A. 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, serial number to be assigned, the disclosure of which is incorporated by reference.

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.

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)).

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.

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.

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^3- and Tb^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.

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); Gosh 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)).

Detection of the probe label may be accomplished by the following approaches. For radioisotopes, detection is by autoradiography, scintillation counting or phospher 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 resolve 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-NiCl₂ for horseradish peroxidase), fluorescence (e.g. 4-methyl umbelliferyl phosphate for alkaline phosphatase) or chemiluminescence (the alkaline phosphatase dioxetane substrate LumiPhos 530 from Lumigen Inc., Detroit MI or AMPPD and CPSD 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.

The oligonucleotide probes for detection preferably range in size between 10 to 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 provides 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 phobes, the hybridization reactions are preferably run between 20^oC. and 60^oC., and more preferably between 30^oC. and 55^oC. 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.

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.

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 1x10⁷ M^-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 1x10⁶ M^-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.

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.

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.

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.

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.

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.

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, wherein calcium phosphate treatment or electroporation may be used for other cellular hosts.

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:

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. Helene 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.

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.

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).

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.

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 hydridize 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.

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.

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.

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.

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.

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.

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.

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.

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.

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 the 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.

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 mitochondrial will repopulate the targeted tissue, normalizing mitochondrial function. This can be accomplished by identifying unique characteristics of mitochondrial 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 oligonucleotide 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 defensive 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.

Mitochondria with defective cytochrome c oxidase activity exhibit impaired electron transport, leading to decreased synthesis of adenosine triphosphate and general bienergetic failure. As a consequence, mitochondria carrying mutated DNA will become enlarged and the intramitochondrial membrane potential increases.

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_i -transport and disrupt respiration. (Maftah et al., FEBS Letters 260(2):236-240 (1990). At these concentrations, NAO is mitochondrial toxin.

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, hydroxy, 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-bis(dimethylamino) acridine chloride salt or 10-N-(10-thiophosphoryl-1-decyl)-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.

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.

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: ##STR6##

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'-tetraethylbenzimidiazolo-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.

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 wherein 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.

Claim 1 of 28 Claims

We claim:

1. A conjugate capable of disabling or destroying a mitochondrion, comprising:

a targeting molecule conjugated to a toxin, wherein the targeting molecule selectively accumulates in a mitochondrion that is selected from the group consisting of (i) a mitochondrion having increased intramitochondrial membrane potential relative to a mitochondrion with intact cytochrome c oxidase activity and (ii) a mitochondrion having an increased level of cardiolipin relative to a mitochondrion with intact cytochrome c oxidase activity.

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