Inventors: de la Monte; Suzanne (Cambridge, MA); Wands; Jack R. (Waban, MA) Assignee: The General Hospital Corporation (Boston, MA) Appl. No.: 340426 Filed: November 14, 1994

Primary Examiner: Wax; Robert A. Assistant Examiner: Longton; Enrique D. Attorney, Agent or Firm: Sterne,Kessler,Goldstein & Fox P.L.L.C. United States Patent 5,948,634 September 7, 1999

Goverment Interests
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY-SPONSORED RESEARCH AND DEVELOPMENT
The present invention was made with U.S. government support. Therefore, the U.S. government has certain rights in the invention.

Parent Case Text
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a continuation-in-part of U.S. application Ser. No. 08/230,139 filed Apr. 20, 1994 now abandoned and U.S. application Ser. No. 08/055,778 filed May 3, 1993 now abandoned. U.S. application Ser. No. 08/230,139 is a continuation-in-part of U.S. application Ser. No. 08/050,559 filed Apr. 20, 1993 now abandoned. U.S. application Ser. No. 08/055,778 is a continuation of U.S. application Ser. No. 07/451,975 filed Dec. 20, 1989 now abandoned which is a continuation-in-part of U.S. application Ser. No. 07/287,207 filed Dec. 21, 1988 now abandoned.

Claims
What is claimed is:

1. An isolated nucleic acid molecule coding for a neural thread protein (NTP), wherein said protein is specifically recognized by monoclonal antibody #2 on deposit with the American Type Culture Collection, Manassas, Va., under accession number HB-12546 or monoclonal antibody #5 on deposit with the American Type Culture Collection, Manassas, Va., under accession number HB-12545.

2. The nucleic acid molecule according to claim 1, wherein the molecule comprises the nucleic acid sequence set forth in SEQ ID NO: 120 which encodes the amino acid sequence set forth in SEQ ID NO: 121.

3. The nucleic acid molecule according to claim 1, wherein the molecule encodes the amino acid sequence set forth in SEQ ID NO: 121.

4. The nucleic acid molecule of claim 1 which is a plasmid.

5. An expression vector comprising the nucleic acid molecule of claim 1.

6. A host cell transformed with the plasmid of claim 4.

7. A method of using the plasmid of claim 4 to prepare an NTP, said method comprising:

(a) introducing said plasmid into a host cell to produce a recombinant host cell;

(b) culturing said recombinant host cell; and

(c) isolating said NTP from said recombinant host cell.

8. A method of producing Neural Thread Protein (NTP), wherein said protein is specifically recognized by monoclonal antibody #2 on deposit with the American Type Culture Collection, Manassas, Va., under accession number HB12546 or monoclonal antibody #5 on deposit with the American Type Culture Collection, Manassas, Va., under accession number HB-12545, wherein said method comprises:

(a) culturing a recombinant host comprising a human gene coding for said NTP; and

(b) isolating said NTP from said host.

9. The method of claim 8, wherein said host is E. coli.

10. The method of claim 8, wherein said gene is contained by a vector.

11. A virion comprising the expression vector of claim 5.

12. An isolated nucleic acid molecule encoding the neural thread protein (NTP) coded for by the AD10-7 DNA molecule present in the DH1 E. coli cells that are on deposit at the American Type Culture Collection, Manassas, Va., under accession no. 69262.

13. The isolated nucleic acid molecule according to claim 1, wherein the NTP has a molecular weight of about 42 kDa.

14. The isolated nucleic acid molecule according to claim 1, wherein the NTP has a molecular weight of about 26 kDa.

15. The isolated nucleic acid molecule according to claim 1, wherein the NTP has a molecular weight of about 21 kDa.

16. The isolated nucleic acid molecule according to claim 1, wherein the NTP has a molecular weight of about 17 kDa.

17. The isolated nucleic acid molecule according to claim 1, wherein the NTP has a molecular weight of about 14 kDa.

18. The isolated nucleic acid molecule according to claim 1, wherein the NTP has a molecular weight of about 8 kDa.

19. An isolated nucleic acid molecule encoding the neural thread protein (NTP) coded for by the G2-2 Pst1DNA molecule present in the DH5 E. coli cells that are on deposit at the American Type Culture Collection, Manassas, Va., under accession no. 69257.

20. An isolated nucleic acid molecule encoding the neural thread protein (NTP) coded for by the G5d-Pst1 DNA molecule present in the DH5 E. coli cells that are on deposit at the American Type Culture Collection, Manassas, Va., under accession no. 69258.

21. An isolated nucleic acid molecule encoding the neural thread protein (NTP) coded for by the 1-9a DNA molecule present in the E. coli cells that are on deposit at the American Type Culture Collection, Manassas, Va., under accession no. 69259.

22. An isolated nucleic acid molecule encoding the neural thread protein (NTP) coded for by the AD3-4 DNA molecule present in the DH1 E. coli cells that are on deposit at the American Type Culture Collection, Manassas, Va., under accession no. 69260.

23. An isolated nucleic acid molecule encoding the neural thread protein (NTP) coded for by the HB4 DNA molecule present in the E. coli cells that are on deposit at the American Type Culture Collection, Manassas, Va., under accession no. 69261.

24. An isolated nucleic acid molecule encoding the neural thread protein (NTP) coded for by the AD2-2 DNA molecule present in the DH1 E. coli cells that are on deposit at the American Type Culture Collection, Manassas, Va., under accession no. 69263.

25. An isolated nucleic acid molecule encoding the neural thread protein (NTP) coded for by the G5d1 Pst1-EcoR1 DNA molecule present in the DH5 E. coli cells that are on deposit at the American Type Culture Collection, Manassas, Va., under accession no. 69264.

26. An isolated nucleic acid molecule encoding the neural thread protein (NTP) coded for by the G2-2 Pst1-EcoR1 DNA molecule present in the DH5 E. coli cells that are on deposit at the American Type Culture Collection, Manassas, Va., under accession no. 69265.

27. The isolated nucleic acid molecule of claim 1, wherein said protein is specifically recognized by monoclonal antibody #2 on deposit with the American Type Culture Collection, Manassas, Va., under accession number HB-12546.

28. The isolated nucleic acid molecule of claim 1, wherein said protein is specifically recognized by monoclonal antibody #5 on deposit with the American Type Culture Collection, Manassas, Va., under accession number 12545.

Description
FIELD OF THE INVENTION

The present invention is in the field of genetic engineering and molecular biology. This invention is directed to recombinant hosts expressing novel proteins associated with Alzheimer's Disease, neuroectodermal tumors, malignant astrocytomas, and glioblastomas. This invention is specifically directed to the recombinant hosts and vectors which contain the genes coding for the neuronal thread proteins. This invention is also directed to substantially pure neural thread proteins, immunodiagnostic and molecular diagnostic methods to detect the presence of neural thread proteins, and the use of nucleic acid sequences which code for neural thread proteins in gene therapy.

BACKGROUND OF THE INVENTION

Alzheimer's Disease

Alzheimer's Disease (AD) is the most frequent cause of dementia in the United States, affecting over two million individuals each year. It is a degenerative brain disorder characterized clinically by loss of memory, confusion, and gradual physical deterioration. It is the fourth most common cause of death. The etiology of the disease is virtually unknown but has been attributed to various viruses, toxins, heavy metals, as well as genetic defects. The disease is at present incurable.

Until quite recently, AD was thought to account for relatively few of the cases generally classified as senile dementia. Other factors can lead to such a condition, including repetitious mild strokes, thyroid disorders, alcoholism, and deficiencies of certain vitamins, many of which are potentially treatable. It can be appreciated, then, that a diagnostic test specific for AD would be very useful for the clinical diagnosis and proper clinical treatment of subjects presenting with symptoms common to all of these conditions.

The brains of individuals with AD exhibit characteristic pathological accumulations of congophilic fibrous material which occurs as neurofibrillary tangles within neuronal cell bodies, and neuritic (or senile) plaques. Neurofibrillary tangles may also be found in the walls of certain cerebral blood vessels. The major organized structural components of neurofibrillary tangles are paired helical filaments. Qualitatively indistinguishable amyloid deposits also occur in normal aged brains but in much smaller numbers with restricted topographical distribution.

There has been considerable recent investigative activity regarding the characterization of proteins found in neuritic plaques and neurofibrillary tangles of AD and other neurologic diseases. One of the amyloid proteins initially described by Glenner et al. has been cloned and sequenced (Glenner et al., Biochem. Biophys. Res. Commun. 120:1131-1135 (1984); U.S. Pat. No. 4,666,829). The A4 amyloid protein found in neuritic plaques and blood vessels has been determined to be a component of a 695 amino acid precursor; a protein postulated to function as a glycosylated cell surface receptor (Masters et al., Proc. Natl. Acad. Sci. USA 82:4245-4249 (1985), Kang et al., Nature 325:733-736 (1987)). In addition, the amyloid protein has been postulated to function as a cell adhesion molecule and as a calcium ion channel protein (Hooper, J. NIH Res. 4: 48-54 (1992); Rensberger, Wayward Protein Molecule May Be Elusive Killer of Brain Cells, The Washington Post, Jan. 25, 1993, .sctn.1, at A3 (1993)). The gene coding for A4 is located on chromosome 21 (Kang et al., ibid.; Goldgaber et al., Science 235:877-880 (1987); Tanzi et al., Science 235:880-885 (1987); St. George-Hyslop et al., Science 235:885-889 (1987)) but apparently is not linked to the familial form of the disease (Van Broekhoven et al., Nature 329:153-155 (1987)). There appears to be little, if any, protein sequence homology between amyloid A4 and .beta. protein, their higher molecular weight precursor, and pancreatic thread protein (PTP) (Gross et al., J. Clin. Invest. 76:2115-2126 (1985)).

A number of other proteins thought to be associated with the disease have been described, including Ubiquitin, ALZ-50, microtubular-associated proteins .tau. and MAP2, and neurofilament protein (see, for example, Manetto et al., Proc. Natl. Acad. Sci. USA 85:4502-4505 (1988); Wolozin et al., Science 232:648-651 (1986); Selkoe, Neurobiol. Aging 7:425-432 (1986); Perry et al., in: Alterations of the Neuronal Cytoskeleton in Alzheimer's Disease, Plenum, New York, pp 137-149 (1987)). More recently, a serine protease inhibitor called .alpha..sub.1 -anti-chymotrypsin has been found in AD amyloid deposits (Abraham et al., Cell 52:487-501 (1988)).

There is currently no useful diagnostic test for AD being practiced clinically. A definitive diagnosis is possible only postmortem, or during life through a brain biopsy, to reveal the presence of the characteristic plaques, tangles, paired helical filaments, and other cerebrovascular deposits which characterize the disorder. Such an invasive surgical procedure is inherently dangerous and is therefore rarely utilized. As a result, the clinical misdiagnosis of AD is estimated to be approximately 20%-30%.

Thread Proteins

The prototype thread protein molecule is pancreatic thread protein (PTP) which bears the unusual physical property of forming insoluble fibrils at neutral pH, but is highly soluble at acid or alkaline pH (Gross et al., supra). PTP is highly abundant, synthesized by pancreatic acinar cells, and secreted into pancreatic juice in concentrations exceeding 1 mg/ml (Id.). An increased thread protein immunoreactivity has been demonstrated in brains with AD lesions, using monoclonal antibodies to PTP (Ozturk et al., Proc. Natl. Acad. Sci. USA 86:419-423 (1989)). In addition, a highly sensitive forward sandwich immunoradiometric assay was used to demonstrate that at least three distinct antigenic epitopes were shared between PTP and the related protein in the brain (Id.) Despite similarities, the pancreatic and neuronal forms of the thread protein are almost certainly distinct since the mRNA molecules and proteins differ in size, and many of the antigenic epitopes which are present in the pancreatic thread protein are not detectable in brain tissue (de la Monte et. al., J. Clin. Invest. 86:1004-1013 (1990); de la Monte et. al., J. Neurol. Sci. 113:152-164 (1992); de la Monte et al., Ann. Neurol. 32:733-742 (1992)).

The central nervous system form of the thread protein, designated hereafter as "neural thread protein" (NTP), has been identified in AD and Down's syndrome brain tissue (Wands et al., International Application Publication No. WO 90/06993). NTP has been found in all AD brains studied where characteristic neuropathologic changes of the disease exist (Id.). The saline- extractable soluble immunoreactivity shares has a molecular weight of approximately 17 to 20 kD (Id.).

Quantitative measurements of NTP immunoreactivity in various regions of AD brains revealed levels varying from 12 to 295 ng/gm tissue (Mean=116 ng/gm tissue) compared to 1-11 ng/gm tissue (Mean=5 ng/gm tissue) in comparable ares of control brains (Id.).

Immunocytochemistry performed with monoclonal antibodies directed against the pancreatic form of PTP demonstrated that NTP is localized within cells, within fine processes within the neuropil, or is extracellular in both AD and Down's syndrome brains (Id.). Two types of cell contain NTP: neurons and astrocytes (Id.). The affected neurons are the large pyramidal type which typically contain the neurofibrillary tangles well known in AD brain (Id.).

That NTP accumulation within neurons is intrinsically important or integrally related to the evolution of AD lesions is corroborated by the presence of identical patterns of immunolabeling for NTP in Down's syndrome brains, but not in control brains (Id.). It is important to note that the same structural abnormalities of AD occur in brains of all middle-age individuals with Down's syndrome, whether or not they are demented. There is also a higher incidence of AD in family members of Down's syndrome patients. Moreover, the regional differences in the densities of NTP-containing neurons parallels the density distributions of neurofibrillary tangles in both AD and Down's syndrome. This provides further evidence that NTP is germane to the pathophysiology of AD. Whether NTP accumulates within neuronal perikarya, as a result of aberrant cellular metabolism or transport is not yet known.

SUMMARY OF THE INVENTION

A need exists for a definitive diagnostic test which can be performed on individuals suspected of having, or being at risk for AD. The present invention satisfies such needs and provides further advantages.

The manner in which these and other objects are realized by the present invention will be apparent from the summary and detailed description set forth below.

Unless defined otherwise, various terms used herein have the same meaning as is well understood in the art to which the invention belongs. All cited publications are incorporated herein by reference.

This invention is directed to recombinant hosts expressing novel proteins associated with Alzheimer's Disease, neuroectodermal tumors, malignant astrocytomas, and glioblastomas. This invention is specifically directed to the recombinant hosts and vectors which contain the genes coding for the neuronal thread proteins (NTP) having molecular weights of about 8 kDa, 14 kDa, 17 kDa, 21 kDa, 26 kDa or 42 kDa. This invention is also directed to the substantially pure neural thread proteins, immunodiagnostic and molecular diagnostic methods to detect the presence of neural thread proteins, and the use of nucleic acid sequences which code for neural thread proteins in gene therapy.

In particular, the invention includes a method for detecting and quantitating an NTP in a human subject, comprising:

(a) contacting a biological sample from a human subject that is suspected of containing detectable levels of an NTP with a molecule capable of binding to the NTP; and

(b) detecting the molecule bound to the NTP.

The invention additionally includes the method as above, wherein the binding molecule is selected from the group consisting of:

(a) an antibody substantially free of natural impurities;

(b) a monoclonal antibody; and

(c) a fragment of (a) or (b).

The invention additionally includes the method as above, wherein the detecting molecule is detectably labeled and where a combination of such binding molecules is used.

The invention additionally includes a method for detecting the presence of a genetic sequence coding for an NTP in a biological sample using a polynucleotide probe derived from a recombinant human NTP of this invention.

The invention additionally includes a method for determining the presence of a condition in a human subject, said condition including, but not limited to, the group consisting of Alzheimer's Disease, the presence of neuroectodermal tumors, the presence of malignant astrocytomas, and the presence of gliomas.

The invention additionally includes a method of diagnosing the presence of AD in a human subject suspected of having AD which comprises:

(a) incubating a biological sample from said subject suspected of containing an NTP with a molecule capable of identifying an NTP; and

(b) detecting the molecule which is bound in the sample, wherein the detection indicates that the subject has AD.

The invention additionally includes a method of diagnosing the presence of neuroectodermal tumors in a human subject suspected of having neuroectodermal tumors which comprises:

(a) incubating a biological sample from said subject suspected of containing an NTP with a molecule capable of identifying an NTP; and

(b) detecting the molecule which is bound in the sample, wherein the detection indicates that the subject has neuroectodermal tumors.

The invention additionally includes a method of diagnosing the presence of a malignant astrocytoma in a human subject suspected of having a malignant astrocytoma which comprises:

(a) incubating a biological sample from said subject, which is suspected of containing an NTP, in the presence of a binding molecule capable of identifying an NTP; and

(b) detecting molecule which is bound in the sample, wherein the detection indicates that the subject has a malignant astrocytoma.

The invention additionally includes a method of diagnosing the presence of a glioblastoma in a human subject suspected of having a glioblastoma which comprises:

(a) incubating a biological sample from said subject, which is suspected of containing an NTP, in the presence of a binding molecule capable of identifying an NTP; and

(b) detecting molecule which is bound in the sample, wherein the detection indicates that the subject has a glioblastoma.

The invention additionally includes the methods as above, wherein a biological sample is removed a human subject prior to contacting the sample with the molecule.

The invention additionally includes the methods as above, wherein detecting any of the molecules bound to the protein is performed by in situ imaging.

The invention additionally includes the methods as above, wherein detecting of any of the molecule bound to the protein is performed by in vivo imaging.

The invention additionally includes the methods as above, wherein the biological sample is reacted with the binding molecule in a manner and under such conditions sufficient to determine the presence and the distribution of the protein.

The invention additionally includes the methods as above, wherein a detectably labeled binding molecule of an NTP is administered to a human subject.

The invention additionally includes the methods as above, wherein the binding molecule is bound to the protein in vivo.

The invention additionally involves an NTP substantially free of any natural impurities and having a molecular weight of about 42 kDa.

The invention additionally involves an NTP substantially free of any natural impurities and having a molecular weight of about 26 kDa.

The invention additionally includes an NTP substantially free of any natural impurities and having a molecular weight of about 21 kDa.

The invention additionally includes an NTP substantially free of any natural impurities and having a molecular weight of about 17 kDa.

The invention additionally includes an NTP substantially free of any natural impurities and having a molecular weight of about 14 kDa.

The invention additionally includes an NTP substantially free of any natural impurities and having a molecular weight of about 8 kDa.

The present invention also particularly relates to the diagnostic methods recited above, wherein the immunoassay comprises two different antibodies bound to a solid phase support combined with a third different detectably labeled antibody in solution.

The invention is also directed to a method of producing an NTP, said method comprising:

(a) culturing a recombinant host comprising a human gene coding for said NTP; and

(b) isolating said NTP from said host.

Additionally, the invention is directed to a substantially pure NTP obtained by the such a process.

The invention is also directed to an 15- to 30-mer antisense oligonucleotide which is complementary to an NTP nucleic acid sequence and which is nonhomologous to PTP nucleic acid sequences, as well as pharmaceutical compositions comprising such oligonucleotides and a pharmaceutically acceptable carrier.

The invention is also directed to ribozymes comprising a target sequence which is complementary to an NTP sequence and nonhomologous to PTP nucleic acid sequences, as well as pharmaceutical compositions comprising such ribozymes and a pharmaceutically acceptable carrier.

The invention is also directed to a method of achieving pharmaceutical delivery of NTP molecules to the brain through acceptable carriers or expression vectors.

The invention is also directed to oligodeoxynucleotides that form triple stranded regions with the various NTP genes (nucleic acid sequences) and which are nonhomologous to PTP nucleic acid sequences, as well as pharmaceutical compositions comprising such oligodeoxynucleotides and a pharmaceutically acceptable carrier.

The invention is also directed to the therapeutic use of NTP-derived molecules or fragments thereof to modify or improve dementias of the Alzheimer's type of neuronal degeneration.

The invention is also directed to methods for the differential diagnosis of sporadic and familial Alzheimer's disease.

DEFINITIONS

In the description that follows, a number of terms used in recombinant DNA technology are utilized extensively. In order to provide a clear and consistent understanding of the specification and claims, including the scope to be given such terms, the following definitions are provided.

Cloning vector. A plasmid or phage DNA or other DNA sequence which is able to replicate autonomously in a host cell, and which is characterized by one or a small number of restriction endonuclease recognition sites at which such DNA sequences may be cut in a determinable fashion without loss of an essential biological function of the vector, and into which a DNA fragment may be spliced in order to bring about its replication and cloning. The cloning vector may further contain a marker suitable for use in the identification of cells transformed with the cloning vector. Markers, for example, provide tetracycline resistance or ampicillin resistance.

Expression vector. A vector similar to a cloning vector but which is capable of enhancing the expression of a gene which has been cloned into it, after transformation into a host. The cloned gene is usually placed under the control of (i.e., operably linked to) certain control sequences such as promoter sequences. Promoter sequences may be either constitutive or inducible.

Substantially pure. As used herein means that the desired purified protein is essentially free from contaminating cellular components, said components being associated with the desired protein in nature, as evidenced by a single band following polyacrylamide-sodium dodecyl sulfate gel electrophoresis. Contaminating cellular components may include, but are not limited to, proteinaceous, carbohydrate, or lipid impurities.

The term "substantially pure" is further meant to describe a molecule which is homogeneous by one or more purity or homogeneity characteristics used by those of skill in the art. For example, a substantially pure NTP will show constant and reproducible characteristics within standard experimental deviations for parameters such as the following: molecular weight, chromatographic migration, amino acid composition, amino acid sequence, blocked or unblocked N-terminus, HPLC elution profile, biological activity, and other such parameters. The term, however, is not meant to exclude artificial or synthetic mixtures of the factor with other compounds. In addition, the term is not meant to exclude NTP fusion proteins isolated from a recombinant host.

Recombinant Host. According to the invention, a recombinant host may be any prokaryotic or eukaryotic cell which contains the desired cloned genes on an expression vector or cloning vector. This term is also meant to include those prokaryotic or eukaryotic cells that have been genetically engineered to contain the desired gene(s) in the chromosome or genome of that organism.

Recombinant vector. Any cloning vector or expression vector which contains the desired cloned gene(s).

Host. Any prokaryotic or eukaryotic cell that is the recipient of a replicable expression vector or cloning vector. A "host," as the term is used herein, also includes prokaryotic or eukaryotic cells that can be genetically engineered by well known techniques to contain desired gene(s) on its chromosome or genome. For examples of such hosts, see Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989).

Promoter. A DNA sequence generally described as the 5' region of a gene, located proximal to the start codon. The transcription of an adjacent gene(s) is initiated at the promoter region. If a promoter is an inducible promoter, then the rate of transcription increases in response to an inducing agent. In contrast, the rate of transcription is not regulated by an inducing agent if the promoter is a constitutive promoter.

Gene. A DNA sequence that contains information needed for expressing a polypeptide or protein.

Structural gene. A DNA sequence that is transcribed into messenger RNA (mRNA) that is then translated into a sequence of amino acids characteristic of a specific polypeptide.

Antisense RNA gene/Antisense RNA. In eukaryotes, mRNA is transcribed by RNA polymerase II. However, it is also known that one may construct a gene containing a RNA polymerase II template wherein a RNA sequence is transcribed which has a sequence complementary to that of a specific mRNA but is not normally translated. Such a gene construct is herein termed an "antisense RNA gene" and such a RNA transcript is termed an "antisense RNA." Antisense RNAs are not normally translatable due to the presence of translation stop codons in the antisense RNA sequence.

Antisense oligonucleotide. A DNA or RNA molecule containing a nucleotide sequence which is complementary to that of a specific mRNA. An antisense oligonucleotide binds to the complementary sequence in a specific mRNA and inhibits translation of the mRNA.

Antisense Therapy. A method of treatment wherein antisense oligonucleotides are administered to a patient in order to inhibit the expression of the corresponding protein.

Complementary DNA (cDNA). A "complementary DNA," or "cDNA" gene includes recombinant genes synthesized by reverse transcription of mRNA and from which intervening sequences (introns) have been removed.

Expression. Expression is the process by which a polypeptide is produced from a structural gene. The process involves transcription of the gene into mRNA and the translation of such mRNA into polypeptide(s).

Homologous/Nonhomologous Two nucleic acid molecules are considered to be "homologous" if their nucleotide sequences share a similarity of greater than 50%, as determined by HASH-coding algorithms (Wilber, W. J. and Lipman, D. J., Proc. Natl. Acad. Sci. 80:726-730 (1983)). Two nucleic acid molecules are considered to be "nonhomologous" if their nucleotide sequences share a similarity of less than 50%.

Ribozyme. A ribozyme is an RNA molecule that contains a catalytic center. The term includes RNA enzymes, self-splicing RNAs, and self-cleaving RNAs.

Ribozyme Therapy. A method of treatment wherein ribozyme is administered to a patient in order to inhibit the translation of the target mRNA.

Fragment. A "fragment" of a molecule such as NTP is meant to refer to any polypeptide subset of that molecule.

Functional Derivative. The term "functional derivatives" is intended to include the "variants," "analogues," or "chemical derivatives" of the molecule. A "variant" of a molecule such as NTP is meant to refer to a naturally occurring molecule substantially similar to either the entire molecule, or a fragment thereof. An "analogue" of a molecule such as NTP is meant to refer to a non-natural molecule substantially similar to either the entire molecule or a fragment thereof.

A molecule is said to be "substantially similar" to another molecule if the sequence of amino acids in both molecules is substantially the same, and if both molecules possess a similar biological activity. Thus, provided that two molecules possess a similar activity, they are considered variants as that term is used herein even if one of the molecules contains additional amino acid residues not found in the other, or if the sequence of amino acid residues is not identical.

As used herein, a molecule is said to be a "chemical derivative" of another molecule when it contains additional chemical moieties not normally a part of the molecule. Such moieties may improve the molecule's solubility, absorption, biological half-life, etc. The moieties may alternatively decrease the toxicity of the molecule, eliminate or attenuate any undesirable side effect of the molecule, etc. Examples of moieties capable of mediating such effects are disclosed in Remington's Pharmaceutical Sciences (1980) and will be apparent to those of ordinary skill in the art.

NTP. The term "NTP" refers to a family of neural thread proteins. The NTP family includes proteins with molecular weights of about 8 kDa, 14 kDa, 17 kDa, 21 kDa, 26 kDa and 42 kDa, as described herein.

Immuno-Polymerase Chain Reaction. A method for the detection of antigens using specific antibody-DNA conjugates. According to this method, a linker molecule with bispecific binding affinity for DNA and antibodies is used to attach a DNA molecule specifically to an antigen-antibody complex. As a result, a specific antigen-antibody-DNA conjugate is formed. The attached DNA can be amplified by the polymerase chain reaction (PCR) using appropriate oligonucleotide primers. The presence of specific PCR products demonstrates that DNA molecules are attached specifically to antigen-antibody complexes, thus indicating the presence of antigen. (Sano et al., Science 258:120-122 (1992)).

For example, Sano et al., supra, constructed a streptavidin-protein A chimera that possesses specific binding affinity for biotin and immunoglobulin G. This chimera (i.e., the "linker molecule") was used to attach a biotinylated DNA specifically to antigen-monoclonal antibody complexes that had been immobilized on microtiter plate wells. A segment of the attached DNA was subsequently amplified by PCR.