Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1997-10-03
2002-02-05
Myers, Carla J. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C536S023500, C536S024100, C536S024310, C536S024320, C536S024330
Reexamination Certificate
active
06344317
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to the detection of nucleic acids associated with disease states. In particular, the invention provides for the detection of nucleic acids in acellular biological fluids as diagnostic assays for chronic illnesses and infectious diseases. Also provided are therapeutic approaches to treating chronic illnesses.
BACKGROUND OF THE INVENTION
Chronic diseases such as cancer, autoimmune diseases, chronic fatigue syndrome and the like afflict millions of people throughout the world. It is known that environmental and other factors (e.g., genotoxic compounds, infectious retroviruses, retroelements and the like) can directly disrupt and/or damage DNA and may play a role in the development of a number of chronic illnesses. The mechanisms by which damage to genetic material leads to the onset of these diseases is not well understood, however. It is known that certain sites in the genome (e.g., fragile sites) are particularly susceptible to such modifications. For instance, it is known that the distribution of insertion sites for retroviruses and retroelements is not random and that fragile sites are often preferred (see, e.g., Craigie
Trends in Genetics
8:187 (June 1992); De Ambrosis et al.
Cancer Genet. Cytogenet.
60:1-7 (1992); Durnam et al. and Romani et al.
Gene
135:153-160 (1993)).
Fragile sites themselves are associated diseases. For instance, expansion of long of blocks of repeated CCG triplets together with methylation of CpG islands in particular fragile sites on the X chromosome have been linked to the fragile X syndrome, an inherited mental retardation (see, e.g., Sutherland and Richards,
Proc. Nat. Acad. Sci. USA
92:3636-3641 (1995).
The detection of nucleic acids from pathogens such as bacteria, parasites and viruses, is a commonly used method for diagnosis of disease. For instance, detection of viral sequences is useful in diagnosis of disease. Enteroviruses are a heterogeneous group of human pathogens and opportunistic agents responsible for a broad spectrum of diseases and make up a large genus within the family Picornaviridae. The genus includes polioviruses, coxsackieviruses, echoviruses as well as a number of uncharacterized enteroviruses isolated from humans and other primates. For a review of taxonomy of Picornaviridae see,
Virus Taxonomy: Classification and Nomenclature of Viruses
Murphy et al., eds (Springer Verlag, 1995).
Like other members of the picornaviridae, enteroviruses are small, single-stranded, nonenveloped RNA viruses. Enteroviruses are distinguished from other members of the picornaviridae by their stability in acid and their fecal-oral route of passage and transmission.
Polioviruses (which exist as at least three serotypes) are the most clinically significant of the enteroviruses worldwide, causing paralytic disease in children in developing countries. Non-polioenteroviruses (NPEV) are also responsible for large numbers of symptomatic infections each year. They are the most common etiologic agents of a number of illnesses including meningitis and nonspecific febrile illnesses. Recent reports have linked NPEV infection with chronic fatigue syndrome (Clements et al.
J. Med. Virol.
45:156-161(1995).
In developed countries, polioviruses have been controlled with the introduction of vaccines in the late 1950's. Vaccines typically contain either inactivated poliovirus, which is administered parenterally or live attenuated poliovirus, which is administered orally. The inactivated vaccines use tissue culture-derived poliovirus which has been inactivated, or killed with formaldehyde. Attenuated virus vaccines are prepared by passage of the virus in cell cultures until it loses its ability to cause the disease. Attenuated live virus replicates in the gut to induce a protective antibody response.
Virus used for these vaccines is typically cultured in African Green Monkey kidney cells. As noted above, a number of poorly characterized enteroviruses have been isolated from primates, including monkeys. Procedures are currently in place to identify monkey cells infected by other viruses (e.g., SV40) before use in culturing polioviruses.
Understanding how these molecular changes lead to disease is not well understood in the art. Increased understanding of the cellular mechanisms, particularly changes in nucleic acids, that occur early in the pathogenesis of these diseases is important to development of useful therapies and diagnostic tools. In addition, identification of viruses, including enteroviruses, in polio vaccine preparations is important to ensure safety of polio vaccines. Moreover, the possibility that new viruses resulting from recombination of poliovirus with other viruses from the monkey cells or the human gut is an obvious public health concern. The present invention addresses these and other concerns.
SUMMARY OF THE INVENTION
The present invention provides methods of screening for a disease state in a patient. The methods comprise providing a sample containing biological material (e.g., biopsies) or biological fluids from the patient (e.g., an acellular biological fluid such as serum or plasma) and contacting the sample with a nucleic acid which specifically hybridizes to a target nucleic acid sequence. The target nucleic acids are then detected. In some embodiments, the target nucleic acid includes sequences from a fragile site in the human genome, in particular, repetitive DNA. In some embodiments the target sequences are derived from Alu sequences in a fragile site. In other embodiments, the target nucleic acid may be a novel composite of microbial origin and in some cases human origin. The target nucleic acid is usually at least about 100 nucleotides in length, sometimes between about 500 and about 1500 nucleotides in length.
The methods are usually used to detect a chronic illness. Examples of chronic illnesses include cancers, such as multiple myeloma. Other diseases include autoimmune diseases, neurodegenerative diseases, heart diseases and the like.
In certain preferred embodiments, the target human nucleic acids are amplified (e.g., by PCR). An exemplary target sequence is provided in SEQ ID NO:23. This sequence can be used in diagnosis of multiple myeloma.
The present invention further provides improved methods for detecting viral nucleic acids in biological samples and polio vaccine preparations. In one embodiment, the invention provides methods for detecting recombinant viral nucleic acids, which comprise nucleic acid sequences from a polio virus and a non-poliovirus, usually a non-polioenterovirus. The methods comprise contacting a biological sample suspected of containing the recombinant viral nucleic acid with a first primer which specifically hybridizes to a conserved sequence in a picornaviral genome and a second primer which specifically hybridizes to a poliovirus nucleic acid sequence. The presence of an amplified product which is a recombinant viral nucleic acid is then detected.
A number of primers may be used in the present invention. For instance, one or both the primers may be one that specifically hybridizes to a 5′ nontranslated region of an picornaviral genome. Since the 5′ nontranslated region is conserved among picornaviruses, the primer will specifically hybridize to most picornaviruses, particularly enteroviruses. Primers PG01 and PG02 (as shown in SEQ ID NO:1 or SEQ ID NO:2 are conveniently used for this purpose. One or both of the primers may specifically hybridize to a P2-P3 region of a poliovirus genome. A preferred primer is one that specifically hybridizes to nucleotides 4922-4941 or nucleotides 5467-5487. Primers PG03 and PG04 (as shown in SEQ ID NO:3 or SEQ ID NO:4) are conveniently used for this purpose. One or both of the primers may also specifically hybridize to a P2 region of a poliovirus genome. A preferred primer is one that specifically hybridizes to nucleotides 4460-4478 or nucleotides 4634-4653. Primers PG07 and PG08 (as shown in SEQ ID NO:5 or SEQ ID NO:6) are conveniently used for this purpose. A preferred combination of primers is
Chronix Biomedical, Inc.
Johannsen Diana
Myers Carla J.
Townsend and Townsend / and Crew LLP
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