| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | February 15, 2000 |
| PATENT TITLE |
Methods and compositions for diagnosis and treatment of pathological conditions related to abnormal dopamine receptor expression |
| PATENT ABSTRACT |
Methods and compositions are provided for diagnosing and treating pathological conditions related to a dopamine receptor abnormality. The methods comprise administering to a patient having such a pathological condition a plasmid encoding an oligonucleotide, antisense to one or more RNA molecules encoding one of the several dopamine receptors, thereby selectively controlling expression of one or more dopamine receptor subtypes, and alleviating the pathological conditions related to their expression. The vectors are targeted to specific regions of the brain via complexation with antibody studded liposomes. |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | March 14, 1997 |
| PATENT REFERENCES CITED |
Milner et al. Selecting effective antisense reagents on combinatorial oligonucleotide arrays. Nature Biotechnology vol. 15 pp. 537-541, 1997. Geisert et al. Transfecting neurons and glia in the rat using pH-sensitive immunoliposomes. Neuroscience Letters vol. 184 pp. 40-43, 1995. Neckers et al. Antisense technology: biological utility and practical considerations. Am. J. Physiol. vol. 265 pp.L1-L12, 1993. Orkin et al. Report and recommendations of the panel to assess the NIH investment in research on gene therapy. Pp. 1-41, 1995. |
| PATENT GOVERNMENT INTERESTS |
CROSS REFERENCE TO RELATED APPLICATIONS This is a continuation-in-part of U.S. application Ser. No. 08/448,386 filed on Dec. 14, 1993, U.S. Pat. No. 5,840,708, which was the National stage of International application No. PCT/US93/12161, filed on Dec. 14, 1993, which is a continuation of U.S. application Ser. No. 07/991,582, filed Dec. 14, 1992, now abandoned. This application claims the benefit of U.S. Provisional Application No. 60/013,440, filed Mar. 15, 1996. -------------------------------------------------------------------------------- |
| PATENT PARENT CASE TEXT | This data is not available for free |
| PATENT CLAIMS |
What is claimed is: 1. A DNA construct encoding a sequence antisense to an mRNA molecule that encodes a predetermined dopamine receptor in a living organism, said construct comprising: a) a 5' promoter element; b) a DNA segment encoding an antisense oligonucleotide comprising a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, said antisense oligonucleotide binding specifically to said mRNA molecule within a region comprising its translational start site; said DNA segment being operably linked to said 5' promoter element such that expression of said antisense oligonucleotide is controlled by said 5' promoter element; and c) a 3' polyadenylation signal sequence, operably linked to said DNA segment. 2. A vector comprising a DNA construct according to claim 1. 3. The DNA construct as claimed in claim 1, wherein said 5' promoter element is selected from the group consisting of cytomegalovirus promoter element, metallothionein promoter element, SV40 promoter element, and heat shock promoter element. 4. The DNA construct as claimed in claim 1, wherein said 3' polyadenylation signal sequence is selected from the group consisting of bovine growth hormone polyadenylation signal sequences and thymidine kinase polyadenylation signal sequences. 5. The DNA construct as claimed in claim 1, further comprising at least one selectable marker gene, said at least one selectable marker gene being disposed within said construct so as not to disrupt expression of said DNA segment and encoding a protein conferring resistance to a selection agent. 6. The DNA construct as claimed in claim 5, wherein said selectable marker gene confers resistance to a selection agent selected from the group consisting of hygromycin, neomycin, ampicillin, spectinomycin, and streptomycin. 7. A recombinant vector encoding an antisense oligonucleotide which binds specifically to a predetermined dopamine receptor-encoding mRNA molecule at a region comprising a translational start site of said mRNA molecule, said vector comprising: a) at least one selectable marker gene encoding a protein conferring resistance to a selection agent, said selectable marker gene being disposed within the vector so as not to interfere with expression of said antisense oligonucleotide; b) a 5' promoter element; c) a DNA segment encoding said antisense oligonucleotide, said antisense oligonucleotide comprising a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, said DNA segment being operably linked to said 5' promoter element such that expression of said antisense oligonucleotide is controlled by said 5' promoter element; d) a 3' polyadenylation signal sequence operably linked to said DNA segment thereby promoting stability of sequences encoded by said DNA segment; e) a ColE1 E. coli origin of replication element; f) a PSV40/ori element; and g) an F1 ori element. 8. The vector as claimed in claim 7, which is pCR3-D.sub.2 AS. 9. The vector of claim 7 complexed with cationic liposomes studded with anti-limbic system associated membrane protein antibody. 10. A recombinant vector encoding an antisense oligonucleotide which binds specifically to a predetermined dopamine receptor-encoding mRNA molecule at a region comprising a translational start site of said mRNA molecule, said vector comprising: a) at least one selectable marker gene encoding a protein conferring resistance to a selection agent, said selectable marker gene being disposed within the vector so as not to interfere with expression of said antisense oligonucleotide; b) a 5' promoter element; c) a DNA segment encoding said antisense oligonucleotide, comprising a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, said DNA segment being operably linked to said 5' promoter element such that expression of said antisense oligomueleotide is controlled by said 5' promoter element; d) a 3' polyadenylation signal sequence operably linked to said DNA segment thereby promoting stability of sequences encoded by said DNA segment; e) a ColE1 E. coli origin of replication element; f) an Epstein Barr virus (EBV) origin; and g) a coding segment for EBV nuclear antigen. 11. The vector of claim 10, which is pCEP4-D.sub.2A S. 12. The vector of claim 10 complexed with cationic liposomes studded with anti-limbic system associated membrane protein antibody. -------------------------------------------------------------------------------- |
| PATENT DESCRIPTION |
FIELD OF THE INVENTION This invention relates to treatment of pathological conditions associated with abnormalities in dopamine receptors. In particular, it relates to diagnosis and treatment of patients having such pathological conditions by administration of oligonucleotides and/or vectors encoding sequences antisense to one or more mRNA molecules encoding one of the several dopamine receptors. BACKGROUND OF THE INVENTION Abnormal activity of the dopamine-responsive nervous system has been implicated in a number of motor and behavioral disorders including Parkinson's disease, Huntington's disease, tardive dyskinesia, certain forms of schizophrenia and other dystonias and dyskinesias. Dysfunctions of the dopamine-responsive system may be caused either by a reduced or increased activity of the system or by the inability of the systems to be modulated by a changing external or internal environment. Dopamine is one of the major catecholamine neurotransmitters in the mammalian brain. Dopamine exerts its effect in part by binding to G protein-coupled dopamine receptors. Pharmacological and molecular biological studies have shown that the dopamine receptor has at least five subtypes, designated D.sub.1-5. The best characterized of these are D.sub.1 and D.sub.2. The D.sub.2 subtype exists in a long and short form, the long form having a larger intracellular loop than the short form. These receptor subtypes appear to be anatomically, biochemically and behaviorally distinct. For example, D.sub.1 and D.sub.2 receptors have different anatomical distributions, in that only D.sub.1 receptors are found in the retina and only D.sub.2 receptors are found in the pituitary, but both D.sub.1 and D.sub.2 are found in the striatum and substantia nigra. D.sub.1 and D.sub.2 receptors are reported to have opposite biochemical effects on adenylate cyclase activity, and stimulation of D.sub.1 and D.sub.2 receptors produces different behavioral responses. See Weiss et al., Neurochemical Pharmacology--A Tribute to B. B. Brody, E. Costa, ed.; Raven Press, Ltd., New York; pp. 149-164 (1989). Recently, three new subtypes of dopamine receptors have been discovered. On the basis of nucleotide and amino acid sequence homology, D.sub.3 and D.sub.4 have been found to be related to D.sub.2, and D.sub.5 is related to D.sub.1. Hence, the dopamine receptor subtypes may be categorized into two subfamilies, D.sub.1 and D.sub.5 being members of the D.sub.1 subfamily, and D.sub.2, D.sub.3 and D.sub.4 being members of the D.sub.2 subfamily. See Sibley et al., Trends in Pharmacological Sciences, 13: 61-69 (February, 1992). The dopamine receptor subtypes can be separately and independently modulated through the administration of selective agonists and antagonists. For example, whereas dopamine and apomorphine are agonists of both D.sub.1 and D.sub.2 receptors, compounds such as SKF 38393 (Setler et al., Eur. J. Pharmacol., 50: 419-30, 1978) is a selective agonist of only D.sub.1 and quinpirole (Tsuruta et al., Nature, 292: 463-65, 1981) is a relatively selective agonist for the D.sub.2 receptor. It should be emphasized, however, that the currently available dopaminergic drugs have only a relative selectivity for the various dopamine receptors. Indeed, there are recent reports suggesting that quinpirole may have a higher affinity for D.sub.3 receptors than for D.sub.2 receptors (see Sokoloff et al., Nature, 347: 146-151 (1990)). It is not surprising, therefore, that the use of specific and nonspecific neuroleptic drugs in the treatment of dopamine-related disorders is contraindicated by the fact that such drugs often produce numerous side effects, presumably due to cross-reactivity with other dopamine receptor subtypes, or even with other classes of neuroreceptors. For example, the therapeutic action of many neuroleptic drugs appears to be due to a blockade of dopamine D.sub.2 receptors. However, patients treated with such drugs often develop tardive dyskinesia, possibly because of the up-regulation of dopamine receptors. A further example is that benzazepines (such as SKF-38393), which are a major D.sub.1 drug class, were recently found to be strongly cross-reactive with the serotonin 5-HT.sub.2 receptor family. Nicklaus et al., J. of Pharmacol. Exp. Ther., 247: 343-48 (1988); Hoyer et al., Eur. J. Pharmacol., 150: 181-84 (1988). Additionally, clozapine is a favored antipsychotic in the treatment of socially withdrawn and treatment-resistent schizophrenics because it does not cause tardive dyskinesia. Clozapine has been found to preferentially bind the D.sub.4 receptor with an affinity ten times higher than to the D.sub.2 or D.sub.3 receptors. Van Tol et al., Nature, 358: 149-152 (1992). However, clozapine treatment results in other side effects that are likely to be related to nonselectivity of the drug. From the foregoing discussion, it can be seen that a need exists for improved selectivity in dopamine receptor drug classes. However, it is difficult to achieve such selectivity with classical pharmaceutical agents because the available drugs likely act on multiple neurotransmitter receptors and because the modulatory responsiveness of the nervous system to such compounds is not well understood. Therefore, it is difficult to separate the effect of drug cross-reactivity from a natural compensatory response of the nervous system to modulation of a specific receptor. Molecular biological techniques provide a useful means to develop highly specific receptor modulatory agents. In the case of dopamine receptors, all five subtypes have been cloned from at least one biological source. Sequence information is available for all five subtypes. The availability of such information enables development of receptor-regulatory agents targeted to pre-translational stages of receptor expression. In particular, knowledge of the nucleotide sequence of dopamine receptor genes and messenger RNAs enables the selection and synthesis of antisense molecules capable of binding to critical targets on dopamine receptor mRNAs, thereby inhibiting or modifying translation. The use of synthetic antisense oligonucleotides for therapeutic purposes was first proposed in 1978 and has been successfully accomplished in vitro and within cultured cells. See Uhlmann et al., Chemical Review, 90: 544-584 (1990). However, successful application of antisense therapy in vivo has been extremely limited. The delivery of antisense oligonucleotides to target cells in vivo is but one obstacle to overcome in developing successful antisense therapeutic agents. Even if biologically significant amounts of antisense molecules reach target cells and bind to selected target sites on mRNA, a subsequent effect on regulation of translation is not guaranteed. Regulation of protein synthesis is highly dependent upon the number of messenger RNA molecules present in the cell that encode a particular protein, as well as the rate of protein synthesis. Furthermore, even if expression of a selected protein can be modulated by an antisense molecule, whether such modulation would have an effect on the associated pathological condition cannot be predicted. There have been a few reports of successful modulation of various pathological conditions by antisense therapy in rodents. Oligonucleotides antisense to the proto-oncogene c-myc have been administered in vivo to rats to suppress the intimal accumulation of rat carotid arterial smooth muscle cells (Simons et al., Nature 359: 67-70 (1992). Antisense oligonucleotides have also been used in vivo in mice. An 18-nucleotide phosphorothioate oligodeoxynucleotide antisense to sequences encoding the interleukin I (IL-1) receptors, when injected subcutaneously into mice, markedly inhibited the infiltration of neutrophils in response to subsequent injections of IL-1. Burch and Mahan, J. Clin, Invest., 88: 1190-1196 (1991). In another study, repeated injection of antisense oligonucleotide (3.times.0.5 nmol per day) conferred 30-70% protection against a normally fatal infection of encephalitis in mice. See Uhlmann et al., supra at 577. It can be surmised from the foregoing examples that antisense technology has great potential in vivo therapy for the treatment of disease. However, this potential, to date, remains largely unexplored. SUMMARY OF THE INVENTION According to one aspect of the present invention, a method is provided for controlling the expression of a pre-determined dopamine receptor in a living organism. The method comprises providing an antisense oligonucleotide or a vector encoding antisense RNA molecules which are capable of binding specifically to an expression-controlling sequence of a nucleic acid encoding the dopamine receptor. The antisense oligonucleotide is administered to the living organism under conditions whereby the oligonucleotide enters cells expressing the dopamine receptor and binds specifically to the expression-controlling sequence of the nucleic acid encoding the dopamine receptor, in an amount sufficient to control the expression of the dopamine receptor. According to another aspect of the present invention, a compound is provided that is useful for controlling expression of a pre-determined dopamine receptor in a living organism. The compound comprises an antisense oligonucleotide analog capable of entering a cell expressing such a dopamine receptor and binding specifically to an expression-controlling sequence of a nucleic acid that encodes the dopamine receptor, in an amount sufficient to control expression of the dopamine receptor. According to another aspect of the present invention, a method is provided for treating a pathological condition related to an abnormality in a dopamine receptor. Examples of such abnormal pathological conditions are Huntington's Disease, tardive dyskinesia, various forms of schizophrenia, dementia, as well as abnormal vascular control, abnormal circadian rhythms and abnormal visual activity. The method comprises administering to a patient having such a pathological condition a pharmaceutical preparation comprising an antisense oligonucleotide analog capable of entering a cell expressing the dopamine receptor and binding specifically to a nucleic acid encoding the dopamine receptor, in an amount sufficient to affect the level of expression of the dopamine receptor, thereby alleviating the pathological condition. According to another aspect of this invention, a pharmaceutical preparation is provided for treating a pathological condition related to a dopamine receptor abnormality. This pharmaceutical preparation comprises, in a biologically compatible medium, an antisense oligonucleotide capable of entering a cell expressing the dopamine receptor and binding specifically to a nucleic acid encoding the dopamine receptor, in an amount sufficient to affect the level of expression of the dopamine receptor. In yet another aspect of the present invention, there is provided an antisense oligonucleotide analog for inhibiting D.sub.2 dopamine receptor agonist-induced rotational behavior in mammals. When administered by intraventricular injection, such an antisense oligonucleotide analog is capable of crossing a biological membrane and binding specifically to a nucleotide sequence comprising a translation start site of mRNA encoding the D.sub.2 dopamine receptor, in an amount sufficient to affect the level of expression of the D.sub.2 receptor. In another aspect of the present invention, there is provided an antisense oligonucleotide analog for inhibiting D.sub.1 dopamine receptor agonist-induced grooming behavior and rotational behavior in mammals. When administered by intraventricular injection, such an antisense oligonucleotide analog is capable of crossing a biological membrane and binding specifically to a nucleotide sequence comprising a translation start site of mRNA encoding the D.sub.1 dopamine receptor, in an amount sufficient to affect the level of expression of the D.sub.1 receptor. According to a further aspect of the present invention, methods are provided for diagnosing pathological conditions relating to a dopamine receptor abnormality. In one such method, a patient whose pathological condition is suspected of being related to a particular dopamine receptor abnormality is administered at least one antisense oligonucleotide capable of crossing a biological membrane and binding specifically to a nucleotide sequence encoding a pre-determined dopamine receptor subtype, in an amount sufficient to affect the level of expression of the particular subtype. It is then observed whether or not the pathological condition is alleviated as a result of administering the antisense oligonucleotides. Alleviation of the pathological condition is thus diagnostic of an abnormality in the pre-determined dopamine receptor subtype. Another diagnostic method provides a way of determining if expression of a pre-determined dopamine receptor subtype in a living organism is related to a pre-determined pathological condition in that living organism. The method comprises first providing a living organism having the pathological condition, as well as providing an antisense oligonucleotide capable of binding specifically to an expression-controlling sequence of a nucleic acid that encodes the dopamine receptor subtype. The antisense oligonucleotide administered to the living organism under conditions whereby the antisense oligonucleotide enters cells expressing the dopamine receptor subtype and bind specifically to the expression-controlling sequence of the nucleic acid that encodes the dopamine receptor subtype, in an amount sufficient to control expression of the dopamine receptor subtype. It is then observed whether or not such control of expression of the dopamine receptor subtype alleviates the pathological condition. Alleviation of the pathological condition indicates that expression of the dopamine receptor subtype is related to the pathological condition in the living organism. According to another aspect of the present invention, the compound is provided for controlling expression of a pre-determined dopamine receptor in cells expressing that receptor. The compound comprises an antisense oligonucleotide capable of entering the cell and binding specifically to an expression-controlling sequence of a nucleic acid that encodes the dopamine receptor, in an amount sufficient to control expression of the dopamine receptor. According to still another aspect of the present invention, a method is provided for determining if a test compound is a specific agonist or antagonist of a pre-determined dopamine receptor subtype (the term "agonist or antagonist" is sometimes substituted herein by the general term "regulator"). According to the methods, cells expressing the pre-determined dopamine receptor subtype are provided; however, the cells may express more than one dopamine receptor subtype. The cells possess at least one detectable biological function =that is related to a response of the pre-determined dopamine receptor subtype caused by the specific regulator. A quantity of the cells are pre-treated with an antisense oligonucleotide capable of entering the cells and binding specifically to an expression-controlling sequence of a nucleic acid that encodes the pre-determined dopamine receptor subtype, under conditions whereby the antisense oligonucleotides enters the cells and binds to the expression-controlling sequence in an amount sufficient to control expression of the specific dopamine receptor subtype. The pre-treated cells, and an equivalent quantity of non pre-treated cells, are exposed to the test compound under conditions promoting the specific regulatory effect, if any, of the test compound. Changes are then detected in the related biological function, if any have occurred, in the pre-treated and non pre-treated cells, to observe whether or not the control of expression of the specific dopamine receptor subtype by the antisense oligonucleotide alleviates the change in the related biological function (if any exists) exerted by the test compound. Any alleviation observed in the pre-treated cells as compared to the non pre-treated cells is indicative that the test compound is a specific regulator (agonist or antagonist) of the pre-determined dopamine receptor subtype. Thus, by observing the effect of a potential agonist or antagonist of a specific dopamine receptor subtype in the presence or absence of the antisense oligonucleotide specific for that receptor subtype, potentially useful dopamine regulating compounds may be screened for their specific agonistic or antagonistic effect on a particular dopamine receptor subtype. In one preferred embodiment of the present invention, the antisense oligonucleotide is an oligonucleotide analog having improved stability and membrane permeability as compared with an unmodified oligonucleotide. In a particularly preferred embodiment of the invention, an antisense RNA molecule is delivered in a DNA vector containing sequences that encode the antisense RNA. The use of double-stranded DNA as a delivery vehicle exploits the greater natural stability of double-stranded as compared to single-stranded nucleic acids. The use of an expression vector that generates multiple RNA copies prolongs expression of the antisense RNA molecules in vivo. Both the antisense oligonucleotide analog and the DNA expression vector encoding the antisense RNA are formulated to be capable of crossing a biological membrane in order to enter cells and thereafter bind specifically with the selected nucleic acid sequence. The selected nucleic acid sequence preferably comprises a translation start site of mRNA encoding the dopamine receptor. The biologically compatible medium is preferably formulated to enhance the lipophilicity and membrane-permeability of the antisense oligonucleotide or plasmid expression vector. The methods and antisense compounds of the present invention provide notable advantages over currently available compounds and methods for treating abnormalities of the dopamine nervous system. Most notably, these methods and compounds provide improved selectivity in the treatment of abnormalities associated with a specific dopamine receptor subtype. Potent, highly specific antisense molecules may be designed, based on knowledge of the nucleotide sequences encoding the dopamine receptor subtypes. Target sequences may be selected which are specific to a pre-determined dopamine receptor subtype, yet critical to expression of the dopamine receptor (e.g., a translation start site). Because such antisense compounds have now been shown to be capable of entering dopaminergic cells and exerting an effect on a pathological condition associated with the dopamine receptor, such compounds and methods will be useful in numerous therapeutic and diagnostic applications relating to the dopaminergic nervous system. |
| PATENT PHOTOCOPY | Available on request |
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