JPH10506003A - Pleiotrophin antisense oligonucleotides - Google Patents

Abstract

  (57) [Summary] Antisense oligonucleotides that hybridize to segments of mRNA corresponding to pleiotrophin cDNA inhibit pleiotrophin synthesis in vitro and in vivo. Also disclosed are pharmaceutical compositions containing the oligonucleotide as an active ingredient.

Description

DETAILED DESCRIPTION OF THE INVENTION Pleiotrophin antisense oligonucleotides Background of the Invention Polypeptide growth factors have been shown to play a physiologically important role in the timely growth of tissues during fetal and neonatal growth, and their expression is therefore closely regulated. Conversely, expression of the polypeptide growth factor gene is unregulated in tumor cell lines as well as in unregulated tumors, and the activity of the corresponding growth factor is autocrine ( autocrine) It appears to be significantly involved in stimuli and paracrine stimuli. See Cross and Dexter, Cell 64: 271 (1991). Pleiotrophin (PTN) is an 18 kD heparin-binding protein that was first purified as a weak mitogen from bovine uterus and as a post-neurite sprouting growth promoter from the brain of newborn rats. Milner et al., Biochem. Biophys. Res. Commun. 165: 1096-1103 (1989); Rauvala, EMBO J .; 8: 2933-2 941 (1989); Li et al., Science 250: 1690-1694 (1990). PTN has been found to belong to the class of heparin-binding growth factors. Lai et al., Biochem. Biophys. Res. Commun. 187: 1113-1121 (1992). The cDNAs for human, bovine and rat PTN have been cloned and sequenced and reveal sequences identical to retinoic acid-induced differentiation factors and retinoic acid-induced heparin-binding proteins from chicken fetuses. It has become. Li et al., (1990), Kadomatsu et al., Biochem. Biophys. Res. Commun. 151: 1312-1318 (1988); Tomomura et al. Biol. Chem. 265: 10765-10770 (1990); Vrios et al., Biochem. Biophys. Res. Co mmun. 175: 617-624 (1991). Previous studies have suggested that PTN transcripts are expressed in a restricted manner in tissues during mouse development and are highly regulated. PTN and the closely related midkine (MK) protein appear to function during the developmental phase of the neutral ectoderm, and the physiological expression of that gene in adults occurs only in very limited areas of the nervous system . Bohlen and Kovesdi, Prog. See Growth Factor Res., 3: 143-57 (1991). PTN has also been implicated in cancer formation. For example, PTN expression is thriving in highly angiogenic melanomas, which activate the growth of SW13 cells on soft agar. Wellstein et al. Biol. Chem. 267: 2582-2587 (1992). PTN expression can also introduce tumors that grow in nude mice, and high levels of PTN mRNA are detected in tissue samples from human lung cancer. Fang et al. Biol. Chem. 267 25889-25897 (1992). In a concurrent study, approximately one-quarter of the tested tumor cell lines showed PTN expression as measured by an RNase protection assay. Carcinogen-induced tumors in rat breast tissue were also positive for PTN expression. A recent study using PTN-targeted hammerhead ribozyme constructs showed that suppression of PTN production in WM852 human melanoma cells suppressed colony formation in soft agar and prevented tumor formation in mice . Czubayko et al. Biol. Chem. See in press (1994). However, other reports have provided conflicting data on the correlation between high concentrations of PTN and neoplasia. See, for example, Garver et al., Am. J. Respir. Cell Mol. Biol. 9: 463-466 (1993) show that PTN expression is significantly higher in healthy lung tissue than in malignant lung tissue. Similarly, PTN mRNA in human cancer tissue was found to be significant in many samples, except for significant levels of expression in PA-1 teratocarcinoma cells and several surgical specimens of Wilms' tumors. It was shown to be barely detectable. Tsutsui et al., Cancer Res. 53: 1281-1285 (1993). It remains unclear whether inhibition of PTN expression may be effective in suppressing tumorigenesis and growth. There are currently no known methods for inhibiting the action of pleiotrophin in cells and detecting the effects obtained on cell growth. Therefore, it is clear that an approach to establish the role of pleiotrophin in tumorigenesis is highly desirable. In particular, the present invention provides a composition and a method for suppressing the cellular effect of pleiotrophin, which has a very high specificity and can inhibit or suppress pathological tissue growth as seen in neoplastic and dysplastic diseases. It is highly desirable to do. Summary of the Invention Accordingly, it is an object of the present invention to provide a method for inhibiting pleiotrophin expression in cells. Another object of the present invention is to provide a method for treating cancer by inhibiting the expression of pleiotrophin. It is a further object of the present invention to provide compositions that inhibit pleiotrophin expression. It is still another object of the present invention to provide antisense oligonucleotides that inhibit pleiotrophin expression by regulating the translation of mRNA corresponding to the pleiotrophin gene. To meet these objectives, there is provided a method of inhibiting PTN expression in cells by introducing an oligonucleotide capable of hybridizing to a single-stranded mRNA encoding human pleiotrophin. In a preferred embodiment, the PTN is human PTN. In a further preferred embodiment, the oligonucleotide is contained in a liposome. Also provided are a set of antisense oligonucleotides that, when introduced into a cell that expresses PTN, inhibit expression of PTN. In other embodiments, at least one antisense oligonucleotide that binds to a single-stranded mRNA segment transcribed from the pleiotrophin gene when introduced into a host cell and inhibits pleiotrophin synthesis in the cell. A composition comprising: In another embodiment, there is provided a pharmaceutically useful preparation comprising at least one PTN antisense oligonucleotide in a sterile pharmaceutically acceptable carrier. In yet another aspect, there is provided a method for treating pathological tissue growth comprising inhibiting the expression of a pleiotrophin gene. In a preferred embodiment, the pathological growth is a dysplasia or neoplastic disease. In yet another embodiment, treating pathological tissue growth in a patient, comprising administering to the patient an amount of at least one PTN antisense oligonucleotide sufficient to inhibit pleiotrophin synthesis in the patient. Therefore, a method is provided. Other objects, features and advantages of the present invention will become apparent from the following detailed description. However, since various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art from the detailed description, the detailed description and specific examples that represent preferred embodiments of the present invention are provided for illustrative purposes only. It should be understood that it is. BRIEF DESCRIPTION OF THE FIGURES FIG. 1 shows the cDNA sequence for human pleiotrophin (SEQ ID NO: 1). FIG. 2 shows the position of the antisense primer in the cDNA sequence for human pleiotrophin (SEQ ID NO: 2). Detailed description of preferred embodiments The present invention relates to methods of inhibiting pleiotrophin synthesis, and thus provides a therapeutic regimen for treating neoplasia and dysplasia. The present invention is based on the use of antisense oligonucleotides that anneal to pleiotrophin-specific single-stranded RNA, thereby inhibiting the production of pleiotrophin. Inhibition of pleiotrophin synthesis suppresses the corresponding growth-stimulatory activity and somewhat mitigates PTN-related neoplastic and dysplastic diseases. In accordance with the present invention, there are provided oligonucleotides designed to hybridize to portions of mRNA encoding pleiotrophin, thereby inhibiting the function of these RNAs. The present invention also provides a pharmaceutically acceptable sterile carrier, as described in Remington's Pharmaceutical Sciences, Drug Receptors and Receptor Theory, 18th ed., Mark Publishing Co., Easton, PA (1990); Also included are pharmaceutical compositions in combination with an effective amount of at least one antisense oligonucleotide according to the present invention. Antisense technology offers a very specific and potent method of inhibiting this gene product. See Stein and Chang, Science 261: 1004-12 (1993). Antisense oligonucleotides ("antisense oligos") are typically short DNA sequences, usually 10 to 50 bases in length, that are complementary to specific regions of the corresponding target mRNA. . Hybridization of antisense oligonucleotides to their target transcripts shows high specificity as a result of forming complementary base pairs. Antisense oligo hybridization is affected by parameters such as transcript length, chemical modification, and secondary structure that can affect the access of the oligo to the target site. See Stein et al., Cancer Research 48: 2659 (1988). When choosing a preferred length for a given oligo, a balance must be made to obtain the most advantageous properties. Short oligos, such as 10-15 mer, show high cell invasiveness, but low gene specificity. In contrast, longer oligos of 20-30 bases exhibit better specificity, but at a lower rate of cellular uptake. Stein et al., PHOSPHOROTHIOATE OLIGODEOXYNUCLE0TIDE ANALOGUES in “Oli godeoxynucleotides-Antisense Inhibitors of Gene Expression” Cohen, ed. See McMillan Press, London (1988). The ability to access the mRNA target sequence is also important, so the loop-forming region present in the targeted mRNA provides a potential target. As used herein, the term `` oligonucleotide '' refers to the nucleic acid portions of oligomers of naturally occurring types, such as the deoxyribonucleotide and ribonucleotide structures of DNA and RNA, as well as artificial, capable of binding to naturally occurring nucleic acids. Includes both analogs. Oligonucleotides of the invention may be based on ribonucleotide or deoxyribonucleotide monomers linked by phosphodiester bonds or by analogs linked by methylphosphonate, phosphorothioate, or other bonds. They may also have monomeric moieties that have altered basic structure or other modifications, but retain their ability to bind to naturally occurring DNA and RNA structures. Such oligonucleotides can be prepared by methods well known to those skilled in the art, for example, using commercially available machines and reagents available from Perkin-Elmer / Applied Biosystems (Foster City, CA). Phosphodiester-linked oligonucleotides are particularly susceptible to nuclease action in serum or inside cells, so in a preferred embodiment the oligonucleotides of the invention have been shown to be nuclease resistant phosphorothioate-linked or It is a methylphosphonate-bound analog. See Stein et al., (1993), supra. One skilled in the art will be able to select other bonds that can be used in the present invention. These modifications can also be designed to improve the uptake and stability of the oligo into cells. See Ghosh et al., Anti-Cancer Drug Design 7: 1 (1992). In another embodiment of the invention, the antisense oligonucleotide is an RNA molecule produced by infecting a target cell with the expression construct. The RNA molecule thus produced is selected to hybridize to the pleiotrophin mRNA, thereby inhibiting translation of the mRNA and inhibiting pleiotrophin synthesis. Hybridization of this oligo with an mRNA target can inhibit the expression of the corresponding gene product by multiple mechanisms. In the "translation arrested" state, the interaction of the target mRNA and the oligo suppresses the action of the ribosome complex, thereby preventing the translation of messenger RNA into protein. Haeuptle et al., Nucl. Acids. Res. 14: 1427 (1986). In the case of a phosphodiester DNA oligo or phosphorothioate DNA oligo, intracellular RNase H can digest the target RNA sequence as soon as the target RNA sequence hybridizes to the DNA oligomer. Walder and Walder, Proc. Natl. Acad. Sci. USA 85: 5011 (1988). As a further mechanism of action in the "translational arrest" state, certain oligonucleotides are capable of forming "triplex" or triple helix structures with double-stranded, standard genomic DNA containing the gene of interest. Apparently prevents transcription by RNA polymerase. Giovannangeli et al., Proc. Natl. Acad. Sci. 90: 10013 (1993); Ebbinghaus et al. Clin. Invest. 92: 2433 (1993). In one preferred embodiment, the PTN oligonucleotide is synthesized according to standard methodology. Phosphorothioate-modified DNA oligonucleotides are generally synthesized using automated DNA synthesizers available from various manufacturers. These devices can synthesize oligonucleotides as long as about 100 nucleotides in nanomolar units. Short oligonucleotides synthesized by state-of-the-art equipment can often be used without further purification. If necessary, the oligo may be purified by polyacrylamide gel electrophoresis or reverse phase chromatography. Sambrook et al., MOLECULAR CLONING: A Laboratory Manual, Vol. 2, Chapter 11, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989). Alternatively, a PTN oligonucleotide in the form of an antisense RNA can be transiently expressed in a suitable cell from a standard DNA expression vector. The PTN DNA sequence can be cloned from a standard plasmid into an expression vector, which has properties that enable it to express endogenous oligonucleotides at higher levels or more effectively. At a minimum, these constructs require a prokaryotic or eukaryotic promoter sequence to initiate transcription of the inserted DNA sequence. Preferred expression vectors are those capable of inducing expression to high levels. This is accomplished in appropriate host cells by the addition of regulatory regions that increase transcription of downstream sequences. Samb rook et al., Vol. See 3, Chapter 16 (1989). For example, a PTN antisense oligo expression vector can be constructed using the polymerase chain reaction (PCR) that amplifies an appropriate fragment obtained from the single-stranded cDNA of plasmid pRc-PTN. Fang et al. Biol. Chem. 267 25889-25897 (1992). FIG. 2 discloses the nucleotide sequence of an oligonucleotide primer suitable for performing a PCR reaction. Oligonucleotide synthesis and purification methods are described in Sambrook et al. And Ausubel et al. (Eds.), Respectively, CURRENT PROTOCOLS IN MOLECULA R BIOLOGY (Wiley Interscience 1987) (hereinafter "Ausubel"). The PCR method is performed by a well-known methodology. For example, see Ausubel, and Bangham, "The Polymerase Chain Reaction: Getting Started," in PROTOCOLS IN HUMAN MOLECULAR GENETICS (Humana Press 1991). Further, PCR kits can be purchased from companies such as Stratagene Cloning Systems (La Jolla, CA) and Invitrogen (San Diego, CA). The PCR product is subcloned into a cloning vector. In this context, a "cloning vector" is a DNA molecule, such as a plasmid, cosmid, or bacteriophage, that is capable of autonomous replication in a prokaryotic host cell. Cloning vectors generally include not only marker genes suitable for use in the identification and selection of cells transformed with the cloning vector, but also foreign DNA sequences without loss of essential biological function of the vector. It contains one or a few restriction endonuclease recognition sites that can be inserted in a detectable manner. Marker genes generally include genes that provide tetracycline resistance or ampicillin resistance. Suitable cloning vectors are described in Sambrook et al., Ausubel, and Brown (ed.), MOLECULAR BIOLOGY LABFAX (Academic Press 1991). Cloning vectors are described, for example, in GIBCO / BRL (Gaithersburg, MD), Clontec Laboratories, inc. (Palo Alto, CA), Promega Corporation (Madison, WI), Stratagene Cloning Systems (La Jolla, CA), Invitrogen (San Diego, CA), and the American Type Culture Collection (Rockville, MD). Preferably, the PCR product is incorporated into a "TA" cloning vector. Methods for producing PCR products with thymidine or adenine overhangs are well known to those skilled in the art. For example, see Ausubel pages 15.7.1 to 15.7.6. Further, a kit for performing TA cloning can be purchased from a company such as Invitrogen (San Diego, CA). The cloned antisense fragment is amplified by transforming the appropriate bacterial cell with the cloning vector and growing the bacterial host cell in the presence of the appropriate antibiotic. See, for example, Sambrook et al. And Ausubel. Subsequently, PCR is performed on the bacterial host cells to screen for the PTN antisense-oriented clone. Methods for performing PCR on bacterial host cells are described, for example, in Hofmann et al., "Sequencing DNA Amplified Directly from a Bacterial Colony," in PCR PROTOCOLS: METHODS AND APPLICATIONS, White (ed.), Pp. 205-210 (Humana Press 1993), and Cooper et al., "PCR-Based Full-Length cDNA Cloning Utilizing the Universal-Adaptor / Specific DOS Primer-Pair Strategy," Id. It is described on pages 305-316. The cloned antisense fragment is cleaved from the cloning vector and inserted into an expression vector. For example, Hind III and Xba I can be used in cleaving the antisense fragment thereof from the TA cloning vector pCR®-II (Invitrogen; San Diego, CA). Suitable expression vectors generally include (1) a prokaryotic DNA element that encodes a bacterial origin of replication and an antibiotic resistance marker that allows for the expression and selection of the expression vector in a bacterial host; DNA elements that regulate the initiation of transcription, such as promoters, and (3) DNA elements that regulate the transcription process, such as transcription termination / polyadenylation sequences. In a mammalian host, the transcriptional control and translational control signals are preferably derived from a viral source, such as, for example, adenovirus, bovine papillomavirus, simian virus, or a similar virus. In these viruses, regulatory signals are involved in specific genes that result in high levels of expression. Suitable transcriptional and translational regulatory sequences can also be obtained from mammalian genes, for example, the actin, collagen, myosin, and metallothionein genes. Transcriptional regulatory sequences contain a promoter region that can successfully initiate RNA synthesis. Suitable eukaryotic cell promoters include the mouse metallothionein I gene promoter (Hamer et al., J. Molec. Appl. Genet. 1: 273 (1982)) and the herpesvirus TK promoter (McKnight, Cell 31: 355 (1982). )), The SV40 early promoter (Benoist et al., Nature 290: 304 (1981)), the Rous sarcoma virus promoter (Gorman et al., Proc. Nat'l Acad. Sci. USA 79: 6777 (1982)), and the cytomegalovirus promoter. (Foecking et al., Gene 45: 101 (1980)). Alternatively, a prokaryotic promoter, such as the bacteriophage T3 RNA polymerase promoter, can be used in regulating fusion gene expression, provided that the prokaryotic promoter is regulated by a eukaryotic promoter. Zhou et al., Mol. Cell. Biol. 10: 4529 (1990), Kaufman et al., Nucl. Acids Res. 19: 4485 (1991). A suitable vector for expression in mammalian cells is the vector pRc / CMV (Invitrogen, San Diego, CA), which provides high levels of constitutive transcription from mammalian enhancer promoter sequences. The cloned PTN antisense vector is amplified in a bacterial host cell, isolated from the cell, and analyzed as described above. Another possible way to use antisense sequences is by gene therapy. Virus-like vectors, usually derived from retroviruses, will prove useful as carriers for the uptake and expression of antisense constructs in tumor cells. Generally, the vectors are non-replicating in vivo and prevent infection of unintended non-target cells. In such cases, a helper cell line is provided that confers a lack of replication competence in vitro, thereby allowing the antisense vector to grow and encapsulate. Another precautionary measure against accidental infection of non-tumor cells involves the use of tumor cell-specific regulatory sequences. Under the control of the sequence, the antisense construct is not expressed in normal tissues. Two previous studies examined the possibility of using antisense oligonucleotides to inhibit the expression of heparin binding growth factor (Kouhara et al., Oncogene 9: 455-462 (1994); Morrison, J. et al. Biol. Chem. 266: 728 (1991)). Have shown that androgen-dependent growth of mouse breast cancer cells (SC-3) is mediated by androgen-induced induction of heparin-binding growth factor (AIGF). Antisense 15mers corresponding to the translation initiation site of AIGF were measured for their ability to interfere with androgen induction in SC-3 cells. At a concentration of 5 μM, the antisense oligonucleotide effectively inhibited androgen-induced DNA synthesis. Morrison has shown that antisense oligonucleotides targeting basic fibroblast growth factor can inhibit the growth of astrocytes in culture. Thus, the general possibility of targeting tumor-associated growth factors has been established. The antisense oligonucleotide according to the present invention may be derived from any part of the open reading frame of pleiotrophin cDNA. Preferably, mRNA sequences that are (i) around the translation start site and (ii) form a loop structure are targeted. Statistical analysis, based on the size of the human genome, has shown that DNA segments approximately 14-15 base pairs in length have unique sequences within the genome. Thus, to ensure the specificity of targeting of pleiotrophin RNA, the antisense oligonucleotide is at least 14 nucleotides in length, preferably 15 nucleotides in length. Thus, oligonucleotides contemplated by the present invention include pleiotrophin cDNA sequences at positions 1-14, 1-15, 1-16, 1-17, 1-18, 1-19, 2 Nucleotides corresponding to positions 、 16, 3-17 and the like are included. All possible oligonucleotides are shown as nucleotides according to formulas n through n + x. Here, n is 1 to 1383, and x is 14, 15, 16, 17, 18, or 19. Not all antisense oligos exhibit a sufficient degree of inhibition or a sufficient level of specificity for PTN targets. Therefore, it is necessary to perform screening to determine oligonucleotides having appropriate antisense properties. There are several ways in which oligos can be screened for inhibition of PTN synthesis. For example, there are numerous cell lines that are active in the synthesis of PTN (eg, HS578T and MDA-MB231 breast cancer cell lines, T98G glioblastoma cells, 1205-LU and WM852 melanoma cell lines). The level of PTN produced by these cells can be measured, for example, by radioimmunoprecipitation, Western blot, RIA, or ELISA. Treatment of PTN-producing cells with an effective antisense oligonucleotide causes a reduction in PTN levels. In addition, it is possible to directly analyze PTN activity. As mentioned above, cell lines with elevated levels of PTN are available. These cells are also characterized by some abnormal behavior, such as colonization in soft agar. In particular, increased endothelial cell proliferation can be measured, which is a useful in vitro model for angiogenesis in vivo. Treating such cells with an effective antisense oligo results in a change in the behavior of the cell, which helps to identify useful oligos. Assays such as those described above are useful as standard models for tumor growth in the body. Antisense oligonucleotides can be tested for efficacy and safety in vivo in animal model systems. Preferred animal systems are those performed with animals that have tumors as closely related as possible to those found in humans. In a preferred embodiment, the mouse is an athymic nude mouse carrying explanted human tumor cells that exhibits signs similar to the clinical signs seen in human cancer. Such mice are a standard animal model used in the development of chemotherapeutic drugs. See, for example, Pitot, Fundamentals of Oncology, 3rd Edition, Marcel Dekker, Inc., New York, 1986, 452. Administration of the antisense oligonucleotide to the subject, either as a bare synthetic oligo or as part of an expression vector, can be via any conventional route (oral, nasal, buccal, rectal, vaginal, or topical), or Subcutaneous, intramuscular, intraperitoneal or intravenous injection can also be used. However, the pharmaceutical compositions of the present invention are advantageously administered in the form of an injectable composition. A typical composition for such purpose includes a pharmaceutically acceptable solvent or diluent, and other suitable physiological compounds. For example, the composition comprises the oligonucleotide and about 10 mg of human serum albumin per milliliter of phosphate buffer containing NaCl. Up to 700 milligrams of antisense oligodeoxynucleotide was administered intravenously to patients over a 10-day process (ie, 0.05 mg / kg / hr), but showed no signs of toxicity (Sterling, Synthetic Antisense Treatment Reported, ”Genetic Engineering News 12: 1, 28 (1992). Other pharmaceutically acceptable excipients include non-aqueous or aqueous solutions and non-toxic compositions, including salts, preservatives, buffers and the like. Examples of non-aqueous solutions include propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters such as ethyl oleic acid. Aqueous solutions include water, alcoholic / aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, and the like. Intravenous vehicles include bodily fluids and nutritional supplements. Preservatives include antimicrobial agents, antioxidants, chelating agents, and inert gases. The pH and exact concentration of the various components of the pharmaceutical composition are adjusted according to routine methods in the art. Preferred pharmaceutical compositions for topical administration are skin creams or transdermal patches. Antisense oligonucleotides or antisense expression vectors can be administered by injection as an oily suspension. Suitable lipophilic solvents or carriers include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate or triglycerides. Further, the antisense oligonucleotide or vector can be combined with a lipophilic carrier, such as one of a number of sterols, including cholesterol, cholate, and deoxycholinic acid. The preferred sterol is cholesterol. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, including, for example, sodium carboxymethyl cellulose, sorbitol, and / or dextran. Optionally, the suspension may also contain stabilizers. Another formulation for administration of antisense PTN oligonucleotides includes liposomes. Liposomal encapsulation provides another formulation for administration of antisense PTN oligonucleotides and expression vectors. Liposomes are microcarriers with one or more lipid bilayers surrounding the aqueous fraction. See, generally, "Bakker-Woudenberg et al., Eur. J. Clin. Microbiol. Infect. Dis. 12 (Supplement 1): S61 (1993)" and "Kim, Drugs 46: 618 (1993)." thing. Liposomes are similar in composition to cell membranes and, as a result, can be administered safely and can be degraded in vivo. Depending on the method of preparation, the liposomes may be unilamellar or multilamellar, and their size may vary within a diameter range from 0.02 μm to more than 10 μm. Various drugs can be encapsulated in the liposome, with the hydrophobic material being distributed in the bilayer and the hydrophilic drug being distributed in the inner aqueous region. For example, "Machy et al., LIPOSOMES IN CELL BIOLOGY AND PHARMACOLOGY in Cell Biology and Pharmacy (John Libbey 1987)" and "Ostro et al., American J. Hosp. Pharm. 46. : 1576 (1989). In addition, by varying the size of the liposomes, the number of bilayers, the composition of the lipids, and the charge and surface properties of the liposomes, the therapeutic availability of the encapsulated drug can be adjusted. Liposomes can adsorb to virtually any type of cell and release the encapsulated drug slowly. Alternatively, the adsorbed liposome may be taken up by endocytosis by phagocytic cells. Following endocytosis, intralysosomal digestion of liposomal lipids and release of the encapsulated drug occur. Scherphof et al., Ann. NYAcad. Sci. 446: 368 (1985). After intravenous administration, normal liposomes preferentially phagocytose the reticuloendothelial system. However, the reticuloendothelial system can be circumvented by several methods, such as saturation with large amounts of liposome particles or selective inactivation of macrophages by pharmaceutical methods (Claassen et al., Biochim. Biophys. Acta 802: 428 (1984)). Furthermore, it has been shown that incorporating phospholipids derived from glycolipids or polyethylene glycol into liposome membranes significantly reduces the uptake by the reticuloendothelial system (Allen et al., Biochim. Biophys. Acta 1068: 131 (1991); Allen et al., Biochim. Biophys. Acta 1150: 9 (1993)). These Stealth® liposomes are present in the circulation for a longer time and are better directed to tumors in animals (Woodle et al., Proc. Amer. Assoc. Cancer Res. 33: 2672 ( 1992)). Clinical trials in humans are ongoing, including stage III clinical trials for Kaposi's sarcoma (Gregori adis et al., Drugs 45:15 (1993)). Antisense oligonucleotides and expression vectors can be encapsulated in liposomes using standard techniques. A variety of different liposome compositions and methods of synthesis are known to those skilled in the art. See, for example, U.S. Pat. Nos. 4,844,904, 5,000,959, 4,863,740, and 4,975,282. These are all included in the present specification by reference. Liposomes can be prepared to target specific cells or organs by altering the phospholipid composition or by inserting receptors or ligands into the liposomes. For example, an antibody specific for a tumor-associated antigen can be incorporated into the liposome along with an antisense oligonucleotide or expression vector to more efficiently direct the liposome to tumor cells. See, for example, Zelphati et al., Antisense Research and Development 3: 323-338 (1993), which describes the use of "immu noliposomes" containing antisense oligonucleotides for treatment of humans. Generally, the dosage of antisense oligonucleotides and vectors encapsulated in liposomes will vary depending on factors such as the patient's age, weight, height, gender, general health, and previous medical history. Will do. Dosage ranges for a particular formulation can be determined by using an appropriate animal model. Dosage and route of administration will also vary with the type of pathological growth. The growth may be dysplasia, such as retinopathy, arthritis, psoriasis, nevus, and virus-induced dysplasia, that is, benign abnormal tissue growth. Proliferation may also be associated with neoplasms such as melanoma, breast, ovarian, prostate, glioblastoma, neuroblastoma, and metastatic disease, ie, tumorigenesis and malignancy. Example 1 Materials and Methods: Cell Line SW-13 cells (human adrenal sarcoma) were obtained from American Type Culture Collection (ATCC, Rockville, MD) and in anchorage independent soft agar as previously described. Used as target cells in colony formation (Fang et al., (1992)). SW-13 / PTN cells are SW-13 cells that have been stably transformed with a PTN-containing clone to overexpress human pleiotrophin as described previously (Fang et al., ( 1992)). SW-13 / MK cells are SW-13 cells that have been transformed to overexpress the human midkine gene (Sale et al., Preparation Procedure (1994)). All SW-13 cell lines were maintained in modified minimal essential medium (IMEM; Biofluid) containing 10% fetal bovine serum (FBS; Biofluids Inc., Rockville, MD). Human metastatic melanoma cells (1205-LU) were obtained from Dr. M. Herlyn of the Wistar Institute, Philadelphia, Pa., And were 80% keratinocyte serum-free medium (KSFM; GIBCO / BRL, Bethesda, MD) and 5% FBS and 1.5 mM CaCl <sub>Two</sub> And maintained in 20% Reibowitz medium (L-15; GIBCO / BRL). Human brain endothelial cells (huBEC) were isolated from primary cultures from the human cerebellum and maintained in 199E medium containing 10% FBS. huBEC was obtained from Dr. P. Costello of Neurosurgery, Washington DC, Georgetown University. Oligonucleotides Phosphorothioate-modified DNA oligonucleotides were synthesized on a Milligen 8750 DNA synthesizer (Millipore, Bedford, Md.) Using a phosphoramidite backbone chemistry to produce a 15-mer (ptn AS1 / It was synthesized as either a SCR1) or a 20 mer (ptnAS3 / SCR3). The first antisense oligo was complementary to the translation initiation codon and the second antisense oligo was complementary to the loop-forming region in the PTN open reading frame. The sequence used is as follows. ptnAS1 (SEQ ID NO: 1) = 5'-GA GCCTGCATTTTTG-3 ', ptnSCR1 (SEQ ID NO: 2) = 5'-ATGCTTACGTTTGCG-3', ptnAS3 (SEQ ID NO: 3) = 5'-CCAGTATGAAAATGAATGCC-3 ', ptnSCR3 (SEQ ID NO: 4) = 5'-C AAGACGATTCCATAGTGAA-3 '. Oligos were solubilized in PBS before addition to cells, and their concentration was determined by optical density (OD <sub>260</sub> ). To confirm that the oligo is complete and full length, <sup>32</sup> The ends were labeled with P, electrophoresed on a polyacrylamide gel, and subjected to autoradiography. ELISA analysis At specific times, the conditioned medium was removed from the treated cells, filtered through a low protein binding membrane to remove debris, and serially diluted 100-200 μl was plated on a 96-well plate (MaxiSorp Nunc, Thomas Scientific, Swedesboro, NJ). After seeding, the wells were dried. Alternatively, the conditioned medium was concentrated, partially purified by heparin affinity chromatography, and then seeded on a plate (Wellstein et al. (1992)). Before each step, each well was washed three times with 200 μl of phosphate buffered saline-0.5% Tween-20 wash solution (PBS / Tween) and four times before the last step. To block non-specific binding, each well was treated with 100-200 μl of PBS / Tween containing 1% BSA for 1 hour at room temperature, and then 100 μl of PTN primary antibody was added at a dilution of 1: 500 (PTN -1 rabbit antiserum was prepared in this laboratory; PTN-HBNF rabbit antiserum was a gift from Dr. P. Bohlen of the Lederle Laboratories, Pearl River, NY). For Midkine ELISA, the MK primary antibody (MK rabbit antiserum was made in our laboratory) was diluted 1: 1000. After incubation at 4 ° C. for 1 hour, 100 μl of secondary antibody (goat anti-rabbit IgG-alkaline phosphatase; Boehringer Mannheim, Indianapolis, Ind.) Was added at a dilution of 1: 3000 and incubated at 4 ° C. for 1 hour. In the final step, 100 μl of substrate solution (pNPP = paranitrophenyl phosphate; GIBCO / BRL; 0.5 mM MgCl 2 <sub>Two</sub> And 1 mg / ml PNpp in 10 mM pH 9.5 diethanolamine) was added and color was allowed to develop for 5-60 minutes depending on protein concentration. The absorbance was measured at 405 nm with a microplate reader (Molecular Devices, Sunnyvale, CA). Soft agar analysis and co-culture experiments Colony formation in soft agar by SW-13, SW-13 / PTN, or SW-13 / MK cells was measured as previously described (Wellstein et al. (1990)). Briefly, 20,000 cells in 0.35% agar (Bactoagar; GIBCO / BR L) were coagulated in a 35 mm dish (Costar Corp., Cambridge) with 1 ml of 0.6% agar coagulated. Placed on top of layer. The test substances were sterilized by filtration, and 500 μl of them were added together with 800 μl of the upper layer unless otherwise indicated. Both layers contained growth medium with 10% FBS. After incubation at 37 ° C. for 1 to 2 weeks, colonies having a diameter of 60 μm or more were counted using an image analyzer. Conditioned media from PTN expressing cells was added to SW-13 cells. Antisense oligos indicated in the text were added to SW-13 / PTN or SW-13 / MK cells. In another experiment, co-culture of PTN-expressing cells and SW-13 cells was examined in a similar manner. However, first, PTN-expressing cells were <sup>Three</sup> From 5 × 10 <sup>Three</sup> Cells were seeded at the bottom of a 35 mm dish at the density of the cells, after which the agar layer and SW-13 cells were added. At the time prior to the addition of agar and SW-13 cells, PTN expressing cells were attached to plastic and treated with antisense oligos for specific times and concentrations. Endothelial cell co-culture huBEC cells were plated at the bottom of a 6-well culture plate (Falcon, Becton Dickinson, Franklin Lakes, NJ) at 5 x 10 per well. <sup>Three</sup> Seeded at a density of 1205-LU cells in a 0.45μm perforated cell culture insert (Falcon) <sup>Four</sup> And treated with antisense oligos, control oligos (random sequences), or phosphate buffered saline (PBS) for 72 hours, then transfer the inserts to huBEC culture plates and allow Culture was continued. The huBEC cells were then detached with trypsin and counted on a particle counter. Alternatively, conditioned medium from oligo-treated 1205-LU cells was harvested and added at different concentrations to huBECs in culture. Six days later, the number of cells was counted. Tumor Growth in Animals Pre-confluent melanoma cells were pretreated with either specific concentrations of antisense oligo DNA, control oligo DNA, or PBS for 72 hours. Thereafter, the cells were trypsinized from the treatment flask, washed three times with a melanoma medium, and then collected. Cells were then injected subcutaneously (1 × 10 5 in 100 μl of medium) into the flank of athymic nude mice (NCr nu / nu; Harlan Sprague-Dawley, Indianapolis, IN). <sup>6</sup> Cells), and tumor diameters were measured every other day as tumors became visible as previously described (Fang et al. (1992)). Example 2 -Selection of the target sequence in the PTN transcript Two different methods were used to select the target region in the PTN. One region was selected based on the fact that targeting an antisense molecule to the translation initiation site of mRNA has been shown to inhibit translation of its protein product. See "Stein et al. (1988), supra." We selected one antisense sequence (ptnAS1, SEQ ID NO: 1) in this region. In the second method, the expected secondary structure of the PTN mRNA was taken into account. The present inventors searched for a loop-forming region within the open reading frame for the predicted secondary structure of PTN mRNA. See "Zuker et al Nucl. Acids Res. 9 :: 133 (1981)." These unique sequences were screened by GENBANK comparison. The antisense oligos were also selected for highly loaded G + Cs that were present at their ends and could improve hybridization characteristics. See Stein et al, loc. Cit. (1989). The control oligos were chemically identified and were the promiscuous sequences of each antisense oligo (ptnSCR1 and ptnSCR3, SEQ ID NOs: 2 and 4). It was determined that these oligonucleotides were free of significant antisense as related to PTN or another region contained in other genes. Sense oligos were generally avoided as control sequences due to the fact that they are chemically different from their respective antisense oligos, to the same extent that they contain nucleotides of different composition. Furthermore, the computer generated PTN mRNA folding pattern predicted that multiple stems of the molecule would be formed. Thus, it appears that self-hybridization is apparently occurring between the complementary strands, and the sense oligos can also act to specifically inhibit PTN by binding to their respective complementary sequences within PTN. Example 3 -Specific inhibition of PTN production in SW-13 / PTN cells In order to detect whether specific antisense inhibition of PTN expression has occurred, we used SWNs transfected with a PTN expression vector. -13 cells were utilized. These cells colonize the agar as a result of the active PTN. See Fang et al. (1992). In these experiments, the formation of SW-13 / PTN cell colonies was suppressed by 64% when treated with 10 μM antisense oligo (ptnAS1, SEQ ID NO: 1) for 48 hours before seeding the cells on soft agar. Similar treatment with an equivalent concentration of control promiscuous oligo (ptnSCR1, SEQ ID NO: 2) or with a carrier did not inhibit the ability of SW-13 / PTN cells to form colonies. No difference was observed between the treatment groups when 3 μM oligo was used. When SW-13 / PTN cells were treated with ptnAS1 at a concentration of 30 μM for 48 hours prior to inoculation on agar, the reduction in colony formation was not greater than that observed when treated with 10 μM. However, treatment with 30 μM ptnSCR1 also inhibited the ability of SW-13 / PTN cells to form colonies. This is probably due to the effect of sequence non-specificity. Antisense inhibition of PTN protein secretion was confirmed by performing ELISA on supernatants obtained from the cells. To test the specificity of the PTN antisense oligo, SW-13 cells infected with the MK expression vector (SW-13 / MK cells) were treated with the PTN antisense oligo and the supernatant analyzed. Analyzed for colony stimulating activity. MK synthesis was also measured by ELISA. SW-13 / MK cells were found to colonize soft agar due to MK expression. See Sale et al. (1994). No inhibition of MK biological activity, ie secretion of MK into SW-13 / MK supernatant, was measured. Thus, although MK is closely involved in PTN (50% sequence identity), it is clear that our PTN antisense oligos are very specific. These data suggest that specific, dose-dependent inhibition of PTN production and secretion can be achieved in vitro. Example 4 -Lack of growth inhibition of melanoma cells expressing PTN in vitro 1205-LU melanoma cells express essentially high levels of PTN and form tumors very aggressively. See Hartmann et al., Manuscript in preparation (1994). These cells were used as a model cell line to determine if PTN plays a role in melanoma growth. It has previously been shown that human melanoma cells express PTN mRNA but not human melanocytes. See Fang et al. (1992). Subsequent studies were conducted to determine whether 1205-cells need a PTN for autocrine action as they proliferate and form colonies, or stimulate the surrounding tissue to paracrine growth In doing so, they were solely used to determine if they were using PTN. Metastatic 1205-LU melanoma cells were found not to require PTN to form colonies on soft agar. More specifically, when these cells are treated with an antisense oligo that inhibits PTN production, they form the same number of colonies at the same size as those cells treated with both messy oligo and PBS . In addition to colony formation analysis, melanoma cell proliferation was measured for one week while treating with either antisense oligos or promiscuous oligos at concentrations up to 20 μM. Regardless of the oligo or dose used for treatment, cells grew to more than 12 times their original density. This suggests that oligoplasts are not toxic to melanoma cells. As described below, oligonucleotide treatment was effective in inhibiting PTN production in these cells. Therefore, it was concluded that PTN did not rate limit the in vitro growth of 1205-LU cells. Example 5 -Effect of antisense treatment on PTN secreted from melanoma and SW-13 cell forming colonies Different growth factors released from melanoma cells, e.g. from the FGF family, stimulate SW-13 as well as endothelial cells Can. Rodeck et al., Cancer Metastasis Rev. 10:89 (1991). Endothelial cells respond to molecules such as, for example, FGFs, PTN, and TGF-α, whereas SW-13 cells respond to FGFs, PTN, as does IL-1,24, but not TFG-α. Do not respond. Conditioned media was collected every 24 hours, the interval between antisense treatments, and analyzed for PTN using (i) PTN-specific ELISA, and (ii) both SW-13 and endothelial cell responses. Inhibition was not detected during the first 24 hours of treatment, probably due to residual PTN protein still not being removed. After 48 hours, the sump showed a specific, dose-dependent decrease, as determined by ELISA, for the amount of secreted PTN. 10 μM antisense oligos inhibited immunoreactive PTN protein by 50% in conditioned media, whereas 10 μM promiscuous oligos did not show any inhibition. It is evident that high doses only cause a modest increase in effect, while causing non-specific inhibition of PTN synthesis, as evidenced by control oligo treatment. From these data, a concentration of 10 μM was chosen for subsequent experiments where a single dose was used. Conditioned medium from 1205-LU cells treated with antisense oligos in equilibrium with the ELISA was added to SW-13 cells seeded on soft agar. As with the ELISA, the sample after 24 hours did not show any significant inhibition of colony formation. Samples after 48 hours (data not shown), and especially at 72 hours, show that antisense oligo treatment reduces colony stimulating activity, which indicates that It indicates that secretion is reduced. At 10 μM, antisense oligos inhibited colony formation of target cells by 40%, while the control, promiscuous oligos, did not show any inhibition. This data was the same with different sets of antisense and promiscuous oligos (ptnAS3 and ptnSCR3, SEQ ID NOs: 3 and 4). This antisense treatment inhibited colony formation by more than 40% compared to controls. It should also be noted that 20 μM ptnAS3 (SEQ ID NO: 3) was 64% inhibited, whereas the corresponding ptnSCR3 (SEQ ID NO: 1) oligo did not show any inhibition. Comparison of ptnAS1 (SEQ ID NO: 1) and ptnSCR1 (SEQ ID NO: 2) shows that ptnAS3 provides greater specificity. These data were included in 1205-LU conditioned media treated using different oligo treatment methods and supported by measuring the amount of PTN measured at different sampling times by ELISA. The ability of 1205-LU cells to directly stimulate SW-13 cells in co-culture with soft agar was also evaluated. SW-13 cells did not show any significant colony formation when the feeder layer was left empty. However, the addition of 1205-LU cells to the feeder layer stimulated colony formation. Pretreatment of 1205-LU feeder cells with 10 μM ptnAS3 for 72 hours resulted in greater than 50% inhibition of stimulatory activity as measured by SW-13 colony formation. Pretreatment of 1205-LU cells with an equal volume of 10 μM ptnSCR3 did not reduce stimulatory activity, confirming that ptnAS3-mediated inhibition is sequence-specific. Further, these data demonstrate the ability of PTN protein secreted from human melanoma cells to stimulate endothelial cell proliferation in a direct paracrine manner. Example 6 -Biological Assay of Supernatants Utilizing Endothelial Cell Proliferation Early on, it was reported that PTN secreted from human tumor cells could stimulate the proliferation of endothelial cells contained in culture. See Fang et al. (1992). In this study, PTN activity in conditioned media was examined by antisense-treated 1205-LU cells. This medium was used when treating endothelial cells simultaneously with SW-13 cells. HuBEC treated with a sample of the conditioned medium for 72 hours showed significantly less growth than the control. When the antisense treatment was actually performed, the activity was suppressed to a background level of PTN activity. The 1205-LU conditioned medium showed a comparable degree of immunoreactivity of PTN as measured by ELISA (data not shown). In addition, 1205-LU cells were seeded in co-culture to directly stimulate target huBEC cells. The same results were obtained when using conditioned medium taken from 1205-LU cells (data not shown). huBEC cells co-cultured with ptnAS1-treated 1205-LU cells and ptnA S3-treated 1205-LU cells showed less proliferation than huBEC cells co-cultured with control 1205-LU cells. Indicated. Endothelial cells co-cultured with 1205-LU cells treated with ptnAS3 showed about 50% growth inhibition per week compared to control (PBS treated) co-culture. However, endothelial cells obtained from the ptnSCR3 group did not show a significant decrease in cell proliferation over the same period. The corresponding ELISA confirmed the inhibition of PTN protein in the conditioned medium for each 1205-LU (data not shown). Endothelial cells not co-cultured with 1205-LU cells did not receive the combined culture activity and were used to determine background proliferation. These data demonstrate not only the ability of PTN protein secreted from human melanoma cells to stimulate endothelial cell proliferation in a paracrine fashion, but also the ability of antisense DNA to reverse this stimulation. Is shown. Example 7 -Inhibition of melanoma tumor formation in nude mice To further clarify the effects of parasecretory stimulation by melanoma cells and PTN secreted from PTN-induced solid tumor formation, , Followed by intramuscular injection into athymic nude mice. 1205-LU cells were pretreated for 72 hours as described above. This metastatic melanoma cell line grows to a detectable tumor within a few days at the site of injection. Four days after injection, the tumor was found to be at least 10 mm in diameter, regardless of pretreatment. This may be based on the fact that tumors grow to a certain size without vascular supplementation for nutritional maintenance. Folk man et al. Biol. Chem. 267: 10931 (1992). As oxygen and other nutrients diffuse, they must infiltrate and become nutrients in these cells during growth and tumor formation in the first few days. Conversely, it is clear that in order for the tumor to continue to grow after this early stage, it must replenish the capillaries and surrounding matrix in a paracrine manner. This is evidenced by an average doubling of tumor size in controls (cells pre-treated with PBS or ptnSCR1) by the end of the first week after injection. In contrast, cells pretreated with antisense (ptnAS1) showed only a slight increase of less than 10% growth over the same period. By 9 days post-injection, control tumors had grown approximately 3-fold in size, whereas antisense-pretreated tumors had a size increase of less than 25%. Due to the eventual absence of oligos from the original cell mass and dilution in the respective tumor cell replication, the effect of antisense inhibition on PTN secretion was `` cleared '' beyond the first week of tumor growth. There is hope to be. " By two weeks after injection, antisense pretreated tumors are indistinguishable in size from control tumors. Although this experiment demonstrates the importance of PTN in the formation of metastatic melanoma tumors in humans, it also suggests the need for continued antisense treatment. First, the results of these studies established the concept that PTN is indeed the rate-limiting growth factor in human melanoma growth.

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Claims (1)

[Claims] 1. Binds to a segment of single-stranded mRNA transcribed from the pleiotrophin gene. Antisense oligonucleotides that inhibit pleiotrophin synthesis in cells Pleiotrophin expression in a cell, including introducing into at least one cell How to inhibit. 2. Pleiotrophin is human pleiotrophin and the cell is a human cell The method of claim 1, wherein 3. Binds to segments of single-stranded mRNA transcribed from the pleiotrophin gene RNA transcript, thereby inhibiting pleiotrophin synthesis in the cell The oligonucleotide is functionally active in a promoter that is active in the cell. 2. The method of claim 1, wherein the method is coupled to 4. Oligonucleotide and promoter contained in one expression vector 4. The method of claim 3, wherein 5. The oligonucleotide is introduced into a pharmaceutically acceptable solvent or diluent The method of claim 1, wherein 6. The method of claim 1, wherein the oligonucleotide is introduced into a liposome. . 7. The method according to claim 4, wherein the oligonucleotide is introduced into the liposome. . 8. The oligonucleotide comprises nucleotides n to n + x in FIG. 1 (SEQ ID NO: 5) , N is an integer from 1 to 1383, and x is an integer consisting of 14, 15, 16, 17, 18, and 19 2. The method of claim 1, wherein the method is selected from the group of: 9. The oligonucleotide is (A) The translation of the mRNA is inhibited by hybridization of the oligonucleotide. That hybridize to human pleiotrophin mRNA under intracellular conditions Lignonucleotides, (B) (i) an antisense oligonucleotide that selectively binds to the translation initiation site (AUG) of the mRNA; Gonucleotides, and      (Ii) an antisense that selectively binds to a conserved loop-forming region of the mRNA. Oligonucleotides, An oligonucleotide selected from the group consisting of (C) (i) 5'GAG CCT GCA TTT TTG3 '(SEQ ID NO: 1), and      (Ii) a group consisting of 5 'CCA GTA TGA AAA TGA ATG CC 3' (SEQ ID NO: 3) R 3. The method of claim 2, which is an oligonucleotide of choice. 10. Transcribed from the pleiotrophin gene when introduced into host cells A protein that binds to a segment of a single-stranded mRNA and inhibits pleiotrophin synthesis in the cell. A composition comprising at least one antisense oligonucleotide. 11. 11. The method according to claim 10, wherein the pleiotrophin is human pleiotrophin. Rigo nucleotides. 12. Binds to a segment of single-stranded mRNA transcribed from the pleiotrophin gene Thereby producing an RNA transcript that inhibits pleiotrophin synthesis in the cell. 11. The oligonucleotide according to claim 10, operably linked to a promoter. 13. Oligonucleotide and promoter contained in one expression vector 13. The oligonucleotide according to claim 12, wherein 14． The oligonucleotide contains nucleotides n to n + x in FIG. 1 (SEQ ID NO: 5). Where n is an integer from 1 to 1383 and x is from 14, 15, 16, 17, 18, and 19. 11. The oligonucleotide according to claim 10, which is selected from a group of integers. 15. Hybridizing to human pleiotrophin mRNA under intracellular conditions; 12. The oligonucleotide according to claim 11, wherein: 16. (A) Antisense oligonucleotides that selectively bind to the translation initiation site of mRNA Otide, and       (B) Antisense that selectively binds to the conserved loop-forming region of mRNA Oligonucleotides, 16. The oligonucleotide according to claim 15, which is selected from the group consisting of: 17． (A) 5'GAG CCT GCA TTT TTG 3 '(SEQ ID NO: 1), and       (B) 5 'CCA GTA TGA AAA TGA ATG CC 3' (SEQ ID NO: 3), 17. The oligonucleotide according to claim 16, which is selected from the group consisting of: 18. At least enough to inhibit pleiotrophin synthesis in patients 11. A disease in a patient, comprising administering to the patient also the composition of claim 10. How to treat physical tissue growth. 19. Pathological tissue growth selected from the group consisting of neoplastic and dysplastic diseases 19. The method of claim 18, wherein the method is performed. 20. Neoplastic diseases include melanoma, breast cancer, ovarian cancer, prostate cancer, glioblastoma, neuroblastoma, and 20. The method of claim 19, wherein the method is selected from the group consisting of: 21. Dysplasia is associated with retinopathy, arthritis, psoriasis, nevus, and virus-induced 20. The method of claim 19, wherein the method is selected from the group consisting of dysplasia. 22. A pharmaceutical composition comprising the composition of claim 10 in a pharmaceutically acceptable sterile carrier. Useful preparations. 23. Pathological tissue, including inhibiting the expression of the pleiotrophin gene How to treat proliferation. 24. Pathological tissue growth selected from the group consisting of neoplastic and dysplastic diseases 24. The method of claim 23, wherein the method is performed. 25. Neoplastic diseases include melanoma, breast cancer, ovarian cancer, prostate cancer, glioblastoma, neuroblastoma, and 25. The method of claim 24, wherein the method is selected from the group consisting of: 26. Dysplasia is associated with retinopathy, arthritis, psoriasis, nevus, and virus-induced 25. The method of claim 24, wherein the method is selected from the group consisting of dysplasia.