MXPA98008578A - Anti-sense inhibitors of factor expression vascular endothelial decrease (vegf / v - Google Patents

Abstract

The present invention relates to the inhibition of expression of vascular endothelial growth factor with oligonucleotides. The oligonucleotides of the present invention are considered to bind to target RNA in a specific sequence form and prevent expression of the encoded gene. Chemical modifications of the oligonucleotides to increase their stability and binding efficiency are described. These modifications increase the stability and efficiency of the oligonucleotides contemplated in this invention. Oligonucleotide compositions can be used in ex vivo therapies for the treatment of macrophages or in vivo therapies by injection, inhalation, topical treatment or other routes of administration.

Description

ANTI-SUFFICIENT EXPRESSION INHIBITORS OF VASCULAR ENDOTHELIAL GROWTH FACTOR (VESF / VPF) CROSS REFERENCE TO RELATED APPLICATIONS This application depends on the priority over a co-pending provisional application that has serial number of US patent. 60 / 015,752 filed on April 17, 1996. DECLARATION REGARDING DEVELOPMENT OR FEDERALLY AUSPED INVESTIGATION Not applicable. BACKGROUND OF THE INVENTION Field of the Invention The present invention relates to cellular inhibition of vascular endothelial growth factor expression with oligonucleotides. The oligonucleotides of the present invention are considered to bind target mRNA in a sequence in a specific manner and prevent expression of the encoded VEGF gene. Chemical modifications of the oligonucleotides are described to increase the stability and binding efficiency of the oligonucleotides. The present oligonucleotide compositions can be employed in ex vivo therapies for macrophage treatments or in vivo therapies by injection, inhalation, topical treatment or other routes of administration. DESCRIPTION OF THE RELATED ART The vascular endothelial growth factor (VEGF = Vascular endothelial growth factor) also known as vascular permeability factor, comprises a family of homodimeric secretory glycoproteins in the size range of 34 to 46 kilodaltons. It is secreted by a variety of cell types in response to hypoxia and certain regulatory factors. Four isotypes of VEGF are known. They arise by alternating separation of mRNA from a single gene (Keck et al., 1989; Leung et al., 1989; Connolly and Plander 1989; Tischer et al., 1991). VEGF is necessary for the formation of blood vessels (angiogenesis) during the growth and development processes, and for tissue repair (Ferrera et al., 1996, Carmeliet et al., 1996, Thomas, 1996, Dvorak et al., 1995a, Folkman. , 1995; Ferrera et al., 1992). This growth factor induces vascular permeability is a chemotactic for monocytes and osteoblasts and is a selective mitogen for endothelial cells. The receptor proteins for VEGF (KDR and Flt-1 in humans) belong to the transmembrane tyrosine kinase family (Terman et al., 1992; de Vries et al. 1992). Activation of the receptor initiates a cascade of events leading to markedly improved rates of vascular endothelial cell proliferation and eventual neovascularization. VEGF is more selective for inducing endothelial cell proliferation than any other protein factor involved in angiogenesis. Unfortunately, under certain conditions the presence of VEGF can have harmful effects on health. Abnormally high concentrations of VEGF are associated with diseases characterized by a high degree of vascularization or vascular permeability. Examples of these conditions include diabetic retinopathy, aggressive cancers, psoriais, rheumatoid arthritis and other inflammatory conditions. (D'Amore, 1994, Dvorak and collaborators 1995 a, b, Folkman, 1995). Compositions and methods for selectively lowering abnormally high VEGF concentrations are desired in order to reduce VEGF mediated neovascularization. These methods and compositions can be used to slow the progression of diseases characterized by vascularization and vascular permeability. One method to reduce VEGF concentrations involves the use of antisense oligonucleotides (Wagner 1994). The central advantage of this technique is the specificity by which inhibition can be achieved. Useful oligonucleotides are considered to bind specific mRNA sequences and interfere with the expression of the encoded genes. Reduced protein expression can result from inhibition of ribosome function, reduced concentrations of translatable substrate mRNA, or other mechanisms. In addition, oligonucleotides can reduce mRNA concentrations by an oligonucleotide-mediated increase in the degradation rate of mRNA molecules. In general, oligonucleotides of about 15 bases are sufficient to provide sequence-specific binding to targeted mRNA targets, although they sometimes bind shorter oligonucleotides.

(Uhlmann and Peyman, 1990). However, antisense oligonucleotides having between 11-30 bases have been employed to reduce protein expression in in vitro experiments. (Reviewed by Uhlmann and Peyman, 1990). A number of obstacles must be overcome before the potential advantages of the antisense treatment strategy for treating disease can be achieved. For example, antisense oligonucleotides are large hydrophilic compounds (~3,000 to 10,000 D) and must cross hydrophobic cell membranes before binding their targets in the nucleus or cytosol. (Uhlmann and Peyman, 1990, Milligan et al., 1993). In this manner, methods are required to facilitate transport of VEGF antisense oligonucleotides through cell membranes. Therapeutic oligonucleotides should also not be toxic and should not interfere with normal cellular metabolism. To minimize these non-specific effects, they must be linked to their connate sequences with high specificity and affinity. Oligonucleotides with a natural phosphodiester backbone are highly susceptible to serum and cellular nucleases. Oligonucleotide sequences with a length of 17 random bases have a half-life of less than three minutes in serum (Bishop et al., 1996). Oligonucleotides with increased stabilities are required before they can be used as therapeutics in the treatment of neovascular disease. Substitution of the phosphodiester group with phosphorothioates to increase the half-lives of oligonucleotides. They must be chemically inert and resistant to nuclease in a variety of chemical environments. However, these oligonucleotides have not previously been shown to inhibit VEGF expression in a selective manner. A disadvantage of the previously known phosphorodithioate oligonucleotides is that they require concentrations above 1 micromolar (μM) to reduce expression of VEGF. (Norara et al., 1995; Robinson et al., 1996). At these concentrations, those oligonucleotides are toxic (Woolf et al., 1992; Stein and Cheng, 1993; Stein and Kreig, 1994, Wagner, 1994; Fennewald et al., 1996) and the effects observed are probably the result of this non-specific toxicity (Fennewald et al., 1995). Novel oligonucleotide inhibitors are required that demonstrate a real antisense effect by inhibiting VEGF expression at non-toxic concentrations. These oligonucleotides will likely have higher association constants and / or increased specificity for their target mRNA sequences than the previous VEGF antisense oligonucleotides. There are several possible explanations for the limited effectiveness of VEGF antisense oligonucleotides. One possibility is that the target mRNA sequences may be confined to macromolecular structures that sterically block the binding of oligonucleotides. For example, mRNA binding proteins and protein translation complexes can block the binding of oligonucleotides. Alternatively, the oligonucleotides may not be able to bind unfavorable conformations of mRNA. In addition, the application of effective target frequencies is variable. Effective target sequences can be located anywhere in target mRNA transcripts and target oligonucleotides to translation initiation codons or to 5 'untranslated regions, they are not always effective. (Agner et al 1993; Fenster et al. 1994).

Non-specific interactions between oligonucleotides and other molecules such as proteins can also lead to variable biological activity (Woolf et al., 1992; Stein and Cheng, 1993). In addition, the oligonucleotides themselves can adopt unexpected tertiary and quaternary structures that bind DNA at unexpected sites. This aberrant union has the potential to produce undesirable biological effects (Chaudhary et al., 1995). Other difficulties have also been encountered in the search for effective antisense oligonucleotides. The affinity of oligonucleotides for their RNA targets, it increases with the length and with the increased G-C content. However, longer oligonucleotides tend to bind RNA sequences not specifically and oligonucleotides. Still further, oligonucleotides with a high G content tend to form G-quatrains, reducing the amount of oligonucleotide-free coil form that is considered required for antisense binding (Bishop et al., 1996). Thus, oligonucleotides that are short and have high affinity for their target sequences that do not form G-quartets are required in spite of having a high G content. As noted previously, the oligonucleotides are large hydrophilic compounds that must cross hydrophobic cell membranes before that link their objectives in the cytosol or nucleus. (Uhlmann and Peyman, 1990; Milligan et al., 1993). However, due to its large size, its hydrophilic nature and negative charge, the oligonucleotides do not efficiently cross cell membranes. In the absence of cell absorption enhancers, oligonucleotides tend to accumulate in perinuclear endosomal compartments of treated cells (Fisher et al., 1993, Guy-Caffey et al., 1995). In cases, the transport of oligonucleotides through the plasma membrane or the membranes of the endosomal compartments limits their rate of internalization and their activity. Therefore, new compositions and methods are required to improve the rate at which the oligonucleotides cross the bi-layer lipids. One class of lipid absorption enhancers includes a positively charged head group that binds nucleic acids, and an interactive membrane tail that is considered to interact with membrane components. These compositions can facilitate the penetration of oligonucleotide from the cell, presumably by transiently disrupting cell membranes. Unfortunately, the activity of many cationic lipid preparations such as Lipofectin ™, a liposomal mixture 1: 1 (mass) of the cationic lipid DOTMA and the fusogenic lipid dioleyl phosphotidylethanolane (DOPE) (Life Technologies, Inc., Gaithersburg, MD), are highly sensitive to factors such as the composition and amount of nucleic acid, the type of target cell and the concentration of serum in the cell growth medium. In addition, some preparations themselves are cytotoxic. These restrictions severely limit the utility of many of their compounds as oligonucleotide delivery agents for therapeutic use in animal systems. Improved delivery systems that are compatible with oligonucleotides must be identified. In summary, the progression of many diseases is associated with increased angiogenesis and vascular permeability caused by the over expression of VEGF. New compositions and methods to specifically reduce VEGF expression will be useful in the treatment of these diseases. Antisense oligonucleotide treatment is an attractive approach due to its potential selectivity. Unfortunately, many of the known VEGF antisense oligonucleotides only work at concentrations that are toxic to cells and exhibit only non-specific effects. In addition, previous antisense oligonucleotides are chemically and biologically labile and those that are more stable tend to have unacceptably low affinities for their target sequences and do not easily penetrate cell membranes and therefore have difficulty in achieving their biological targets. Finally, oligonucleotides with high G content tend to form G quatrains. New antisense oligonucleotide compositions are required that are non-toxic and have increased affinity for their target mRNA sequences. These compositions should have improved biological stability, including increased resistance to degradation by nucleases. In addition, useful oligonucleotides should not be added independently of their sequences. New compositions are also required that facilitate the transport of oligonucleotides through cell membranes. SUMMARY OF THE INVENTION The present invention provides compositions and methods for slowing the progression of diseases associated with increased angiogenesis and vascular permeability. The present antisense oligonucleotide compositions are markedly superior to previous oligonucleotides to selectively inhibit the expression of VEGF by producer cells and are intended for use in the treatment of these diseases. The selectivity of the present invention is provided by antisense oligonucleotides that specifically bind mRNA, VEGF molecules and block the expression of VEGF.

The present invention provides oligonucleotides and methods for producing and using them, with chemical modifications to increase their affinity and specificity for target mRNA sequences. The present oligonucleotides have improved biological stability and high affinities for their target sequences. Oligonucleotides are relatively inert to chemical and biological attacks in both hydrophobic and hydrophilic environments and resist aggregation independently of their sequence. The present invention provides VEGF antisense oligonucleotides that are both effective and non-toxic. Specifically, this invention is for novel oligonucleotide compositions which, when used to treat cells at concentrations less than 1 micro-molar, cause a decrease in cellular VEGF production. At these concentrations, the present anti-sense oligonucleotides are non-toxic and do not interfere with cellular metabolism. The invention provides compositions and methods that allow oligonucleotides to easily penetrate cell membranes to achieve their biological targets. This is achieved by providing methods for producing and using antisense oligonucleotides with cell absorption enhancers. The cell absorption enhancers are non-toxic, are compatible with VEGF antisense oligonucleotides and facilitate the efficient penetration of oligonucleotides through cell membranes. For purposes of this invention, the term "oligonucleotide" includes nucleic acid polymers and chemical structures that resemble nucleic acid polymers. Equivalents of ribose or of oxiribose can be substituted in the structures provided that the base portions connected to the structure can maintain the hydrogen bonds required for specific binding to the target sequences. Similarly, oligonucleotides may contain chemical equivalents of the phosphodiester backbone such as phosphodiester linkages. In addition, oligonucleotides can include base portions that are chemically modified. Specifically, oligonucleotides may include but are not limited to (propynyl or hexinyl) uridine with 5 carbon atoms or cytidine residues, 6-aza-uridine or cytidine residues and pyrimidines both with modifications of 5 carbon atoms and 6-aza. The term "VEGF" is intended to include all proteins in the class known as vascular endothelial growth factors. The term VEGF includes at least 4 known human isotopes that are considered to arise upon alternative cleavage of mRNA and any homologous protein having similar biological function. Known proteins include those that are encoded from mRNA species known in the art as VEGF 206, VEGF 185, VEGF 165 and VEGF 121. Antisense oligonucleotides of the present invention are prepared as follows. A sequence of about 15 to 30 nucleotides and preferably about 19 nucleotides is identified in an mRNA that encodes VEGF. The sequences of VEGF mRNA molecules are known in the art. The RNA sequence can be anywhere in any mRNA that codes for any protein in the VEGF family of proteins. More preferred are antisense oligonucleotides that are complementary to the mRNA encoding human VEGF 206, VEGF 185, VEGF 165 and VEGF 121. Oligonucleotides that bind sequences found in all VEGF mRNAs are preferred (See Table 1).

TABLE 1 Oligonucleotides Anti-VEGF I.D. Description Modification Sequence T3051S. antisentldo to mRNA 18S • 203 + S'-f * c * g * * l: * g *** * «* 3 • i» * c * a * t * c * c *** t * g-3, Total KT (phosphorothioate) DNA X30S39s var. from T3ß «iS S * -f« C * g * C * $ * g *** U »** 3 * a * c * a * ü * c * C * a * tr * -?« total? T, C5-propyr.-pyrimidine DNA fies-t? Sec. MRNA 2 »* -222 5l -C * g * a *« * tf * g * g *** 0 * g * g * C *** 9 * tJ * «* g * C«: - 3 «total vr , C5 propyny pyrimidines »0S l! sec. MRNA 2.13-25 $ 5- -0 ** ^ * tf * c * C * ff »g * a * a *« * g *. { ? * 3 * 0 * C * C *? T-3 * total BT, C5-propynyl pyrimidines 3CW4 * ?! var of T3f 63S 5 «.p * c * § * c * B * g - ** W ** - ga * C * aa * C» C «» * 0 «-g-3 '* BD links 10 T3ß84f« var. of igtfjji 5 '-3 * C * g-C »0-f-a * 0 ** - g-a * e ** - rjf * C * C *** s * g - 3» S Pí >; links * 3 S «s t var. of T3 «SJ S * .9 ^« g * C * D * g -. **! í ** «g - ** C * a * l? * C * C *« * 0 * 0-3 '2 KB links «0S? Fii sec. MRNA 224 -2 * 2 S «-3« ***** g *** lf * f * £ f * C »C * a * C * C *** g« g * g «tf * C-3 total i * T, CS «propynyl pyrimidines T30Í77: sec. MRNA «Sßg-424 5 * - ** ^ * g *» * a * g * C * W * C »a * ü * c * 0 * C * Ü * C * C * ü ** - 3 'total W «CS- propyl pyrimidines T3087Í. sec. MRNA 522-54.0 S »-tf« ** C * a * C * 9 * W * C * ll * a * C * s * g *** 0 * C * «* 0 * g * -3» total ST , CS- propynyl pyrimidines T30i7S? sec. MRNA 5 * 5-533 5 '* tl * a * «* C» tf »€ * a *« * g * C * ÍJ * g! * C * C * U * C * gs * C * C * -3i «Total ST, CS- propyl pyrimidines« D8fß; sec. MRNA 171-189 5 «-C« C * »* 0 * g * a *** C * e * W« c *** C * C *?, * Cí * í? * «J * fl-3 ' Setal SET »CS- propynyl pyrimidines 15 T30l87? sec. MRNA 17C-1Í4 S'-g »» * C * a * £ l * C * C *** s * er *** a * c * t * t * C * a * ß «c-3» total PT . CS- propynyl pyrimidines T30IS8. sec. MRNA 119-217 5 »-g * gr * a * u * g * g * c *« * f * ü * a * * C * D «g * C * g * C * s-3 'total? T, CS-propini! pyrimidines • n yes mRNA 195-213 S * -g «* C * a * s * O * a * s * C * O * er * C * e (* C * W * 0 *» ** ü * a -3 <total PT, C5 * propini! Pirimldlnas 30SSO; var of T3M3§ 5'-g * C * g * C * t * ga '* «e * a * f *** C *** l: * * C *** * g-.3, total PT, CS- propinil only C: - T3M> 1; var. Of T30S39 total i? T, CS -propinil only U t30íí52i var. Of T3063S »S '- g * s * g * c * t * 3 * a * 0 * a «g *** c * a * U * C *« *** t «f-3 'total PT, 4 CS- propinü pyrimidines W06I3: var. of T30OS 5 «-g * C * g * e * go * g *« * ü * # * g * »» C *** t * C * c *** a * g-3 'total STt S CS- propynyl pyrimidines 20 Yes »« - S2 »« i var. Of T30 (539 S '-g «C * g * C * OgaO * ag -» - C * ?? - Uí «CC *» * «» 0 -3 «[JU -I> ip- l ß POÍ CS-propynyl, extreme lipid S96-S297: var. of Y30Í3Í * S «-g * .C * s * C * 0-§-a-tl ** - 3 - * - C ** - and« CC * a * lJ * g-3 (M «l - Píreno S «; CS-propinyl.

T3fi $ ftß. var. of T30S15 S, -g * C * g * C * O * a «U * 3 *** C * a * rj * C * C *** li * 0-3» total PT, CS - hexinil piri idlnas DNA T386 «¡2- not mating S« -g * C * g * C * 0 * »* C * a * * a * C * a * 1l * íJ * C * A * lJ * g-3 'total P? , CS-propinyl pyrimidines, DNA version of T30.f39 > T3d8d7: DNA 'sense of TXMíS 5' «c * a * t * g * § * a * t * g * t * c * t *« ** t * c * a * g * c * g * c ~ 3 'total phosphodiester, DNA T30807 * RNA' sense 'of TSß €? SS, -c «a * e * g * g * a * t * f * c * c * t« a * t * c ** »9 * c «g * c-3 'total phosphodiester, RNA 25 + Human VEGF RNAra Sequence from Leung et al., Science, 246: 1306, 1989. Initiator Codon at base 57. * Phosphorothioate linkage-1C phosphodiester linkage, U represent modified bases A series of complementary or "antisense" oligonucleotides is prepared. For purposes of this invention, "antisense" means that the oligonucleotides have sequences complementary to mRNA sequences in a manner that will bind those sequences through specific hydrogen bonding patterns. However, an antisense oligonucleotide can have mismatches or imperfect hydrogen bonding patterns, provided that the oligonucleotide has anti-VEGF activity at concentrations below 1 micromolar. Antisense oligonucleotides contemplated in this invention include modifications that improve their biological stability. Biological stability is enhanced by incorporating nuclease-resistant bonds such as phosphorothioate linkages between various or all nucleotide residues. The present oligonucleotides also include chemically modified bases at various or all pyrimidine sites. These modified bases include C5-propynyl pyrimidines, C5-hexinyl pyrimidines or 6-aza-pyrimidines or combined C5 and 6-aza pyrimidine derivatives and can further stabilize the oligonucleotides of the present invention. Antisense oligonucleotides contemplated in this invention include modifications that improve their affinity for binding to the target sequences. The affinity for union is improved by incorporating different chemical portions based on pyrimidines. The present oligonucleotides include chemically modified bases at various or all pyrimidine sites. These modified bases include C5-propynyl pyrimidines, C5-hexinyl pyrimidines or combined derivatives of C5 and 6-aza-pyrimidine. The antisense oligonucleotide binding can be to current mRNA or to chemically synthesized RNA sequences that are identical to the sequences found in VEGF mRNAs. This union can be demonstrated in a variety of ways. A method for observing binding is described in Example III. This method involves mixing antisense oligonucleotides with chemically synthesized RNA sequences of the same length, allowing the antisense oligonucleotide to anneal at an initial stage of heating and cooling, and observing the absorbance change of the mixture at 260 nm upon heating. The binding can also be measured by other methods such as nuclease protection experiments, oligonucleotide extension experiments, NMR, gel electrophoresis or other techniques well known to those skilled in the art. For purposes of this invention "improved binding affinity" or "stability" means that the oligonucleotide has a higher melting temperature (Tm), when tested with its target RNA sequence than an oligonucleotide without the modification. Melting point assays as described in Example III, are employed for this determination. Chemical modifications that increase the binding affinity of target mRNA / antisense oligonucleotide sequence duplexes are contemplated for use by the present invention. In general, antisense oligonucleotides having a Tm above 45 ° C are contemplated in the described assay. Oligonucleotides having a Tm above 50 ° C are particularly preferred. Certain oligonucleotides of the present invention include chemical modifications that enhance their activity against previously known VEGF antisense oligonucleotides. Improved activity means that lower concentration of oligonucleotides are required to inhibit VEGF expression in vivo. Although the invention is not intended to be limited by the mode of action of these modifications, increased binding affinity and biological stability are considered at least partially responsible for the increased activity of the oligonucleotides currently contemplated. Specific chemical modifications as established above, are employed to increase the activity of the present oligonucleotides. Antisense oligonucleotides that are contemplated in this invention are also non-toxic at concentrations less than about 1 μM. Toxicity is measured according to the method set forth in Example V. Antisense oligonucleotides of the present invention reduce VEGF production in treated cells. In one method, the cells are treated by placing them in direct contact with the oligonucleotide compositions, such that the oligonucleotide can be internalized in the cell and reach its target RNAra sequence. Prior to treatment, the oligonucleotide is dissolved or suspended in a liquid or incorporated into a solid. Suitable liquid and solid formulations are known in the art and can be selected by well-known methods. Formulated oligonucleotides are placed in direct contact with cells. In other methods, the formulated oligonucleotides are placed in such a way that oligonucleotides can reach their target cells through diffusion, dispersion or the like. The present invention does not require that the oligonucleotide formulation directly contact the target cells. The invention only requires that the oligonucleotide reach the target cells. For example, an oligonucleotide can enter the bloodstream but diffuse out of the blood before reaching target tumor cells, arthritic cells or the like. Alternatively, the oligonucleotide can be mixed in a powder that is applied directly to the skin and diffuses to the underlying cells. Cells treated with antisense oligonucleotides present produce at most, about 90% VEGF that is produced by untreated cells under the same conditions. This effect is observed when concentrations of oligonucleotide solution are below about 1 micro molar (μM). A method for measuring production of reduced cellular VEGF is described in Example VI. However, other methods can be employed to detect the reduction in VEGF production, if they are as sensitive as the method described in Example V. The percent of VEGF produced by the treated cells is determined by measuring the amount of VEGF produced by cells. untreated and treated cells. The percentage is equal to the amount produced by the treated cells divided by the amount produced by the untreated cells multiplied by 100. The untreated and treated cells are claimed to be approximately identical in all respects except with respect to the presence or absence of formulations of oligonucleotides. In this way, the cells used in the assay are of the same type, passage number, phenotype and are in the same growth stage. The cells are grown under identical conditions including identical media (except for changes due to the presence or absence of the oligonucleotide formulation itself), temperature and atmosphere. Under these conditions, cells treated with the antisense oligonucleotides contemplated by this invention may produce approximately 90% of the VEGF as produced by identical untreated cells, when antisense oligonucleotides are used at concentrations up to 1 μM in solutions or a molar percent. similar if it is used in solid formulations. Preferred oligonucleotides incorporate certain chemical modifications that increase their resistance to non-nucleolytic degradation. Chemical modifications contemplated in these modalities are modifications of the common natural origin chemistries found in oligonucleotides. Certain chemical moieties contemplated in this invention include phosphorothioate linkages. These can be placed between some states the nucleoside residues. The most preferred oligonucleotide contains phosphorothioate and 8 phosphodiester bonds. In addition to nucleotide bonds, chemical modifications to the base portions may increase resistance to nuclease degradation. More specifically, modifications to pyridine rings including C5-propinyl or hexinyl groups and / or 6-aza pyridine modifications are contemplated. One method to measure nuclease resistance is by determining the half-life of oligonucleotides in blood serum. This is achieved by standard methods well known in the art. For purposes of the present invention, a chemical moiety decreases the rate of degradation of antisense oligonucleotides by nucleases, if the oligonucleotide has longer serum half-life with the portion that would have without the portion. Oligonucleotides containing phosphorothioate have half-lives of more than 24 hours, whereas their counterparts that only contain phosphodiester bonds have serum half-lives of less than 3 hours. Oligonucleotides containing chemical modifications in their pyrimidine rings are contemplated. Preferred oligonucleotides contain either C5-propynyl pyrimidines, C5 -hexinyl pyrimidines and / or 6-aza pyrimidine. These modifications increase Tms biological stability and activity. The syntheses of nucleotide precursors containing these modifications are described in Example I. The synthesis of oligonucleotides from these certain protected nucleotides is by standard phosphoramidite chemistry well known in the art. Certain embodiments of the present invention are directed to compositions for improving cell absorption that improve oligonucleotide activity. In general, these compositions improve the transport of a liquid through the bi-layer of the liquid. In some modalities, the oligomere is covalently conjugated to a lipophilic molecule. This improves the association of oligonucleotide membrane and permeability properties, such as cholesterol, fatty acids and other lipophilic ends. These molecules can be chemically linked to oligonucleotides by standard methods well known in the art. In other embodiments, absorption enhancers such as cationic lipids or liposomal preparations may be employed. These agents are attractive because of their versatility. These embodiments have the advantage that the same delivery vehicle can be used to administer a mixture of oligonucleotide. One embodiment specifically contemplates the use of the liposomal preparation Cellfectin ™. Other embodiments include a class of polyamino lipid absorption improvers, including spermidine-cholesterol (SpdC). This latter compound has the advantage of functioning particularly well, even in the presence of serum. Compositions and methods for preparing antisense oligonucleotides with cell absorption enhancers are described in Examples IV, VI and VII. Certain oligonucleotides contemplated in the present invention are in salt form. A salt form is one in which the oligonucleotide is associated with positively charged (cationic) atoms or molecules. Suitable cations include but are not limited to sodium, potassium, ammonium, spermidine or polyaminoalipids such as spermidine-cholesterol and the like. Certain embodiments contemplated in the present invention comprise a liposome. Suitable liposomes are well known in the art. Certain liposome compositions specifically contemplated by the present invention include Cellfectin ™. Other compositions include spermidine-cholesterol mixed with DOPE. Liposomal preparations are prepared by methods well known in the art. Certain embodiments of the present invention contemplate supplies of their oligonucleotide compositions through sustained delivery systems, including but not limited to polymeric delivery devices, for example polycaprolactone or mixtures of polycaprolactone with methoxypolyethylene glycol.

Methods and compositions for incorporating the present antisense oligonucleotides into sustained delivery systems are well known in the art. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. Representative modified bases of the invention that are used to replace the natural bases in the synthesis of antisense oligonucleotides. Figure 2. Synthetic scheme for the preparation of 5- (1-hexinyl or propinyl) -6-aza-2'-deoxyuridine phosphoramidite (see also Figure 1). Figure 3. Effects of antisense oligonucleotides on VEGF production by normal human keratinocyte in culture. Figure 4. Effect of oligonucleotide (SEQ ID NO: 2) administered with or without Cellfectin ™ on VEGF expression by keratinocytes. Figure 5. Effect of oligonucleotide (SEC ID DO NOT. 27) administered with or without Cellfectin ™ in expression of VEGF by keratinocytes. Figure 6. Effect of absorption enhancers • Cell different in the activity of T30639 (SEQ ID NO. 2) with keratinocytes. Figure 7. Short-term cellular exposure to oligonucleotide formulations of the invention and long-term inhibition of VEFG expression.

Figure 8. Short-term cellular exposure to oligonucleotide formulations of the invention and long-term inhibition of VEFG expression. Figure 9. Long-term cellular exposure to oligonucleotide formulations of the invention and long-term inhibition of VEFG expression. Figure 10. Effect of chimeric VEGF antisense oligonucleotides, highly modified in VEGF expression, in the presence or absence of absorption enhancer. DESCRIPTION OF PREFERRED MODALITIES The preferred embodiment includes an antisense oligonucleotide that binds a sequence common to multiple mRNA molecules encoding VEGF and prevents the expression of VEGF in vitro. Preferred oligonucleotides contain phosphorothioate linkages instead of various phosphodiester linkages and other chemical modifications that increase the affinity of the oligonucleotide for its target mRNA sequence. In the preferred compositions, oligonucleotides are formulated as cellular enhancement enhancers that improve their ability to cross the cell membrane. Oligonucleotides of the present invention may be in the length range from about 17 residues to 30 residues long. Preferred oligonucleotides are 19 nucleotides long. Their sequences are chosen based on their complementarity to the mRNA molecules that encode the VEGF genes. The region of the mRNA molecule that is complementary to the oligonucleotide is called the target sequence. Preferred antisense oligonucleotides are complementary to target sequences found in each of four known mRNA molecules, VEGF including VEGF 206, VEGF 185, VEGF 165 and VEGF 121. Oligonucleotides containing chemical modifications are contemplated that enhance their binding affinity to target mRNA . Preferred oligonucleotides already contain C5-propynyl pyrimidines, C5 -hexinyl pyrimidines and / or 6-aza-pyrimidines. Preferred modifications increase the temperature at which the oligonucleotide dissociates from its target sequence. The synthesis of nucleotide precursor containing these modifications is described in example I. Oligonucleotide synthesis of protected nucleotides is by standard phosphoramidite chemistry and is well known in the art. Preferred oligonucleotides incorporate certain chemical modifications that increase their resistance to nucleolytic degradation. Although the invention is not limited by the mechanism by this resistance, it is considered that the chemically modified nucleotides resist nuclease digestion by interfering with oligonucleotide binding in the nuclease substrate binding cavity. Preferred nuclease-resistant oligonucleotides contain phosphorothioate linkages between at least some of the nucleotide residues. The most preferred oligonucleotide contains phosphorothioate and 8 phosphodiester bonds. In the preferred compositions, the oligonucleotides of the present invention are formulated or mixed with cell absorption enhancers that increase their ability to penetrate cell membranes. The cellular absorption enhancers contemplated for use in. This invention includes dioleyl phosphotidylethanolamine, Cellfectin ™, spermidine-cholesterol and the like. Particularly preferred is a 1: 1 mixture per mass of spermidine-cholesterol and dioleyl phosphotidylethanolamine. This formulation is mixed with 10 nanomolar to 1 micro-molar concentrations of oligonucleotide, according to standard methods well known in the art. Oligonucleotide compositions contemplated by the present invention are chosen based on in vivo activity. Preferred compositions are not substantially cytotoxic for cells with oligonucleotide concentrations up to 1 micro-molar. Standard cytotoxicity assays as described in Example I are employed to effect this determination. The present compositions should also demonstrate an ability to reduce the production of cellular VEGF at concentrations below 1 micro-molar. The present invention has been described in terms of particular embodiments that are found or proposed to comprise preferred modes for the practice of the invention. It will be appreciated by those skilled in the art that, in light of the present disclosure, numerous modifications and changes may be made to the particular embodiments exemplified without departing from the intended scope of the invention. All these modifications are intended to be included within the scope of the appended claims. EXAMPLES Example I: Method for the preparation of modified bases for incorporation into synthetic oligonucleotides: The modified bases that increase the binding affinity and / or specificity of the synthetic oligonucleotides are illustrated in Figure 1. The synthetic scheme for preparing 5- (1 -hexinyl or propynyl) -6-aza-2'-deoxyuridine phosphoramidite is illustrated in Figure 2. This synthesis provides the building block for preparing the antisense oligonucleotides containing 6-aza-U. Similar schemes have been used to synthesize C-aza-C. The detailed synthetic methodology for the preparation of 5- (1-hexinyl) -6-aza-2'-deoxyuridine phosphoramidite is described below. In a similar manner, 5-propynyl derivatives are prepared from the 5-iodo derivative 7. 3 ', 5'-Di-0-p-toluoyl-5-iodo-6-aza-2'-deoxyuridine (7A): Chlorotrimethylsilane (0.5 ml) is added to a suspension of 5-iodo-6-azauracil (5.8 g, 33.47 mmol) in 1, 1, 1, 3, 3, 3-hexamethyldisilazane (HMDS, 80 ml) and the mixture is heated at reflux for 6 hours. The reaction mixture is cooled to room temperature and HMDS evaporated in vacuo. The residue is dried under high vacuum for 4 hours. The dried silyl derivatives are dissolved in dichloromethane (60 ml). 1-chloro-2-deoxy-3, 5-di-Op-toluoyl-b De? ri tro-pentofuranose (6, 16.3 g, 42 mmol) and zinc chloride (0.046 g, 3.35 mmol) are added to this solution and The mixture is stirred under an argon atmosphere for 24 hours. The reaction mixture is diluted with dichloromethane (250 ml) and the dichloromethane solution is washed with saturated aqueous NAHC03 solution (100 ml). The aqueous layer is extracted with dichloromethane (4 x 100 ml) and the combined organic layer is dried Na2SO4) and evaporated. The residue is purified by column chromatography on silica gel (4 x 15 cm) and the product eluted in dichloromethane containing 0-5% methanol. The dry anomeric product weighs 15 g. Pure b-anomer is obtained by triturating with a mixture of dichloromethane and methanol (4: 1, 200 ml). The solid is collected by filtration and evaporation. 10.5 pure b-anomer is retained after repeating this grinding process, m.p. 204-205 ° C. 1 H NMR in (DMSO-de): d 2.35, 2.37 (2s, 6 H, 2 CH3), '2.80 (m, 2, H, C2, H and C2"H), 4.43 (s, 3 H, C4, H, C5, H2), 5.55 (br s, 1 H, C3, H), 6.39 (t, J = 6.0 Hz, 1 H, C ±,?.), 7.28 (t, 4 H, Tol), 7.85 (t, 4 H, Tol), 12.42 (br s, 1 H, NH). Anal cale, for C24H24IN307: C, 48.58; H, 4.08; N, 7.08. Found: C, 48.85; H, 3.80; N, 6.92. 3 ', 5' -Di-Qp-toluoyl-5- (1-hexinyl) -6-aza-2'-deoxyuridine (8A): 3 ', 5'-Di-toluoyl-5-iodo-6-aza- 2'-deoxyuridine (7.83 g, 6.5 mmol) is dried by co-evaporation with dry DMF (25 ml) and dissolved in DMF (30 ml) at which Cul (0.25 g, 1.3 mmol), triethylamine 1.82 ml, 13 mmoles), 1-hexin (2.23 ml, 19.5 mmol) and tetrakis (triphenylphosphine) palladium (0.75 g, 0.65 mmol) are added under an argon atmosphere. The reaction mixture is stirred at room temperature for 18 hours and an additional 0.5 g of tetrakis (triphenylphosphine) palladium is added. After 48 hours the solvent is evaporated and the residue is co-evaporated with toluene. The residue is purified by silica gel column chromatography and the product is eluted in dichloromethane containing 0-5% ethyl acetate to give 0.9 g of the title compound m.p. 198-200 ° C.

^ -H NMR in (DMSO-d6): d 0.87, (t, 3 H, CH3), 1.45 (m, 4, H, 2, CH2), 2.37, 2.39 (2s, 6 H, 2 CH3), 2.45 (m, 3 H, CH2 and C2"H), 2.84 (m 1H, C2, H), 4.45 (s, 3 H, C4, H, C5, H2), 5.59 (br s, 1 H, C3, H ), 6.49 (t, J = 6.3 Hz, 1 H, C?, H), 7.31 (dd, 4 H, Tol), 7.88 (dd, 4 H, Tol), 12.40 (br s, 1 H, NH) .

Analysis calculated for C30H33N3O7: C, 65.80; H, 6.07; N, 7.68. Found: C, 65.61; H, 5.73; N, 7.29. 6-Aza-5- (1-hexinyl) -2'-deoxyuridine (9a): A mixture of 3 ', 5'-Di-Op-toluoyl-5- (1-hexinyl) -2'-deoxyuridine (8a, 0.8 g, 1.47 mmol), methanol (55 ml) and sodium methoxide (25% solution in methanol, 1.28) is adjusted to room temperature for 2 hours. The reaction is neutralized by the addition of Dowex 50X8 (H +) resin. The resin is removed by filtration and the filtrate is evaporated. The residue is purified by silica gel column chromatography using dichloromethane containing 0-4% in methanol as eluent to give 0.38 g (84%) of the title compound, as a very hygroscopic solid. - "- H NMR in (DMSO-ds): d 0.88, (t, 3 H, CH3), 1.47 (m, 4, H, 2, CH2), 2.02, (m H, C2" H) 2.33 (m ÍH, C2, H) 2.46 (m, 2 H, CH2), 3.40 (m, 2 H, C5, H2), 3.68 (m, 1 H, C4, H,), 4.21 (br s, 1 H, C3, H), 4.61 (t, 1 H, C5.0H), 5.17 (d, 1 H, C3.0H), 6.49 (dd, 1 H, C?, H), 12.27 (br s, 1 H, NH) 5 '-0- (4-4'-Dimethoxytrityl) -6-aza-5- (1-hexinyl) -2'-deoxyuridine (10a): 4,4'-dimethoxytrityl chloride (0.51 g, 1.5 mmol ) is added to a solution of 5- (1-hexinyl) -6-aza-2'-deoxyuridine (0.38 g 1.24 mmol) in dry pyridine (10 ml) After stirring for 6 hours, an additional 0.5 g of DMT C 1 is added and the reaction mixture is stirred overnight.The reaction mixture is diluted with dichloromethane (100 ml) and the organic solution is washed with water (20 ml) The aqueous layer is extracted with dichloromethane and the The combined organic is dried (Na2SO4) and evaporated The residue is co-evaporated with toluene (10 ml) and purified by chromatography on a silica gel column (2 x 1 0 cm) The product is eluted with dichloromethane containing 0-1.5% methanol. Yield 0.45 g. E NMR in (DMSO-d6): d 0.85, (t, 3 H, CH 3), 1.37 (m, 4, H, 2, CH 2), 2.08, (m, 3 H, CH 2"H), 2.37 (m 3H, CH2 and C2, H) 3.06 (m, 2 H, C5, H2), 3.72 (s, 6 H, 2 OMe), 3.87 (m, 1 H, C4, H), 4.20 (m, 1 H, C3, H) 5.23 (d, 1 H, C3, OH), 6.35 (dd, 1 H, C- ^ H), 6.83 (m, 4 H, DMT), 7.16-7.25 (m, 9 H, DMT) 12.30 (br s, 1 H, NH). 5 '-0- (4-4'-Dimethoxytrityl) -6-aza-5- (1-hexinyl) -2'-deoxyuridine-3' -0- (2-cyanoethyl) - N, N -diisopropylphosphoramidite (lia) : 9 5'-0- (4-4'-Dimethoxytrityl) -6-aza-5- (1-hexinyl) -2'-deoxyuridine (10a) upon reaction with 2-cyanoethyl-N, N-diisopropylphosphoramidite in dichloromethane in the presence of N, N-diisopropylethylamine provides the phosphoramidite ly by well-known methods. EXAMPLE II Design v. Synthesis of olionucleotide: Antisense oligonucleotide sequences that can ligate complementary mRNA target sequences shared by all cleaved variants of VEGF mRNA were chosen. The sequence of exemplary synthetic oligonucleotides is illustrated in Table 1. To improve their binding affinity for mRNA targets, the oligonucleotides were synthesized with pyrimidines having C5-propynyl or C5-hexynyl groups, as illustrated in Figure 1 (agner and collaborators). Other modified bases including 6-aza-dU and 6-aza-dC, were also contemplated.

(Fig. 2). Combinations of these modifications were also contemplated. Oligonucleotide T30691 (SEQUENCE ID No. 27) which was complementary to the antisense oligonucleotide (SEQ ID NO: 2) is used as a control in the following experiments. It was the same size and contains the same main structure and base modifications as T30639 (SEQUENCE ID NO.2). Example III: Tm analysis of heteroduplex RNA-oligonucleotide antisense interaction: The temperature (Tm) of antisense oligonucleotide RNA duplexes is used to estimate binding affinity. The T is measured in a diode array spectrophotometer equipped with a temperature controlled cell carrier (Hewlett Packard Model 8452). The antisense oligonucleotide is mixed with target synthetic RNA of the same size (each at 1 μM) in a buffer containing 2 mM sodium phosphate., pH 7.0, 18 mM NaCl and mM EDTA. The solution, prepared in a spectrophotometer cell, is heated at 90 ° C for 10 minutes, cooled to 20 ° C for 10 minutes and equilibrated for 10 minutes to allow duplex formation. To measure the melting temperature (Tm) of the duplex, the cell is slowly heated from 20 ° C to 80 ° C at a rate of 1 ° C / min and the absorbance at 260 nm is measured as a function of temperature. An increase in absorbance signals the fusion or separation of the duplex in single-stranded oligomers. The Tm of duplex formation is obtained from the melting curve data using equations described by standard methods. (Puglisi and Tinoco, 1989). The Tm data are illustrated in Table 2. Incorporation of bases modified with C5-propyne or bases modified with C5-hexinyl in phosphorothioate oligonucleotides, leads to marked increases in Tm values over unmodified oligonucleotides. This was indicative of a significant improvement in the affinity of the antisense oligonucleotide for its target sequence. Table 2 T30807 (DNA / Antisense) T30615 T30639 (unmodified) (Propinyl) Tm (° C) 43 53 AG at 37 ° C (Kcal / mol) -11.5 -13.4 Ah (Kcalmol) -13.6 -91.6 AS (eu) -402 -252 T30808 (RNA * antisense) T30615 T30639 Tm (° C) 43 57 AG at 37 ° C (Kcal / mol) -11.0 -14.3 AH (Kcallmol) -128 -88.3 AS (eu) -378 -239 Table 2 (cont.) T30807 (DNA / Antisense) T30688 T306? 2 (hexinil) (Propinyl does not correspond) Tm (° C) 49 345 AG at 37 ° C (Kcal / mol) -11.9 -8.1 Ah (Kcalmol) -79.8 -103 AS (eu) -219 -306 T30808 (antisense RNA) T30688 T30692 tm (° c) 53.5 43.5 AG at 37 ° C (Kcal / mol) -13.5 -11.3 AH (Kcalmol) -90.7 -119 AS (eu) -249 -347 Example IV: Preparation of improvers of a so: The unassisted absorption of antisense oligonucleotides by cells was low (Fisher et al., 1993, Guy-Caffey et al., 1995). To improve cell penetration, a number of commercially available absorption enhancers as well as novel inolipid polyacrylates synthesized by the inventors is employed (Gao et al., 1989; Guy-Caffey et al. 1995). Example V: Cytotoxicity assays: Cells were seeded at a density of 500 cells / well in a 96-well plate. One day after coating, the cells were exposed to oligonucleotide formulations diluted in series (4 wells per dilution). After 1 or 4 days of exposure, the effect on cell viability is determined by a non-radioactive assay system (Cell Titer 96 aqueous cell proliferation assay, Promega Corp.). No toxicity was observed by the present oligonucleotides were at concentrations lower than 1 μM. EXAMPLE VI Cellular Test of Oligonucleotides: The activity of antisense oligonucleotides, their modified counterparts and various formulations were evaluated using cultured human keratinocytes, a cell line that secretes VEGF under normal culture conditions (Ballaun et al., 1995; Frank et al., 1995). . Cells were coated in 48-well plates at a density of 50-100,000 cells / well / 0.5 ml of KGM medium (Clonetics). An ELISA-based protein assay system (R & D Systems) is used to measure the levels of VEGF protein in the cell supernatant. Preliminary measurements show that when NHEK cells are grown in the recommended medium, 50,000 cells reconstructed in 0.5 ml of medium produce approximately 150-200 pg of VEGF in 15 hours (ie -300-400 pg / ml in the control supernatant not treated). Cells were also incubated for 15 hours with the oligonucleotide formulation. Three of four anti-VEGF oligonucleotides demonstrate activity in the range at 0.2 μM, in the presence of 10 μg / ml of Cellfectin ™. The control sense oligonucleotide has no effect (not shown). The result is illustrated in Figure 3.

For the evaluation of the antisense effect, oligonucleotides were administered to cells in the presence or absence of absorption enhancers. In preliminary experiments, phosphorothioate oligonucleotides without base modification were effective at concentrations lower than 1 μM and there was no significant effect observed in the absence of carriers (data not shown). At concentrations above 1 μM, the oligonucleotides tend to inhibit non-specifically VEGF expression (data not shown). These non-specific effects were known in the art (Stein et al., 1993; Wagner, 1994). To avoid these non-specific effects, oligonucleotides were mixed with absorption enhancers. Cellfectin ™, a liposomal preparation of tetrapalmi t ilespermine (TM-TPS) with dioleyl phosphotidiethanolamine (DOPE) (TM-TPS / D0PE in a 1: 1.5 mass ratio of Life Technologies, Inc.), was more effective and less toxic than others commercially available supply agents tested. Oligonucleotides formulated with liposomal preparations of the polyaminolipid SpdC (Guy-Caffey et al., 1995; SpdC / DOPE, 1: 1 by mass), were even more effective. In typical cell culture experiments, oligonucleotides (10 nM to 1 μM) were dissolved in water -20-40 μl of an aqueous solution of absorption enhancer at room temperature and incubated for approximately 10 minutes. This solution was mixed with 0.5 ml of warm growth medium and added to cells. The cells were incubated for 15 hours in the presence of the oligonucleotide. After incubation, the supernatant is collected and is already used immediately for ELISA or stored at -80 ° C for future analysis (no significant difference was observed in VEGF levels between supernatant samples never frozen, or frozen and thawed). As illustrated in Figure 4, the antisense oligonucleotide T30639 (sequence ID No. 2) was more active in the presence of Cellfectin ™, while the "sense" control oligonucleotide T30691 (SEQUENCE ID No. 27) had little effect except at the highest concentration employed as illustrated in Figure 5. Figure 6 shows the effect of administering 0.1 μM or 0.2 μM oligonucleotide (SEQUENCE ID NO.2) with various formulations of cationic lipid SpdC, spermidine-cholesterol ( Guy-Caffey et al., 1995); DC-Chol (Gao and Huang, 1991); CS, cholate-esper idina; DCS, deoxycholate-spermidine; cf, Cellfectin ™ (Life Technologies, Inc.). Liposomal preparations of each cationic lipid were prepared by mixing with the fusogenic lipid DOPE (mass ratio 1: 1) and stored after use until used. The liposomes were resuspended in 5% dextrose (at 1 mg / ml) before use and stored at 4 ° C for use in two weeks. Oligonucleotides were mixed with the cationic liposomal preparations just before cell treatment as described above. Figures 7 to 9 show the results of cell incubation with varying concentrations of the antisense oligonucleotides T30639 (SEQUENCE ID NO.2), or its phosphodiester phosphorothioate version T30848 (SEQUENCE ID No. 6). (See Table 1). Figure 7 shows the 0.1 μM effect of oligonucleotide, Figure 8 shows the effect of 0.2 μM of oligonucleotide and Figure 9 was for 0.4 μM of oligonucleotide. In each experiment, cells were treated for 4 hours in medium supplemented with the antisense oligonucleotide pre-mixed with SpdC / DOPE. Then the medium is replaced with fresh medium without supplementation. Graph 1 shows the percentage inhibition in VEGF production, 16 hours after the oligonucleotide composition is washed from the culture, the graph is 40 hours after washing the oligonucleotide and graph 3 is 64 hours after the washing of the oligonucleotide. The amount of VEGF level in the harvested medium is then determined. Cell morphology at the end of the incubation period of approximately 3 days was normal. The long-term effects of the oligonucleotide on VEGF production are set forth in Figures 7 to 9. In the graphs, the symbol (?) Is for 0.1 μM T30848 (SEQUENCE ID No. 6). The symbol () is for 0.1 μM T30639 (SEQUENCE ID No. 2). Figure 10 shows the results in similar experiments with oligonucleotides derivatized with lipophilic groups. S96-5296 (SEQUENCE ID NO: 20) is modified at the 3 'end by a C16 lipid group and contains phosphodiester 8 and phosphorothioate bonds. S96-5297 (SEQUENCE ID No. 21) has the same main structure and is extremely modified with a 3'-pyrene portion. These hydrophobic portions aid in the absorption and activity of the oligonucleotides when mixed with Cellfectin ™ and the phosphodiester linkages increase the activity of the oligonucleotides. The symbol () is for S96-5296 (SEQUENCE ID NO. 20), the Symbol () is for S96-5296 (SEQUENCE ID NO. 20) with 10 ug / ml of Cellfectin ™, the symbol (or) is for S96-5297 (SEQUENCE ID No. 21) with 10 ug / ml of Cellfectin ™, the symbol () is for symbol (or) is for S96-5297 (ID DE) SEQUENCE NO. 21) with 10 ug / ml of Cellfectin ™, the symbol (??) is for 0.2 μM T30639 (SEQUENCE ID NO.12) with 10 ug / ml of Cellfectin ™. Example VII Anti-VEGF activity of antisense oligonucleotides: Phosphorothioate containing antisense oligonucleotides without base modifications, seems to have no significant effect on the cellular production of VEGF, except for some non-specific sequence-independent inhibition at concentrations exceeding 1 μM (data not shown). The results were consistent with other studies showing that low submicromolar doses of simple oligonucleotide phosphorothioate inhibitors were ineffective and at high levels, the same oligonucleotides can exert non-specific effects on cellular metabolism (reviewed by Stein and Cheng, 1993; ). However, oligonucleotides containing phosphorothioate contain pyrimidines comprising C5-tip (Wagner, 1993) specifically inhibit the cellular production of VEGF. See Figure 3. These modified oligonucleotides have melting temperatures that were about 15 ° C higher than their unmodified counterparts. See table 2. This suggests that the modified oligonucleotides bind their targets with higher affinity than the non-modified forms. Optimal oligonucleotide at the ratio of Cellfectin ™: The cellular absorption of the cationic liquid-oligonucleotide mixture is partially determined by the chemical nature of each component in the formulation, partly by its relative concentration and mass proportions and partly by the endocytic properties of the cell objective. With T30639 (SEQUENCE ID NO.2) co-administered with Cellfectin ™ to NHEK cells, the ratio of oligonucleotide to TMTPS 1: 3 (by mass) results in optimal activity. In a related experiment, the concentration of the oligonucleotide is altered while the ratio of the cationic lipid and oligonucleotide is maintained and the effect of the VEGF expression is measured with respect to the "sense" control T30691 (SEQUENCE ID No. 27). Oligonucleotide T30639 (SEQUENCE ID NO.2) showed specific anti-VEGF activity, while the control oligonucleotide had no effect (see Figures 4 and 5). Effect of formulations of idina-cholesterol + DOPE or DC-chol- + DOPE (liposome preparations: 1: 1 by weight) in oligonucleotide efficacy: A number of absorption enhancers are used with nucleic acid therapeutics (Behr, 1994, Guy-Caffey et al., 1995, Lewis et al., 1996). One of these compounds was sperimidine-cholesterol conjugate (SpdC) (Guy-Caffey et al., 1995). This compound was not toxic to cells at concentrations significantly higher than those required by this invention and was not toxic to rodents when treated for up to one week. Another cationic lipid, DC-Chol (Gao and Huang, 1991), is approved for therapeutic use in gene therapy and was relatively non-toxic to the cellular system. Liposomal test preparations of SpdC / DOPE and DC-Chol / DOPE (spdC or DC-Chol with dioleoyl phosphotidylcholine) at mass proportions in the range of 1:05, 1: 1, 1: 1.5, and a 1: 2 ratio at 1: 1 seems more effective in anti-VEGF assays with NHEK cells. See Figure 5. Compositions with cationic reagents appear to be 20-40 more active than those with Cellfectin ™ (Figure 6). Short duration of exposure to oligonucleotide formulation is sufficient to observe long-term inhibitory effect in VEGF production: VEGF expression is reduced after relatively brief exposures to the compositions described in this invention. For example, "4-hour incubations demonstrate more anti-VEGF activity than what is observed with oligonucleotide exposure overnight." See Figures 7 to 9. Surprisingly, the effect lasted at least 3 days, the entire duration of the experiment , Figures 7 to 9. Other experiments showed that anti-sense oligonucleotides with phosphorothioate-phosphodiester main and medical structures were potent inhibitors of VEGF expression., the chimeric variants of T30639 (SEQUENCE ID No. 2) containing 10 phosphorothioate and 8 phosphodiester linkages and lipid end modifications S96-5296 (sequence ID No. 20) and S96-5297 (sequence ID No. 21) demonstrated excellent activity in the presence of SpdC / DOPE. (Figure 7). Inhibition of VEGF lasted more than three days after only a four hour incubation (Figure 7). In the absence of SpdC, the chimeric oligonucleotide backbones do not affect VEGF expression. In this way, oligonucleotides with lower phosphorothioate linkages can have improved efficacy and reduced non-specific effects. Absorption seems crucial to the efficacy of the oligonucleotide. Example VIII VEGF Inhibition Jn Vivo A. Specific Objectives Increased expression of vascular endothelial growth factor (VEGF = Vascular Endothelial Growth Factor) has been implicated in the advancement of ocular neovascularization associated with proliferative diabetic retinopathy, neovascular glaucoma and many other blinding conditions. Retinal ischemia leads, to increased synthesis of the angiogenic protein VEGF, which triggers the proliferation of vascular endothelial cells, resulting in the formation of an abnormally large number of blood vessels in the retina, optic nerve and iris. So far, there are no accepted therapeutic treatments for this condition. Our overall objective is to apply a rational design and test procedures to identify inhibitors of potentially novel antisense oligonucleotides, therapeutic, of VEGF expression, with the aim of treating retinal ischemia associated with neovascularization in humans. Our recent in vitro data in human cell culture systems indicate that we can prepare specific oligonucleotide formulations that inhibit cellular VEGF expression by more than 50% in the subrachromolar concentration range. Our goal for this purpose is to extend our findings in vi tro in a neovascularization rat model associated with VEGF. Our specific objectives are: 1.- To synthesize a "library" of antisense oligonucleotides directed against rat VEGF mRNA. There are three major cleavage variants and 1 minor VEGF. Ten oligonucleotides will be targets for sequences in the common version of RNAs. They will also contain nuclease-resistant backbones and modified bases to improve binding affinity with mRNA. 2.- Establish monolayer of rat cells and spheroidal models to evaluate the activity and toxicity of the anti-sense oligonucleotides and their formulations. The C6-glioma cell line will be used for this because it has been widely used to study VEGF function. The spheroid (mass of cells) will be useful to estimate if our oligonucleotides are able to penetrate cellular layers. 3. - Evaluate the efficacy of oligonucleotides using various cell absorption enhancers. Compounds developed in Aronex will be compared with commercially available agents. 4.- Develop a proof-of-concept test to obtain data to support the antisense mechanism. In this way, an experiment in vi tro is designed to test whether we can specifically target just an isotope of VEGF, to answer the question if our olinucleotides are truly working in the predicted mode. This will help in the future design of antisense oligonucleotides. 5. - Use of an in vivo rat model of iris neovascularization to test the most promising antisense compounds. These studies will be carried out in collaboration with Dr. Anthony Ada is at Harvard. Upon completion of the proposed studies, we hope to have quantitative information regarding the in vivo efficacy of 1-2 oligonucleotides. Some information regarding dosage, mechanism of potential action, cellular availability and potential toxicity will also be known. If any of the oligonucleotides reduces vascularization and / or VEGF expression by 20% in vivo, we would consider that it would be a positive development and we would proceed to more detailed studies in animal models during phase 11 of the investigation. Oligonucleotide is chosen, the risk of nonspecific binding to other RNAs would be unacceptably high and choosing a sequence with high G content may lead to undesirable G quartet formation, which reduces the availability of available free coil form by binding (Bishop et al. , nineteen ninety six) . An alternative approach, which we propose to take, is to use selectively modified oligonucleotides containing C-propynyl pyrimidines, a modification that leads to very efficient binding without significant toxicity (Wagner et al., 1993).; Fenster et al., 1994). We have recently shown that propynyl modification seems to work especially well (see preliminary results). Approaches to improve oligonucleotide nuclease resistance: Oligonucleotides with a natural phosphodiester backbone, are highly susceptible to serum and cellular nucleases. We have determined that a base sequence oligonucleotide 17 has a half-life of less than 3 minutes in serum (Bishop et al., 1996). An alternative is to use oligomers with phosphorothioate backbone (Stein et al., 1991), a modification that markedly improves the half-life of serum oligonucleotides by one day or more. The use of phosphorothioate linkages is considered to direct some non-specific effects at high levels, but as discussed above, we propose to synthesize specially modified oligonucleotides that work at very low concentrations, thus reducing the risk of non-specific interaction. Oligonucleotides with alternating main structures have been tested but all have had rather more non-specific effects than phosphorothioate oligonucleotides. Approaches to improve cellular uptake of oligonucleotides: Studies of subseclular distribution show that cells treated with fluorescent oligonucleotides accumulate in perinuclear endosomal compartments (Guy-caffey et al., 1995). The speed limiting step in the internalization process appears to be transport of oligonucleotides through the plasma membrane or the membranes of the endosomal compartments. There are two potential ways to improve the transport of oligonucleotides through the membrane lipid bilayer. In the first approach, the oligomer is covalently conjugated to a compound that improves its permeability and membrane association properties, for example, by conjugating cholesterol (Letsinger et al., 1989).

We have recently identified a novel property modification, a lipophilic end ferroxene (tether) (see experimental design) that appears to markedly improve the efficacy of antisense oligonucleotides. Alternatively, absorption enhancers such as cationic lipids or normal phosphorus preparations may be employed. These agents are attractive because of their versatility-the same delivery vehicle can be co-administered with a variety of oligonucleotides. The design of these cationic lipids incorporates a subsequently charged head group that is linked to the nucleic acid and an interactive membrane tail that is proposed to interact with fusogenic lipids and / or destabilize the cell membranes. The activity of many preparations of cationic lipids (such as lipofectin) are influenced by factors such as composition and amount of nucleic acid, cell type and the concentration of serum in the cell growth medium. In addition, some preparations are cytotoxic. These restrictions severely limit the utility of many of these compounds as delivery agents for therapeutic oligonucleotides in animal systems, and there continues to be a tremendous demand for effective absorption enhancers. We have synthesized a novel delivery vehicle, spermidine-cholesterol (SpdC) that improves cellular uptake and membrane permeability of oligonucleotides, even in the presence of serum (Gay-Caffey et al., 1995). Formulations that use this compound will be evaluated as part of the proposed studies. Our goal is to identify antisense formulations that inhibit VEGF expression by cells in the eye, with concomitant reduction in angiogenesis associated with disease. These studies were pointed out by our recent discovery that chemically modified antisense oligonucleotides can have potent inhibitory activity at sub-micromolar doses. In addition, the development of an animal model of neovascularizacoon associated with VEGF by Dr. Adamis, will allow us to test the most effective compounds in vivo. Preliminary Results: Compendium: The aim of our preliminary experimental series of experiments has been to discover and / or test general principles and approaches to improve the activity of antisense oligonucleotides in cell-based models of VEGF expression. Primarily, our objective has been: ° To develop quantitative cell based assays to inhibit the effect of antisense oligonucleotides on VEGF protein levels.

° Synthesize oligonucleotides that contain structural modifications that lead to improve the binding affinity for target mRNA, greater nuclease resistance and greater specificity. ° Test formulations using novel absorption enhancers (some developed in Aronex) to increase cell internalization and membrane penetration of the administered oligonucleotide. Oligonucleotide designs: Because we expect one of our compounds to be tested eventually in humans, that work was initiated using antisense oligonucleotides directed against human VEGF mRNAs. To achieve maximum inhibition of expression, we chose antisense oligonucleotides that are complementary to sequences shared by all VEGF mRNAs (Table 3). Antisense oligonucleotides: (Codón AUG primer in sequence mRNA 57) T30638: mRNA sec. 87-105 5 x -a * g * a * g * C * a * g * C * a * a * g * C * g * a * g * g * C * t-3 T30639: mRNA sec. 185-023 5 v -g * C * g * C * U * g * a * U * a * g * a * C * a * U * C * C * a * U * g-3 T30640: mRNA sec. 204-222 5 -C * g * a * U * U * g * g * a * U * g * g * C * a * g * U * a * g * C * t-3 T30641: mRNA sec. 232-250 5? -U * a * C * U * C * C * U * g * g * a * a * g * a * U * g * U * C * C * a-3 Structure of total phosphorothioate (*) to confer resistance to nuclease. C-5 propynyl pyrimidines to improve binding affinity to RNA target. Table 3 Antisense oligonucleotides directed against human VEGF. In coincidence with initial reports (Wagner et al., 1993) found that the base substitutions of C-5 propinyl pyrimidine, increase Tm of duplex formation, an indicator of the affinity of the strands between them, from -60 ° C for an unmodified oligonucleotide to more than 80 ° C. To use as controls, we synthesized a "sense" oligonucleotide of the same size and modifications T30691. Cellular oligonucleotide test: The activity of antisense oligonucleotides, their modified counterparts and various formulations, was evaluated using cultured human keratinocytes, a primary cell line that secretes VEGF. Cells were repeated at a density of 50,000 cells / well of a 46-well plate in 0.5 ml of KGM medium (Clonetics). The level of VEGF secreted in the growth medium is measured by an enzyme-linked immunosorbent assay (ELISA) (R & amp; amp;; D Systems). The assay is linear over the range of 5 to 1000 pg / ml. Our measurements show that when NHEK cells are grown in the recommended medium, 100,000 cells coated in 0.5 ml in the medium, produce approximately 150 pg of VEGF 15 hours (approximately 300 pg / ml in the control untreated supernatant). The majority of the cell-based assays were performed using a commercially available cationic liposome formulation, commercially available as a transfection agent for cell gene delivery (Cellfectin, from Life Technologies). Other commercial preparations of transfection agents are either toxic or relatively ineffective (7 tested). A curious effect of Cellefectin is that when only cells are administered, as a control, it currently improves the production of VEGF. The reason for this is not known. More recently, we have begun to use spermicidal-cholesterol formulations (Guy-Caffey et al., 1995). In the very first experiments, using antisense oligonucleotides, phosphorothioate without 5-propynyl modifications, we did not observe effect on VEGF levels (up to 5 μM of extracellular concentration, in the presence or absence of absorption enhancers). At higher levels, there was a small amount of what appears to be non-specific inhibition (data not shown). When pyrimidines containing 5-propyne (Wagner et al., 1993) are replaced by cytosines and thymidines, there was an improvement of 20 ° in the Tm measured in vivo, indicating that the oligonucleotide can be linked to its synthetic synthetic RNA with much greater affinity than the unmodified variant (data not shown). We tested the effect of these oligonucleotides in the presence or absence of absorption enhancers, and the ratio of absorption enhancer to oligonucleotide was also varied, in an effort to identify an optimal formulation, We observed that different oligonucleotides have different effects on VEGF levels .

Figure 3. Three of four oligonucleotides had activity in the range of 0.2 μM, in the presence of 10 μg / ml of Cellfectin ™. The control sense oligonucleotide had no effect (not shown). The most potent of the test oligonucleotides, T30639, has since been used for subsequent optimization studies. An optimal ratio of oligonucleotide to absorption enhancer: In a follow-up experiment, we maintained the ratio of oligonucleotide (T30639 antisense and T30691 sense of control) to the cationic lipid component of cF at mass ratio 1: 3 and the effect on production was measured of VEGF. Again, T30639 + cF showed specific anti-VEGF activity while the control oligonucleotide had no effect, Figure 11.

Figure 11. Effect of oligonucleotide formulation Cellfectin (1: 3) on expression of VEGF. At high concentrations, there seem to be non-specific effects (not shown). It is important to note that we have made no attempt to separate the free absorption enhancer from the bound material. This probably happens de facto when we change the relative relationships of one to the other. Effect of oligonucleotide size: In the next experiment, we asked if it would be feasible to reduce the size of the oligonucleotide while maintaining the specific anti-VEGF activity. Shorter olympics are also cheaper to synthesize. However, using the same NHEK assay, we found that the base 19 oligonucleotide was more effective than the base 16 or 14 derivatives, all of the oligonucleotides administered with 10 μg / ml of Cellfectin. Changing the ratio of Cellfectin to oligonucleotide does not alter the relative activity (not shown). Effects of formulations of spermidine-cholesterol + DOPE or DC-Chol + DOPE (liposomal preparations, 1: 1 by weight) on oligonucleotide efficacy: We have recently begun to explore alternatives to Cellfectin, which can be somewhat toxic to cells after long-term exposure. An absorption enhancer, spermicide-cholesterol conjugate, (SpdC) (Guy-Caffey et al., 1995), has found that it is not toxic to cells at the levels used, and that there is no detectable toxicity in rodents treated throughout the week. . It has been tested for that cationic-Chol lipid (Gao et al., 1991), for clinical trials of gene therapy, and has very low toxicity in cellular systems. Preliminary data indicate that formulations of these novel lipids were 20 to 40% more potent than Cellfectin in parallel experiments. A short duration of exposure to oligonucleotide formulation is sufficient to observe long-term inhibitory effect on VEGF production: In all the above experiments, we have been incubating the cells with the absorption enhancer plus oligonucleotide overnight. We then asked whether a shorter duration of cell exposure to the oligonucleotide can achieve the same level of anti-VEGF activity. We discovered that undoubtedly, a wash after four hours and a return to a medium not supplemented fresh does not diminish the ante-VEGF activity (Figure 12). Even more, the effect lasted for up to three days (duration of the experiment).

Time (hours) after addition of 0.2 μM T30639 + 10 μg / ml cF. Figure 12. Treatment for four hours of oligonucleotide + cF, then replacement with simple medium. The inhibition continues for almost three days. There was no significant inhibition by the control sense oligonucleotide (not shown). In fact, the level of inhibition in relation to the control (without oligonucleotide) was much better than previously observed. One reason for this may be that in the simple incubation experiment, the -VEGF protein continues to be synthesized from pre-existing mRNA (not yet blocked by the antisense oligomer) and accumulated in the medium. By replacing the medium to four hours, this "bottom" VEGF source is removed. These data have an important implication in the in vivo test system, due to the long-term antisense effect suggests that the drug does not have to be administered frequently. This aspect will have to be verified in the in vitro and in vivo composite assays. Conjugated oligonucleotide-ferrocene: We recently discovered that a metallocene-modified oligonucleotide formulated with an absorption enhancer is the most effective VEGF inhibitor in our in vitro assays, with very little toxicity in the concentration range used (Figure 13). The oligonucleotide formulated with Cellfectin has specific anti-VEGF activity at 20 μm concentration. The ferrocene end has been designed to improve the membrane association of the oligonucleotide (D. Mulvey, Aronex, personal com). Furthermore, we postulate that the lipophilic iron portion can help in being a cellular target and transmembrane movement of the oligonucleotide, probably by exploiting the active transport system of the cell. Handle work of the mechanism by which the modification is beyond the scope of this fact and is the subject of a separate study. However, the fact that we have observed high activity with ferrocene-modified oligonucleotides suggests that this avenue should be explored as we tested oligonucleotides for testing in the in vivo model.

Figure 13. The potent antisense effect of the conjugate-ferrocene variant of antisense T0639 As described in detail in the experimental design section, the adult rat model of iris neovascularization provides a means to test the activity of antisense oligonucleotides in a manner quantitative In this trial, rats are placed in a hypoxic chamber for 1 to 21 days, and the increase in iris vascularization is quantified by digital imaging. As shown by the data (equal to 14, there is clear progression in the degree of vasculature with increased length of incubation.The level of retinal RNA also rises but not in the same proportion (Figure 15) .Stimulated by our preliminary data and the availability of the rat model, data, we now propose to obtain a similar proof of concept in an in vivo model of angiogenesis.

C. Relevant Experience The principal investigator, Nilabh Chaudhary, PhD in Philosophy, he has broad-based experience in cellular and molecular biology. He received his Ph.D. in biochemistry from the University of Western Ontario, London, Canada, 1984. He was subsequently awarded a Damon Runyon-Walter Winchell Cancer Fund Graduate Scholarship to continue his post-doctorate studies in Cell Biology in the Doctor's Laboratory. Gunters Biobel at Rockefeller University, New York,. In 1986 he was appointed associate of the Howard Hughes Medical Institute in the same laboratory. Dr. Chaudhary met with Triplex (recently renamed Aronex) in 1992 as a research scientist to start a program in Cell Biology aimed at developing techniques to improve the nuclear and cellular absorption of nucleic acid therapies. He has collaborated closely with a team of organic chemists to design, synthesize and test numerous modifications of oligonucleotides and reagents for improved resolution. Dr. Chaudhary has co-authored scientific papers on the function-structure relationship of potentially therapeutic oligonucleotides and has designed approaches to improve their cellular internalization and efficacy. He has experience in the design of cell-based assay systems, immunoclinical techniques, protein micro-quantification, nucleic acid purification and molecular cloning techniques, sub-cellular fractionation, lipid and membrane protein isolation, and fluorescence icrocosm. Anthony P. Adamis, M.D., is a collaborator and consultant in this project. He is an assistant professor in the Department of Ophthalmology at Harvard Medical School and a research associate in the Department of Surgery at the Children's Hospital of Boston. In 1994, Dr. Adamis and his colleagues demonstrated for the first time a causative link between increased ocular VEGF levels, angiogenesis, and progression of proliferative diabetic retinopathy, a primary cause of blindness. In studies conducted since then, he has confirmed the physiological role of hypoxia in stimulating VEGF expression, which leads to neovascularization in the eye. His scope of research experience includes clinical studies in patients, development of diseased rodent models of cell-based proof-of-concept trials and use of molecular biology, and molecular biology and cloning techniques. He has published more than 20 documents with 10 in the field of angiogenesis. D. Methods and experimental design Summary of approach: Our objective in this proposal is to demonstrate the efficacy of antisense anti-VEGF compounds in an animal model of angiogenesis. In preparation for this, we propose to conduct a series of in vi tro experiments that will guide us to the most promising antisense formulation for in vivo testing. We will begin to classify a "library" of 10 candidate antisense oligonucleotides (19 bases) directed to the rat VEGF mRNA. Oligonucleotides will contain C5-propynyl pyrimidine to improve binding affinity of target mRNA, and phosphorothioate internucleotide bonds to achieve nuclease resistance. For cellular tests, we plan to use cells from C6 rats (glioma) that respond to hypoxia by producing copious amounts of VEGF mRNA and proteins. Assays performed in a 96-well format will be used to classify the activity of the various control or antisense oligonucleotide preparations. The time course of its effect on the level of VEGF secreted in the extracellular medium will be verified by ELISA. To improve cellular uptake, oligonucleotides with novel absorption enhancers will be co-administered. Different proportions of nucleic acids and lipids will be approved. In addition, the two "best" antisense sequences will be chosen to conjugate to a 3'-lipophilic extreme ferrocene, a modification that may contribute to cellular entry of the antisense oligonucleotide. Also, the effecy of the two best oligonucleotides (or their formulations) at VEGF mRNA levels will be determined by Northern blotting (and compared with the effect of appropriate controls). In an effort to provide evidence for the antisense mechanism, a separate series of in vitro experiments is planned. C6 cells will be treated with antisense oligonucleotides, specially designed to be isotype specific VEGF, that is to target only one or two species of VEGF mRNA (three major one minor in the rat). RNase protection assay will be used to measure the relative effects of each species of VEGF mRNA. In principle, if the antisense effect is truly sequence specific, only the expression of the target isotype will be deregulated. Oligonucleotides of different sequence will be ineffective. The cellular toxicity of the most effective antisense compounds will be tested in two different cell lines, and the two less toxic formulations will be tested in C6 spheroid cell models, designed to determine whether oligonucleotides can penetrate through cell layers. The levels of VEGF mRNA in successive layers of cells in the spheroid will be determined in hybridization in si tu. The utility of absorption enhancers and extremes will also be verified in this model. The most effective anti-angiogenic anti-VEGF oligonucleotide activity is then assessed in animals using a rat eye model of iris neovascularization. Albino rats will be placed in chambers with low oxygen content (up to two weeks) and vascularization in the iris verified by a quantitative non-invasive digital imaging procedure. In this model, increased vascularity is increased after only 1 to 2 days of hypoxia. The oligonucleotide (or test formulations) will be introduced directly into one eye of the rat, with the other eye viewing it as an untreated one as a control. After up to one week of exposure, any effect on vascular growth will be quantified. Changes in VEGF protein levels (in the vitreous if possible) and levels of mRNA in the retina, will be verified by ELISA and Northern spotting, respectively. Any side effect will be noted. Depending on the initial results, a multi-dose experiment will be attempted. Effects of control oligonucleotides will also be verified. The less toxic, more effective formulation (greater than 20% inhibition of neovascularization) will then be tested in a prolonged series of animal tests, as part of phase II of these studies. The selection of control antisense oligonucleotides: To achieve maximum blockade of VEGF expression, we synthesize antisense oligonucleotides that bind 10 of the common region of all VEGF isotypes in the rat (Conn et al. 1990). 10 oligonucleotides selected essentially randomly, without hairpin motifs or sections rich in G, will be synthesized for testing in the first round of classification. 5 will be rat specific and 5 sequences will be chosen to bind human mRNA equally. It is not clear whether the evolutionarily conserved sites are "good" or "bad" antisense targets although the most recent evidence suggests that there is no preferred forecast location for ignition targets. All synthetic oligonucleotides will contain 5-propynyl pyrimidines (to improve binding affinity for targeting) and phosphorothioate linkages (nuclease resistance) (Wagner et al.1993; Fenster et al., 1994). We already have several "irrelevant" oligonucleotides that we use as controls, but if necessary, we will synthesize a control oligonucleotide of the same size and base composition as the antisense sequence. The oligonucleotides will be synthesized, purified (more than 95% by HPLC) and characterized by The Oligonucleotide Synthesis Group in Aronex. Oligonucleotides for mechanism of action studies: To obtain data that supports the antisense mechanism of action, several oligonucleotides (approximately 4, depending on efficacy) specific to the 20-mer isotype will be prepared. An oligonucleotide directed against a sequence found only in VEGF-165 mRNA will not bind to VEGF-120 mRNA. Similarly, an oligonucleotide of base 20 complementary to the cleavage binding of VEGF-120 (ie, 10 bases per exon) should not be able to bind well to VEGF-165. For use as a control, oligonucleotides with inverted sequences will be synthesized (two halves will be inverted). The effect of these oligonucleotides on VEGF expression will be determined by comparing the relative levels of the various mRNAs. We will use the RNAse protection levels to quantify the relative levels of each mRNA (Ambion, Austin, TX). The probes to do this (in the approximately 150 to 250 base long range) have already been prepared using specific primers from rat mRNA sequences and RT-PCR technology (Perkin-Elmer). Cell culture: The biological classification will be carried out in glial C-6 cells derived from rat glioma.

The predominant isoforms of VEGF in this cell line are VEGF-165 (amino acids) and VEGF-120 (46% each), while VEGF-188 only represents approximately 8% (Bacic et al., 1995). This cell line has been widely used to investigate structure and function of VEGF. To induce VEGF synthesis by stimulating with hypoxia, cells were placed in a low oxygen chamber (GasPak Plus anaerobic culture chamber) (BBL Microbiology Systems) with palladium and hydrogen catalyst to remove all oxygen (Stein et al., 1995). . Typical incubation times will be in the range of 6 to 18 hours. Alternatively, cultures will be exposed to 100-300 μM cobalt chloride, which interferes with the heme-dependent hypoxia response system and activates a hypoxia response factor that induces transcription of VEGF mRNA. Evaluation of antisense oligonucleotide activity in vi tro. - Cells C-6, broken down in monolayers, will be kept in Dubelcco medium with 5% fetal bovine serum and antibiotic. For the preliminary test of anti-VEGF oligonucleotides, cells will be coated at a density of 10,000 or 20,000 cells / well, in a 96-well dish. After one day of recovery, the cells will be treated with oligonucleotide (in .25 ml of medium). Two types of media will be tested, the C6 medium containing only regular Opti in (Life Technologies), the reduced serum medium that is often used to improve transfection efficiency, by reducing interference by serum components. After varying periods of incubation in the hypoxia chamber, the supernatant will be transferred to a fresh plate for further analysis by ELISA. As a rule, when new formulations are tested, we examine cell morphology through a microscopy to look for unusual changes or any obvious signs of toxicity. For RNA analysis, a large number of cells (> 2xl06 to 107 cells in T75 flask) will be treated with a select number of formulations. After oligonucleotide treatment (and exposure to hypoxia, etc.) the supernatant will again be saved for ELISA, and RNA will be isolated and analyzed using methods described below. ELISA VEGF assay: No commercial equipment is available yet for rodent systems, so we are designing one that uses antibodies that are known to react well with rat VEGF (RD1-1020 or RD1-4060 from Research Diagnostics, Inc., and another from R &D Systems). Other antisera to VEGF are also available, so that we will choose the best combination, ELISA reagents (second antibody bound to enzyme, substrate) have been purchased from Pierce. RNA extraction, Northern blotting, RNAse protection assays and hybridization probes: mRNA size VEGF is in the range of 3.8 to 4 kilobases, primarily due to the long untranslated region. For RNA analysis, total RNA will be treated or untreated cells by the RNAzol method (Tel-Test, Inc., Friendswood, TX). To use as probes, specific VEGF segments corresponding to the common region and isotype-specific probes have already been generated by combination of reverse transcriptase polymerase chain reaction (RT-PCR equipment, Perkin Elmer) using specific VEGF primers and C6 RNA followed by size selection of cDNAs that originate different mRNAs, and selective amplification using specific isotope primers. These probes will be cloned in PCRII plasmid cloning vectors (Invitrogen) and the sequence will be confirmed by sequencing (sequential, USB). The PCRII vector allows the RNA polymerase-dependent production of radio labeled RNA probes for use in RNAse protection assays (Ambion, Austin TX). A beta-actin probe will be used to normalize RNA levels. For Northerm staining, RNA (up to 20 μg) will be fractionated on formaldehyde gels, transferred to nylon, and probed with radio labeled probes according to standard procedures, with beta-actin to normalize RNA levels. In all RNA assays, phosphorus imaging (Fuji Phosphorimager) will be used to quantify the relative levels of radioactivity. Experiments to support the mechanism of antisense action: The antisense oligonucleotides in this study are complementary to the mRNA sequence encoding VEGF mRNA. However, its inhibitory effect on the biological system does not necessarily prove a mechanism of antisense action. In fact, recent analyzes indicate that many oligonucleotides can interfere in a non-specific manner with the cellular metabolisome, especially concentrations above 1 μM (reviewed by Stein and Cheng, 1993). Proof of antisense mechanism is disappointingly difficult, and has not really been shown except by circumstantial evidence. Our current experiment, although it is indirect, has been designed to obtain evidence of probable antisense mechanism. Briefly, we have synthesized "isotype-specific" antisense oligonucleotides that will be employed in a rat cell-based assay to selectively inhibit the expression of a single VEGF variant. Rat C6 cells (origin of glioma) produce three main types of VEGF isotypes. RNAse protection assays have shown that approximately 45% of VEGF in C6 cell line is variant of 120 amino acids, 45% is variant to 165aa, and the rest is variant 188aa (Bacic et al., 1995). Cells will be treated with antisense oligonucleotide specific for one or more isotypes, and then mRNA transcription will be stimulated by hypoxia or by the addition of 200 μM cobalt chlorine, which mimics hypoxia. After 9 hours of exposure, cellular levels of VEGF mRNA will be verified by RNAse protection assay (Ambion, Austin, TX). Very important, isotype-specific probes (length of approximately 100 to 200 bases) that we have already generated by RT-PCR techniques will be used to quantify the level of each gf mRNA species and levels will be normalized with respect to beta-actin. If the antisense mechanism is operative, and single-type specific oligonucleotide is employed, only the expression of VEGF will be reduced relative to others. On the other hand, an oligonucleotide complementary to a common reaction of all VEGFs will reduce the expression of all VEGF. Synthesis of ferrocene-conjugated oligonucleotides: After the first round of classification, the most active oligonucleotide with the least side effects will be connected through the 3-terminus to a ferrocene end, in the hope of further increasing the absorption and specific activity of the oligonucleotide ( see the preliminary results; T30781 (+ ferrocene) vs. ' T30639 (not modified)). If effective, the activity of this oligonucleotide will be compared to that of a random sequence control containing the same endpoint. It is assumed that the ferrocene portion can allow the oligonucleotide to exploit the active transport or permeation system (iron?) Of the cell, but the mechanism has not yet been studied. Use of absorption enhancers: In most cases, to facilitate cell entry, oligonucleotides will be administered to cells in the presence of cationic lipid reagents. Revealed as transfection agents to deliver the gene, many cationic lipids are now commercially available, but only Cellfectin (Life Technologies) is found to be consistently effective in our assay (of 7 tested major lipids). Cellfectin is a liposomal mixture 1:15 (weight / weight) of polyamnolipid tetrapalmityl-spermidine and the phospholipid dioleyl phosphotidyl etalonamine (DOPE). Another lipid we are working on is DC-Chol, developed by Leaf Huang (Gao and Huang, 1991), and approved for clinical trials for gene therapy (Rgene Therapeutics, The Woodlands, TX). In addition, we have designed and synthesized (Guy-Caffey et al., 1995) a series of novel polyamidolipid absorption enhancers, which markedly increase the cellular uptake of oligonucleotides, even in the presence of serum and without significant associated toxicity. The most effective of these is SpdC (spermidine-cholesterol). When i.v. in mice, SpdC improves the oligonucleotide supply to many tissues and no toxicity is observed when SpdC is injected into mice at concentrations as high as 100 / mg / kg / day for one week (data not shown). We have prepared liposomal preparations of SpdC / DOPE and DC-chol / DOPE (SpdC or DC-chol with dioleyl phosphotidylcholine) in mass proportions in a range of 1: 0.5, 1: 1, 1: 1.5, and 1: 2. The proportion that works most effectively in our previous anti-VEGF assays in NHEK cells was 1: 1, for both cationic lipids. We propose to investigate the usefulness of these preparations to improve the activity of anti-VEGF oligonucleotides in the C6 cell line, in C6 spheroids and eventually in the eye model. The exact mechanism by which these absorption methods work is still controversial. Mixing the cationic lipid with the nucleic acid, almost always produces "micro-precipitates that enter the cells efficiently." To maintain consistency, and probably to discover an underlying principle, we will check the particle size when using a Coulter particle sizing apparatus. Formulations for in vivo injection .- We have used the thermal formulation loosely in this proposal to convey the message of using a multicomponent mixture to deliver drugs, however, as we approach the in vivo testing stage, it would be prudent to design optimal formulation for injection in the eye, especially if absorption enhancers are involved Other parameters to consider would be the amount of antisense and concentration, salinity, particle size, pH and vehicle Dr. Joe Wyse will help us in choosing and preparing the optimal formulation. Cytotoxicity assays: Cells will be planted at a time 500 cells / well in a 96 well plate. One day after cover, the cells will be exposed to oligonucleotide formulations diluted in series (4 wells per dilution). After a day or four days of exposure, the cell viability effect will be verified using a non-radioactive assay system (Cell Titer 96 Aqueous Cell Proliferation Assay from Promega Corp.). For the most potent oligonucleotide, this assay will be performed on three separate cell lines (including C6, NHEK, and fibroblast cell line). Evaluation of antisense activity in spheroids: C6 cells, normally developed in monolayers (4.5 g glucose / 1, DMEM, 5% FCS plus antibiotics) can be induced to grow in spheroids or cell aggregates of approximately 0.4 to 0.8 mm. It would be informative to know if our antisense oligonucleotides formulated with lipids or otherwise can pass through the cell layers of a spheroid and still have biological activity. To prepare spheroids, the method described by Stein et al. (1995) will be used. C6 cells will be transferred from confluent cultures to non-adherent bacteriological dishes, and will develop for 48 hours. Emerging spheroblasts will be transferred to centrifuge flasks, grown for an additional 10 days (80 rpm), and the spheroids will be sized uniformly by sedimentation through a 10 ml pipette. Growth will continue for an additional six weeks, with a change of medium each day sautéed. The flask will be washed by drag each day with 95% air + 5% C02 to ensure adequate oxygenation and pH. The spheroids will be treated with antisense formulations or appropriate controls, exposed to hypoxia to induce VEGF synthesis for up to 1 day, and then the level of VEGF mRNA in spheroidal sections, will be examined by in situ hybridization. For this, the spheroids will be fixed with 4% paraformaldehyde, freeze, sectioned in 10 μm thickness, and processed for in situ hybridization with RNA or DNA probes tagged with 35S for VEGF generated as previously described. The processed section will be subjected to contrast dye with hematoxylin and stained or dyed cosine. After several days of autoradiography (Guy-Caffey et al.) The plates will be examined (photographed) by dark field illumination and bright field. The VEGF RNA distribution indicates the degree of inhibition achieved by the antisense oligonucleotide. Ideally, all layers will show low level of VEGF mRNA. More likely, the surface layers will have less VEGF, either because the drug does not penetrate the cell layers or because the cells were more hypoxic to the center and produced more VEGF. If the supply is only in the surface layers, we will try to design new supply approaches. Evaluation of the anti-angiogenic activity of antisense oligonucleotides VEGF in vivo. The adult rodent model and iris neovascularization associated with VEGF: Adult rats in a hypoxic atmosphere, stimulate new growth of blood vessels in the iris. Neovascularization correlates in time with the upregulation of VEGF mRNA levels in the retina. The sequence of ocular events reproduces closely those seen in the simian model of rubeosis iridis and human iris neovascularization, where ischemic retinal VEGF is known that is causal in the development of iris neovascularization. It is our intention to use this model to test the activity of antisense compounds that can reduce angiogenesis. The animal experimentation, which is carried out in the Adamis laboratory, involves animal handling and surgery, photography, computer quantification and Northern analysis of VEGF mRNA. Sprague-Dawley rats (albines) of colony Kingston, 350 to 400 adult grams (female), are placed in hypoxic chambers (1% oxygen) for 1-21 days. Biomicroscopic examination and camera photography with standardized slit are done before and after the incubation period. Signs of tortuosity and dilatation of progressive iris vessels as well as increased development of vascular density in the hypoxic atmosphere. The iris vessels are clearly visible in albino animals. Standardized photographs of the iris are scanned or digitized in a computer program (NIH image, 1.54D software) and the area of neovascularization is quantified in a masked form through pixel analysis. The vascularity of the iris shows a progressive increase until day 14. Immunostaining of Factor VIII and proliferation of cellular nuclear antigen (PCNA) has confirmed endothelial cell proliferation starting on day 2; which shows that the increased vascularity represents angiogenesis. Isolated retinas are prepared for staining or Northern staining, show that hypoxic animals increase levels of stable state retinal VEGF mRNA. In summary, adult rats in a hypoxic atmosphere stimulate new vessel growth in the iris. It is our intention to use this model to test the effect of candidate anti-VEGF formulations. The experiment to determine the effect of interrupted hypoxia in neovascularization: The previous rat model has only been used for continuous exposure to hypoxia. Because the model can prove to be more useful if hypoxia can be interrupted, for example for repeated drug administration, we would like to characterize the degree of iris neovascularization for animals withdrawn from hypoxia for short periods of time on a daily basis. We speculate that the removal will synergize neovascularization since neonatal rats treated in this way have a greater neovascular response compared to animals with constant oxygen (Reynaud and Dorey, 1994). Hypoxic reoxygenation of retinal produces reactive oxygen intermediates that are known to increase in retinal VEGF mRNA protein (Kuroki et al., 1995). Rats will be placed in hypoxic chambers (10% oxygen) for 1, 3, 5, 7, 14 and 21 days (n = 3 per point! In time, 18 total). They will retire for one hour per day and expose to normal ambient oxygen (21%). At the end of the incubation period, standard iris photographs will be taken. The animals will be sacrificed and the anterior half of the eye is prepared by PCNA staining and light microscopy. The retinas will be isolated and prepared for Northern staining with a labeled randomized primer-labeled VEGF 558 bp cDNA 32. The upregulation of retinal VEGF mRNA will be correlated with immunohistochemical photographic documentation for iris neovascularization over the 21-day time period. The area of vascularity will be quantified from standardized photographs and compared with animals placed in uninterrupted hypoxia. From this experiment, we will be able to estimate the maximum number of times that the animal can take from the hypoxia chamber and dose without compromising the epoxic effect. Evaluation of the antisense effect in vivo. On day 0, baseline photographs will be taken. An eye of an anesthetized animal will be randomized to receive a simple intravitreal injection of the VEGF antisense compound. The other eye will be left untreated. Doses will be given for in vitro studies but they will be administered in 20 μl or less. The animals will be placed in hypoxia without interruption for 7 days. In the first test, the animals will be transferred by formulation (4 formulations: oligo alone, oligo + ends, and each with absorption enhancer). The objective will be to see if these compounds have any acute effect. If there is marked reduction in vascularization, a large number of animals will be used.

The table below summarizes the statistical rationale for the amount that may be required. In total, it is expected that approximately 20 rats will be used per treatment. Table 1. Number of rats required: Ninety percent of the rats in this model develop neovascularization. Considering a level of significance of 0.05 (alpha = 0.05) and a power of 0.8 (l-beta = 0.80), the number of eyes per treatment group can be determined for the varying effectiveness of an angiogenesis-blocking agent.

Group Number of Treatment Control Groups Treatment Required 90 40 38 90 50 24 90 60 17 90 70 12 Inhibition of neovascularization for these experiments will be defined as a 20% decrease in the area of vascularization in eyes treated against of control . If the considered effectiveness of a particular agent is high, the percentage of eyes that develop neovascularization of the iris that is in this treatment group will be low, and the number of eyes required for statistical significance will decrease dramatically. On day seven, the animals will be photographed and sacrificed. The retinas will be reolected and prepared for Northern spotting (individually). Stable-state mRNA VEGF will be quantified after normalization to the 285 ribosomal RNA signal, using a Phosphorimager (Molecular Dynamics). The vascularity of the iris will be quantified and compared between treated and control eyes. F. Procedure for handling vertebrate animals: All procedures with animals will be carried out at Children's Hospital, Harvard Medical School, followed by the lines established by the Resolution of the Association for Research in Vision and Ophthalmology on the use of animals for research and guidelines on the use of animals for research and guidelines established by the Massachusetts Eye and Ear Infirmary Animal Care Commitee (Committee for Animal Care of Eyes and Eyes Nursing of Massachusetts). In total, 120 Sprague Dawley rats of Kingston female albino clones will be used. They will be anesthetized and injected intravitrally with 20 μl of oligonucleotide or control formulation, using a 30 gauge needle. Gentaminicin Sulfate will be applied following the injection. The animals will be kept for up to 30 weeks in a 10% oxygen atmosphere. Following the termination of the treatment, they will be sacrificed by inhalation of C02. All the experiments are consistent with the OPRR guides. I. Literature Cited Adamis, A.P. et al., 1994, Arch. Ophthalmol. 118: 445-450 Adamis, A.P. et al., 1993, Biochem. Biphys. Res. Comm., 193: 631-638. Adamis, A.P. et al., 1996, Arch. Ophthal. 114: 66-71 Bacic, et al., 1995, Pharm. News 2: V, (abst) Bishop, et al., 1996, 3. Biol. Chem. 271: 5698-5703. Conn., (3, et al., 1990, Proc. Nati, Acad. Sci. USA 87: 2628-2632.

DvAmore, P.A., 1994, Investigative Ophthal. Vis. Sci. : 3974-3979. de Yries, C, et al., 1992, Science, 255: 989-991. Fenster, S.D. et al., 1994, Endocrine reviews, 13: 18-31. Ferrera, N., et al., 1992, Endocrine Reviews 13: 18-31 Guy-Caffey et al., 1995, J. Biol. Chem. 270: 31391-31396 Gao, X., and Huang, L. 1991, Biochem. Biophys. Res. Comm. 179: 280-285 Miller, J. et al., Am. J. Pathol. 145: 574-584. Milligan, J.F., et al., J. Med. Chem., 36. 1923-1937. Píate, K.H. et al., 1992, Nature 359: 845-848. Shima, D.T. et al., 1995, Molecular Med. 2: 64-75 Shweiki et al., 1992, Nature 362: 841-844. Stein, C.A. and Cheng, Y.C., 1993, Science 261: 1004-1012. Ter an et al., 1992, Biochem. Biophys. Res. Comm. 187: 1579-1586. Uhlmann, F., and Peyman, A., 1990, Chem. Rev. 90: 543 Wagner, R.W. et al., 1993, 260: 1510-1513. Wagner, R.W., 1994, Nature 372: 333-335. REFERENCES CITED The following references in the proportion that offer procedural details to those established here, are specifically incorporated herein by reference. Ballaun, C, Weninger, W., Uthman, A., Weich, H., Tschachler, E. (1995) J. Invest Ital. Dermatol. 104, 7-10.

Behr, J. (1994) Ital, Bioconjugate Chem. 5,382-389 Bishop, J. S., Guy-Caffey, J.K. , Ojwang, J.O., Smith, S.R., Hogan, M.E., Cossu, P.A., Rando, R.F., Chaudhary, N. (1996) J. Biol. Chem 271, 5698-5703 Carmeliet, P., Ferreriera, V., Breier, G., Pollefeyt, S., Kieckens, L., Gertsenstein, M., Fahrig, M., Vandenhoeck, A., Harpal, K., Eberhardt, C, Declercq, C, Pawling J., Moons, L., Collen, D., Risau, W., Nagy, A, (1996) Nature 380, 435-439 Chaudhary, N., Bishop, JS, Jayaraman, K., Guy-Caffey, JK, in Delivery Strategies For An isense Oligonucleotide Therapeutics, S. Akhtar, Ed (CRC Press, Boca Raton, 1995) pp. 39-60. Connolly, D. T., Plander, J.V. (1989) J. Biol. Chem. 264, 20017-20024 D'Amore, P.A. (1994) Invest. Ophthalmol. Vis. Sci. 35, 3974-3978 de Vries, C, Escobedo, J.A. Ueno, H., Houck, K., Ferrera, N., Williams, L.T. (1992) Science 255, 989-991 Dvorak, H.F., Brown, L.F., Detmar, M., Dvorak, A.M. (1995a) Am. J. Pathol. 146, 1029-1039 Dvorak, H.F., Detmar, M., Claffey, K.P., Nagy, J.A., van der Water, L., Senger, D.R. (1995b) Int. Arch. Allergy Immunol. 107, 233-235 Fenster, S.D., Wagner, R.W., Froehler, B.C., Chin, D.J. (1994) Biochemistry 33, 8391-8398 Ferrera, N., Houck, K., Jakeman, L., Leung, D.W. (1992) Endocr, Rev. 13, 18-32 Ferrera, N., Carver-Moore, K., Chen, H., Dowd, M., Lu, L., O'Shea, K. S., Powell-Braxton, L., Hillan, K.J., Moore, M.W. (1996) Mature 380, 439-442 Fisher, T.L., Terhorst, T., Cao, X., Wagner, R.W. (1993) Nuc. Acids Res. 21.3857 Folkman, J. (1995) Nat. Med. 1, 27-31 Frank, S., Hubner, G., Breier, G., Longaker, M. T., Greenhalgh, D.G., Werner, S. (1995) J. Biol. Chem. 270, 12607-12613 Gao, X., Huang, L. (1991) Biochem. Biophys. Res. Commun. 179, 280-285 Gao, X., Huang, L. (1991) Biochem, Biophys. Res. Commun. 179, 280-285 Guy-Caffey, J.K., Bodepudi, V., Bishop, J.S., Jayraman, K., Chaudhary, N. (1995) J. Biol. Chem. 270, 31391-31396 Keck, P.J., Hauser, S.D., Krivi, G., Sanzo, K., Warren, T., Feder, J., Connolly D.T. (1989) Science 246, 1309-1312 Ledley, F. (1994) Curr. Opin. Biotechol 5, 626-636 Leung, D. W., Chachianes, G., Kuang, W.J., Goedddel, D.V., Ferrera, N. (1989) Science 246, 1306-1309 Lewis, JG, Lin, K, Y., Kothavale, A., Flanagan, WM, Matteucci, MD, DePrince, RB, Mook, J., RA, Hendren, RW, Wagner, RW (1996) Proc. Nati Acad. Sci. USA 93, 3176-3181 Milligan, JF, Matteucci, MD, Martin, JC (1993) J. Med. Chem 36, 1923-1937 Nomura, M., Ya agishi, S., Harada, S., Hayashi, Y., Yamashima, T., Yamashita, J., Yamamoto, H. (1995) J. Biol. Chem. 270, 28316-28324 Puglisi, J, D., Tinoco, J., I. (1989) Methods Enzymol . 180, 304-325 Robinson, G.S., Pierce, E.A., Rook, S.L., Foley, E., Webb, R., Smith, L.E.H. (1996) Proc. Nati Acad. Sci. USA 93, 4851-4856 Stein. C.A., Cheng, Y.-C (1993) Science 261, 1004-1012 Stein, C.A., Kreig, A.M. (1994) Antisense Res. Dev. 4.67-69 Ter an, B.I., Dougher-Vermazen, M., Carrion, M.E., Dimitrov, D., Armellino, D.C., Gospodarowicz, D., Bohlen, P. (1992) Biochem Biophys. beef. Commun. 187, 1579-1586 Thomas, K.A. (1996) J. Biol. Chem. 271, 603-606 Tischer, E., Mitchell, R., Hartman, T., Silva, M., Gospodarowicz, D., Fiddes, J.C., Abraham, J.A. (1991) J. Biol. Chem 266, 11947-11954 Uhlmann, E., Peyman, A. (1990) Chemical Reviews 90, 543-584 Wagner, RW, Matteucci, MD, Lewis, JG, Gutierrez, AJ, Molds, C. , Froehler, B.C. (1993) Science 260, 1510-1513 Wagner, R.W. (1994) Nature 372, 333-335 Winternitz, C.I., Jackson, J.K., Oktaba, A.M., Burt, H.M. (1996) Pharm. Res. 13, 368-375 Woolf, T.M. Melton, D.A., Jennings, C.G.B. (1992) Proc. Nati Acad. Sci. USA 89, 7305-7309 LIST OF SEQUENCE (1) GENERAL INFORMATION: (i) APPLICANT: Chaudhary, Nilabh Rao, T. Sudhakar Revankar, Ganapathi R. Cossium, Paul A. Rando, Robert F. Peyman, Anusch Uhlmann, Eugen (ii) TITLE OF THE INVENTION: ANTISENTIAL INHIBITORS EXPRESSION OF VASCULAR ENDOTHELIAL GROWTH EXPRESSION (VEgF / VPF) (iii) SEQUENCE NUMBER: 21 (iv) ADDRESS FOR CORRESPONDENCE: (A) RECIPIENT: Conley Rose & Taylor, P.C. (B) STREET: 600 Travis, Suite 1850 (C) CITY: Houston (D) STATE: texas (E) COUNTRY: E.U.A. (F) CP: 77002-2912 (v) COMPUTER LEGIBLE FORM: (A) TYPE OF MEDIA: Flexible disk (B) COMPUTER: PC COMPATIBLE WITH IBM (C) OPERATING SYSTEM: Wind? WS95 (D) PROGRAM (SOFTWARE) : Microsoftword 7.0a (vi) CURRENT REQUEST DATA: (A) APPLICATION NUMBER: (B) SUBMISSION DATE: (C) CLASSIFICATION: (vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: (B) DATE OF PRESENTATION: (viii) INFORMATION OF AGENT / ATTORNEY: (A) NAME: McDaniel, C. Steven (B) REGISTRATION NUMBER: 33,962 (C) REFERENCE / NUMBER OF FILE: 1472-07200 (ix) TELECOMMUNICATIONS INFORMATION: (A) TELEPHONE: 713 / 38-8010 (B) FAX: 713 / 238-8008 (2) INFORMATION FOR NO. SEC ID: 1 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs. (B) TYPE: synthetic nucleic acid (C) HEBRA: Simple (D) TOPOLOGY: Linear (E) ANTI-SENSE: YES (iv) ANTI-SENSE: YES (ix) CHARACTERISTICS: (A) NAME / KEY: misc_feature ( B) LOCATION: 1-19 (D) OTHER INFORMATION: / note = "phosphorothioate link between each residue" (xi) SEQUENCE DESCRIPTION: NO. SEC ID : 1 GCGCTGATAG ACATCCATG 19 (2) INFORMATION FOR NO. SEC ID: 2 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs. (B) TYPE: synthetic nucleic acid (C) HEBRA: Simple (D) TOPOLOGY: Linear (iv) ANTI-SENSE: YES (ix) CHARACTERISTICS: (A) NAME / KEY: misc_feature (B) LOCATION: 1-19 ( D) OTHER INFORMATION: / note = "phosphorothioate link between each residue C5 -propinyl pyrimidines" (xi) SEQUENCE DESCRIPTION: NO. SECTION ID: 2 GCGCUGAUAG ACAUCCAUG 19 (2) INFORMATION FOR NO. SEC ID: 3 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs. (B) TYPE: synthetic nucleic acid (C) HEBRA: Simple (D) TOPOLOGY: Linear (iv) ANTI-SENSE: YES (ix) CHARACTERISTICS: (A) NAME / KEY: misc_feature (B) LOCATION: 1-19 ( D) OTHER INFORMATION: / note = "phosphorothioate link between each residue C-5 propynyl pyrimidines" (xi) SEQUENCE DESCRIPTION: NO. SECTION ID: 3 CGAUUGGAUG GCAGUAGCCT 19 (2) INFORMATION FOR NO. SEC ID: 4 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs. (B) TYPE: synthetic nucleic acid (C) HEBRA: Simple (D) TOPOLOGY: Linear (iv) ANTI-SENSE: YES (ix) CHARACTERISTICS: (A) NAME / KEY: misc_feature (B) LOCATION: 1-19 ( D) OTHER INFORMATION: / note = "phosphorothioate link between each residue C5-propynylpyrimidines" (xi) SEQUENCE DESCRIPTION: NO. SEC ID: 4 (2) INFORMATION FOR NO. SEC ID: 4 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs. (B) TYPE: synthetic nucleic acid (C) HEBRA: Simple (D) TOPOLOGY: Linear (iv) ANTI-SENSE: YES (ix) CHARACTERISTICS: (A) NAME / KEY: misc_feature (B) LOCATION: 1-19 ( D) OTHER INFORMATION: / note =, "phosphorothioate link between each residue C5-propynylpyrimidines" (xi) SEQUENCE DESCRIPTION: NO. SEC ID: 4 UACUCCUGGA AGAUGUCCA 19 (2) INFORMATION FOR NO. SEC ID: 5 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs. (B) TYPE: synthetic nucleic acid (C) HEBRA: Simple (D) TOPOLOGY: Linear (iv) ANTI-SENSE: YES (ix) CHARACTERISTICS: (A) NAME / KEY: misc_feature (B) LOCATION: 6-7; 9-10; 10-11; 13-14 (D) OTHER INFORMATION: / note = "phosphorothioate link between the indicated residues" (xi) SEQUENCE DESCRIPTION: NO. SEC ID : 5 GCGCUGAUAG ACAUCCAUG 19 (2) INFORMATION FOR NO. SEC ID: 6 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs. (B) TYPE: synthetic nucleic acid (C) HEBRA: Simple (D) TOPOLOGY: Linear (iv) ANTI-SENSE: YES (ix) CHARACTERISTICS: (A) NAME / KEY: misc_feature (B) LOCATION: 3-4; 5-6; 6-7; 9-10; 10-11; 13-14 (D) OTHER INFORMATION: / note = "phosphorothioate link between the indicated residues" (xi) SEQUENCE DESCRIPTION: NO. SECTION ID: 6 GCGCUGAUAG ACAUCCAUUG 19 (2) INFORMATION FOR NO. SEC ID: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs. (B) TYPE: synthetic nucleic acid (C) HEBRA: Simple (D) TOPOLOGY: Linear (iv) ANTI-SENSE: YES (ix) CHARACTERISTICS: (A) NAME / KEY: misc_feature (B) LOCATION: 6-7; 10-10 (C) OTHER INFORMATION: / note = "phosphorothioate link between the indicated residues" (xi) SEQUENCE DESCRIPTION: NO. SEC ID : 7 GCGCUGAUAG ACAUCCAUG 19 (2) INFORMATION FOR NO. SEC ID: 8 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs. (B) TYPE: synthetic nucleic acid (C) HEBRA: Simple (D) TOPOLOGY: Linear (iv) ANTI-SENSE: YES (ix) CHARACTERISTICS: (A) NAME / KEY: misc_feature (B) LOCATION: 1-19 ( C) OTHER INFORMATION: / note = "phosphorothioate link between all residues, C5 -propynyl pyrimidines" (xi) SEQUENCE DESCRIPTION: NO. SEC ID: 8 GAAGAUGUCC ACCAGGGUC 19 (2) INFORMATION FOR NO. SEC ID: 9 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs. (B) TYPE: synthetic nucleic acid (C) HEBRA: Simple (D) TOPOLOGY: Linear (iv) ANTI-SENSE: YES (ix) CHARACTERISTICS: (A) NAME / KEY: misc_feature (B) LOCATION: 1-19 ( D) OTHER INFORMATION: / note = "phosphorothioate link between the indicated residues, C5 -propynyl pyrimidines" (xi) SEQUENCE DESCRIPTION: NO. SEC ID: 9 AGGAAGCUCA UCUCUCVCUA 19 (2) INFORMATION FOR NO. SEC ID: 10 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs. (B) TYPE: synthetic nucleic acid (C) HEBRA: Simple (D) TOPOLOGY: Linear (iv) ANTI-SENSE: YES (ix) CHARACTERISTICS: (A) NAME / KEY: misc_feature (B) LOCATION: 1-19 ( D) OTHER INFORMATION: / note = "phosphorothioate C5 -propinyl pyrimidines" (xi) SEQUENCE DESCRIPTION: NO. SEC ID: 10 UACACGUCUG CGGAUCUUG 19 (2) INFORMATION FOR NO. SEC ID: 11 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs. (B) TYPE: synthetic nucleic acid (C) HEBRA: Simple (D) TOPOLOGY: Linear (iv) ANTI-SENSE: YES (ix) CHARACTERISTICS: (A) NAME / KEY: misc_feature (B) LOCATION: 1-19 (D) OTHER INFORMATION: / note = "C5-propynyl pyrimidine phosphorothioate linkages" (xi) DESCRIPTION OF SEQUENCE: NO. SECTION ID: 11 UAACUCAAGC UGCCUCGCC 19 (2) INFORMATION FOR NO. SEC ID : 12 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs. (B) TYPE: synthetic nucleic acid (C) HEBRA: Simple (D) TOPOLOGY: Linear (iv) ANTI-SENSE: YES (ix) CHARACTERISTICS: (A) NAME / KEY: misc_feature (B) LOCATION: 1-19 ( D) OTHER INFORMATION: / note = "phosphorothioate bonds; C5-propynyl pyrimidines "(xi) SEQUENCE DESCRIPTION: SEC ID NO: 12 CCAUGAACUU CACCACUUC 19 (2) INFORMATION FOR SECTION ID: 13 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: synthetic nucleic acid (C) HEBRA: Simple (D) TOPOLOGY: Linear (iv) AN I-SENSE: YES (ix) FEATURE: (A) NAME / KEY: misc_feature (B) LOCATION: 1-19 (D) OTHER INFORMATION: / note = "phosphorothioate linkages, C5 -propynyl pyrimidines" (xi) SEQUENCE DESCRIPTION: SECTION ID NO .: 13 GACAUCCAUG AACUUCACC 19 (2) INFORMATION FOR ID NO. SEC.: 14 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: synthetic nucleic acid (C) HEBRA: Simple (D) TOPOLOGY: Linear (iv) ANTI-SENSE: YES (ix) ) CHARACTERISTICS: (A) NAME / KEY: misc_feature (B) LOCATION: 1-19 (D) OTHER INFORMATION: / note = "phosphorothioate bonds, C5 -propinyl pyrimidines" (xi) SEQUENCE DESCRIPTION: SEC ID NO. .: 14 GGCUGGC AGU AGCUGCGCU 19 (2) INFORMATION FOR NO. SEC ID : 15 (i) SEQUENCE CHARACTERISTICS:, (A) LENGTH: 19 base pairs. (B) TYPE: synthetic nucleic acid (C) HEBRA: Simple (D) TOPOLOGY: Linear (iv) ANTI-SENSE: YES (ix) CHARACTERISTICS: (A) NAME / KEY: misc_feature (B) LOCATION: 1-19 ( D) OTHER INFORMATION: / note = "phosphorothioate bonds, C5 -propinyl pyrimidines" (xi) SEQUENCE DESCRIPTION: NO. SEC ID: 15 GGAUGGCAGU AGCUGCGCU 19 (2) INFORMATION FOR NO. SEC ID: 16 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs. (B) TYPE: synthetic nucleic acid (C) HEBRA: Simple (D) TOPOLOGY: Linear (iv) AN I-SENSE: YES (ix) CHARACTERISTICS: (A) NAME / KEY: mise feature (B) LOCATION: 1- 19 (D) OTHER INFORMATION: / note = "phosphorothioate bonds, C5 -propinyl C residues" (xi) SEQUENCE DESCRIPTION: NO. SEC ID : 16: GCGCTGATAG ACATCCATG 19 (2) INFORMATION FOR NO. SEC ID: 17 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs. (B) TYPE: synthetic nucleic acid (C) HEBRA: Simple (D) TOPOLOGY: Linear (iv) ANTI-SENSE: YES (ix) CHARACTERISTICS: (A) NAME / KEY: misc_feature (B) LOCATION: 1-19 ( D) OTHER INFORMATION: / note = "phosphorothioate bonds, C5 -propinyl U residues" (xi) SEQUENCE DESCRIPTION: NO. SEC ID: 17: GCGCUGAUAG ACAUCCAUG 19 (2) INFORMATION FOR NO. SEC ID : 18: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs. (B) TYPE: synthetic nucleic acid (C) HEBRA: Simple (D) TOPOLOGY: Linear (iv) ANTI-SENSE: YES (ix) CHARACTERISTICS: (A) NAME / KEY: misc_feature (B) LOCATION: 1-19 ( D) OTHER INFORMATION: / note = "phosphorothioate bonds, C5 propinyl pyrimidines in residues 8, 12, 14 and 15" (xi) SEQUENCE DESCRIPTION: NO. SEC ID: 18: GCGCTGAUAG ACAUCCATG 19 (2) INFORMATION FOR NO. SEC ID : 19 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs. (B) TYPE: synthetic nucleic acid (C) HEBRA: Simple (D) TOPOLOGY: Linear (iv) ANTI-SENSE: YES (ix) CHARACTERISTICS: (A) NAME / KEY: misc_feature (B) LOCATION: 1-19 ( D) OTHER INFORMATION: / note = "phosphorothioate bonds, C5 propinyl pyrimidines in residues 2.5 8, 12, 15 and 18" (xi) SEQUENCE DESCRIPTION: NO. SEC ID : 19: GCGCUGAUAG ACATCCAUG 19 (2) INFORMATION FOR NO. SEC ID : 20 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs. (B) TYPE: synthetic nucleic acid (C) HEBRA: Simple (D) TOPOLOGY: Linear (iv) ANTI-SENSE: YES (ix) CHARACTERISTICS: (A) NAME / KEY: misc_feature (B) LOCATION: 1-19 ( D) OTHER INFORMATION: / note = "phosphorothioate bonds, C5 extreme end-bound propynyl pyrimidines (lipid tether") (xi) SEQUENCE DESCRIPTION: SECTION ID NO: 20: GCGCUGAUAG ACAUCCAUG 19 (2) INFORMATION FOR NO SEC ID: 21 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: synthetic nucleic acid (C) HEBRA: Simple (D) TOPOLOGY: Linear (iv) ANTI-SENSE: YES (ix) CHARACTERISTICS: (A) NAME / KEY: misc_feature (B) LOCATION: 1-19 (D) OTHER INFORMATION: / note = "phosphorotioat links between residues 1-5, 8-9, 12-13, 14- 15, and 16-19; 3'-terminates pyrene; C5-propynyl pyrimidines "(xi) SEQUENCE DESCRIPTION: SECTION ID NO .: 21: GCGCUGAUAG ACAUCCAUG 19 (2) SECTION ID NO. : 22 (i) SEQUENCE CHARACTERISTICS: (A) LON GITUD: 19 base pairs. (B) TYPE: synthetic nucleic acid (C) HEBRA: Simple (D) TOPOLOGY: Linear (iv) ANTI-SENSE: YES (ix) CHARACTERISTICS: (A) NAME / KEY: misc_feature (B) LOCATION: 1-19 ( D) OTHER INFORMATION: / note = "phosphorothioate bonds, C5 hexinyl pyrimidines" (xi) SEQUENCE DESCRIPTION: NO. SEC ID: 22: GCGCUGAUAG ACAUCCAUG 19 (2) INFORMATION FOR NO. SEC ID: 23 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs. (B) TYPE: synthetic nucleic acid (C) HEBRA: Simple (D) TOPOLOGY: Linear (iv) ANTI-SENSE: YES (ix) CHARACTERISTICS: (A) NAME / KEY: misc_feature (B) LOCATION: 1-19 ( D) OTHER INFORMATION: / note = "phosphorothioate bonds, C5-propynyl pyrimidines" (xi) SEQUENCE DESCRIPTION: NO. SEC ID : 23: GCGCUGACAG ACAUUCAUG 19 (2) INFORMATION FOR NO. SEC ID: 24 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs. (B) TYPE: synthetic nucleic acid (C) HEBRA: Simple (D) TOPOLOGY: Linear (iv) ANTI-SENSE: NO (xi) DESCRIPTION OF SEQUENCE: NO. SEC ID : 24: CATGGATGTC TATCAGCGC 19 (2) INFORMATION FOR NO. SEC ID: 25: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs. (B) TYPE: synthetic nucleic acid (C) HEBRA: Simple (D) TOPOLOGY: Linear (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: NO. SEC ID: 25: CATGGATGTC TATCAGCGC 19 (2) INFORMATION FOR NO. SEC ID: 26: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs. (B) TYPE: synthetic nucleic acid (C) HEBRA: Simple (D) TOPOLOGY: Linear (iv) ANTI-SENSE: YES (ix) CHARACTERISTICS: (A) NAME / KEY: misc_feature (B) LOCATION: 1-19 ( D) OTHER INFORMATION: / note = "phosphorothioate bonds, C5-propynyl pyrimidines residues C "(xi) SEQUENCE DESCRIPTION: SEC ID NO: 26: AGAGCAGCAA GGCGAGGCT 19 (2) INFORMATION FOR SECTION ID: 27: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: synthetic nucleic acid (C) HEBRA: Simple (D) TOPOLOGY: Linear (iv) ANTI-SENSE: YES (ix) FEATURE: (A) NAME / KEY: misc_feature (B) LOCATION: 1-19 (D) OTHER INFORMATION: / note = "phosphorothioate linkages, C5-propynyl pyrimidines residue C" (xi) SEQUENCE DESCRIPTION: SECTION ID NO: 27: CAUGGAUGUC UAUCAGCGC 19

Claims (48)

1. CLAIMS 1.- Antisense oligonucleotide that reduces production of cellular VEGF, in cells treated with the antisense oligonucleotide at concentrations below about micromolar, - the treated cells do not produce more than about 90% of the VEGF produced by cells if treated.
2. 2. The antisense oligonucleotide according to claim 1, characterized in that the antisense oligonucleotide binds to RNA sequences that are found in mRNA coding for VEGF.
3. 3. The antisense oligonucleotide according to claim 1, characterized in that the antisense oligonucleotide binds to an RNA sequence which are found in at least one of two of the mRNAs encoding VEGF.
4. 4. - The antisense oligonucleotide according to claim 1, characterized in that the antisense oligonucleotide binds to an RNA sequence found in VEGF 205 mRNA.
5. 5. - The antisense oligonucleotide according to claim 1, characterized by "that the antisense oligonucleotide binds to an RNA sequence found in VEGF 185 mRNA.
6. 6. The antisense oligonucleotide according to claim 1, characterized in that the antisense oligonucleotide binds to an RNA sequence found in VEGF 165 mRNA.
7. 7. The antisense oligonucleotide according to claim 1, characterized in that the antisense oligonucleotide binds to an RNA sequence found in VEGF 121 mRNA.
8. 8. - The antisense oligonucleotide according to claim 1, characterized in that it comprises a chemical portion that decreases the degradation rate of antisense oligonucleotide by nucleases.
9. 9. - The antisense oligonucleotide according to claim 1, characterized in that the oligonucleotide comprises a phosphorothioate group and a phosphodiester group.
10. 10. The antisense oligonucleotide according to claim 1, characterized in that it comprises a pair of adjacent residues connected through a chemical portion that resists degradation by nucleases.
11. 11. The antisense oligonucleotide according to claim 8, characterized in that it comprises the portion that decreases the rate of degradation of the antisense oligonucleotide by nucleases is a phosphorothioate group.
12. 12. - The antisense oligonucleotide according to claim 8, characterized in that each oligonucleotide residue is linked through a phosphorothioate group.
13. 13. - The antisense oligonucleotide according to claim 1, characterized in that the oligonucleotide comprises a nucleotide residue selected from the group consisting of C5-propynyl uridine, C5-propynyl cytidine, C5-hexinic uridine, C5 hexinyl cytidine, 6-aza uridine and 6-aza cytidine.
14. 14. The antisense oligonucleotide according to claim 1, characterized in that the oligonucleotide comprises a phosphorothioate group and a nucleotide residue selected from the group consisting of C5-propynyl uridine, C5 propynyl cytidine, C5-hexinyl uridine, C5 hexynyl cytidine, -az uridine and 6-aza cytidine.
15. 15. The antisense oligonucleotide according to claim 1, characterized in that it comprises a C5 residue propinyl uridine.
16. 16. The antisense oligonucleotide according to claim 1, characterized in that it comprises a C5 residue propinyl uridine and a phosphorothioate group.
17. 17. The antisense oligonucleotide according to claim 1, characterized in that it comprises a C5 residue propynyl cytidine.
18. 18. - The antisense oligonucleotide according to claim 1, characterized in that it comprises a C5 residue propynyl cytidine and a phosphorothioate group.
19. 19. The antisense oligonucleotide according to claim 1, characterized in that it comprises a C5 residue, hexinyl cytidine and a phosphorothioate group.
20. 20. The antisense oligonucleotide according to claim 1, characterized in that it comprises a C5 residue, hexinyl cytidine.
21. 21. The antisense oligonucleotide according to claim 1, characterized in that it comprises a C5 residue, hexinyl cytidine and a phosphorothioate group.
22. 22. The antisense oligonucleotide according to claim 1, characterized in that it comprises a residue 6 aza-deoxy uridine.
23. 23. The antisense oligonucleotide according to claim 1, characterized in that it comprises a residue 6 aza-deoxy uridine and a phosphorothioate group.
24. 24. The antisense oligonucleotide according to claim 1, characterized in that it comprises a residue 6 aza-deoxy uridine.
25. 25. - The antisense oligonucleotide according to claim 1, characterized in that it comprises a residue 6 aza-deoxy uridine and a phosphorothioate group.
26. 26. A composition characterized in that it comprises an antisense oligonucleotide that reduces the production of cellular VEG, in cells treated with the antisense bligonucleotide, concentrations lower than approximately 1 micromolar and the treated cells produce at most approximately 90% of the VEG produced by cells without treat, the composition further comprises a cell absorption enhancer.
27. 27. The composition according to claim 26, characterized in that the cellular absorption enhancer has a lipophilic portion.
28. 28. The composition according to claim 27, characterized in that the lipophilic portion comprises cholesterol.
29. 29. The composition according to claim 1, characterized in that the oligonucleotide also comprises an ionic bond with a cation to form a salt.
30. 30. The composition according to claim 29, characterized in that the cation is a cationic lipid.
31. 31. The composition according to claim 30, characterized in that the cationic lipid is a polyamino lipid.
32. 32. The composition according to claim 31, characterized in that the polyaminolipid e spermidine-cholesterol.
33. 33. - The composition according to claim 26, characterized in that the cellular absorption enhancer comprises a liposome.
34. 34. - The composition according to claim 33, characterized in that the liposome comprises Cellfectin ™.
35. 35.- The composition according to claim 33, characterized in that the liposome comprises spermidine-cholesterol in a mixture with DOPE.
36. 36.- An antisense oligonucleotide that binds VEG mRNA and comprises a phosphorothioate group and a nucleotide residue selected from the group consisting of C5-propynyl uridine, C5 propynyl cytidine, C5 -hexinyl uridine, C5 hexinyl cytidine, 6-azuridine and 6- aza cytidine, wherein the antisense oligonucleotide has a duplex melting temperature of at least about 5 ° C above the melting temperature of an identical antisense oligonucleotide lacking chemically modified pyrimidine residues; the antisense oligonucleotide residue reduces production of cellular VEGF in cells treated with and antisense oligonucleotide at concentrations lower than about 1 micro-molar, the treated cells yields no more than about 90% of the VEGF that is produced by cells if treated.
37. 37. A composition comprising an antisense oligonucleotide that binds VEGF mRNA, comprising a phosphorothioate group and a nucleotide residue selected from the group consisting of C5-propynyl uridine, C5-propynyl cytidine, C5-hexinic uridine, C5 hexinyl cytidine, -aza uridine and 6-aza cytidine, e wherein the antisense oligonucleotide has a duplex fusion temperature of at least about 5 ° C over the melting temperature of an identical antisense oligonucleotide, lacking chemically modified pyrimidine residues; the antisense oligonucleotide residue reduces production of cellular VEGF, and cells treated with the antisense oligonucleotide concentrations less than about 1 micro-molar, the treated cells produce no more than about 90% of the VEG that is produced by untreated cells; the composition also comprises a polymeric sustained release compound.
38. 38.- In an antisense oligonucleotide containing phosphorothioate bonds and ligated to mRNA encoding VEGF, a better characterized in that it includes a nucleotide residue in the antisense oligonucleotide selected from the group consisting of C5-propynyl uridine, C5-propynyl cytidine, C5-hexinyl uridine, C hexinyl cytidine, 6-aza uridine and 6-aza cytidine, wherein the improvement increases the antisense oligonucleotide duplex fusion temperature by at least about 5 ° C.
39. 39.- A method for reducing cellular VEGF production in a cell, which comprises contacting a cell with an antisense oligonucleotide according to claim 1, the cell producing at most approximately 90% of the VEGF that is produced by a non-contacting cell, the The oligonucleotide concentration is less than about 1 micro-molar.
40. 40.- An antisense oligonucleotide selected from the group consisting of sequence numbers ID 2 through 22 ..
41. 41.- A composition comprising an antisense oligonucleotide having a sequence ID No. 2.
42. 42. - A composition which comprises an antisense oligonucleotide having a sequence ID No. 3.
43. 43. - A composition comprising an antisense oligonucleotide having a sequence ID No. 4.
44. 44.- A composition comprising an antisense oligonucleotide having a sequence ID No. 6.
45. 45. - A composition comprising an antisense oligonucleotide having a sequence ID of No. 10.
46. 46.- A composition comprising an antisense oligonucleotide having a sequence ID of No. 20.
47. 47.- A composition which it comprises an antisense oligonucleotide having a sequence ID No. 21.
48. 48. A composition comprising an antisense oligonucleotide having a sequence ID No. 22.