CN1222192A - Antisense inhibitors of vascular endothelial growth factor (VEFG/VPF) expression - Google Patents

Abstract

The present invention relates to the inhibition of vascular endothelial growth factor expression with oligonucleotides. The oligonucleotides of the present invention are thought to bind to target nRNA in a sequence specific manner and prevent expression of the encoded gene. Chemical modifications of the oligonucleotides for increasing their stability and binding efficiency are disclosed. These modifications increase the stability and the efficiency of the oligonucleotides contemplated in this invention. Oligonucleotides 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

The Antisense Suppression thing of vascular endothelial growth factor expression

The cross reference document of related application

Ask right of priority according to a temporary transient common pending application, the sequence number 60/015,752/ of its application, April 17 1996 submission date in this

Statement about joint study or exploitation promoter

Inapplicable

Background of invention

Invention field

The present invention relates to utilize the cell of the vascular endothelial growth factor expression of oligonucleotide to suppress.Oligonucleotide of the present invention can combine in sequence-specific mode with target mRNA, and hinders the VEGF expression of gene of its coding.Disclose to improving the oligonucleotide chemically modified that stability and joint efficiency are done.This oligonucleotide composition can be used for exsomatizing and treats scavenger cell, perhaps is used for interior therapeutic by injection, suction or topical therapeutic or other route of administration.

The specification sheets of correlation technique

Vascular endothelial growth factor (VEGF) is also referred to as vascular permeability factor, comprises the secretion glycoprotein of gang's homologous duplex, and its molecular weight is between 34-46Kda.It can be by polytype cell response anoxic and specific regulatory factor and secretes.The present known phenogen that four kinds of VEGF are arranged.They are to be formed by different cut modes by the mRNA of same genes encoding.(people such as Keck, 1989; People such as Leung, 1989; Connolly and Plander, 1989; People such as Tischer, 1991)

VEGF for the formation of g and D process medium vessels and organized renewing be essential (Ferrera waits the people, 1996; People such as Carmeliet, 1996; Thomas, 1996; People such as Dvorak, 1995a, b; Folkman, 1995; People such as Ferrera, 1992).The permeability of this growth factor-induced blood vessel is monocyte and osteoblastic chemokine, and is a kind of selectivity mitogen of endotheliocyte.The receptor protein of VEGF (in the people for KDR and Flt-1) belongs to strides film family tyrosine kinase (people such as Terman, 1992; People such as de Vires, 1992).The activation of acceptor can cause and causes vascular endothelial cell growth speed to improve significantly and a series of incident that final neovascularity generates.VEGF is more selective in the inducing endothelial cell growth than the rho factor that other participates in the blood vessel generation.Unfortunately, the existence of VEGF may have and impair healthy effect under specific circumstances.

The VEGF of abnormality high concentration is usually relevant with the disease that the vascularization and the vascular permeability of height increase to feature with some.These diseases comprise diabetic retinopathy, invasive tumor, psoriasis, rheumatoid arthritis and other some inflammation situation (D ' Amore, 1994; People such as Dvorak, 1995a, b; Folkman, 1995).Therefore need some compositions and method to be used for optionally reducing abnormal high VEGF concentration to reduce the neovascularization of VEGF mediation.It is the disease process of feature that these method and compositions can be used to slow down with neovascularization and vascular permeability.

A kind of method that is used to reduce VEGF concentration comprises utilizes antisense oligonucleotide (Wagner, 1994).The key advantages of this technology is to realize specific inhibition.Effectively oligonucleotide is considered to be incorporated on the special sequence of mRNA, and disturbs the expression of its encoding gene.The minimizing of protein expression may be owing to the inhibition to the rrna function, has reduced the concentration of the substrate mRNA that can transcribe or other mechanism.In addition, oligonucleotide can reduce the concentration of mRNA by a kind of increase of oligonucleotide mediated promotion mRNA molecular degradation speed.Usually the oligonucleotide that is about 15 bases just enough provides sequence-specific combination the with target RNA target spot, though shorter oligonucleotide sometimes also can be in conjunction with (Uhlman and Peyman, 1990).Yet the antisense oligonucleotide length that is used to reduce protein expression in experiment in vitro is generally 11-30 base (summary Uhlman and Peyman, 1990).

The potential advantages of antisense therapy scheme to be used for the treatment of diseases means, also must overcome a series of obstacles.For example, antisense oligonucleotide be some big hydrophilic compounds (about 3,000 to 10,000D), and before they are incorporated into target in tenuigenin or the nucleus, must pass hydrophobicity cytolemma (Ulhmann and Peyman, 1990; People such as Miligan, 1993).Therefore, the transporting method that needs promotion VEGF antisense oligonucleotide to pass cytolemma.It also must be nontoxic treating used oligonucleotide, and can not disturb normal cellular metabolism.In order to reduce these non-specific effects, they combine specificity and the avidity that height must be arranged with the sequence of its identification.

Oligonucleotide with natural phosphodiester skeleton is extremely sensitive to serum and nucleus enzyme.The long transformation period of oligonucleotide in serum of 17 bases at random was less than 3 minutes people such as (, 1996) Bishop.The stability that before being used as treatment neovascularization treatment of diseases means, needs to increase oligonucleotide.Utilize thiophosphatephosphorothioate to replace the transformation period that phosphodiester group can improve oligonucleotide.They should be chemically inert, and under the number of chemical environment nuclease resistance are arranged.Yet this oligonucleotide never shows the expression that suppresses VEGF in a selective manner before this.

A kind of defective of previously known thiophosphatephosphorothioate oligonucleotide is, for the concentration of this oligonucleotide of expression that reduces VEGF will reach 1 μ M (people such as Nomura, 1995; People such as Robinson, 1996).Under this concentration, these oligonucleotide toxic (people such as Woolf, 1992; Stein and Cheng, 1993; Stein and Kreig, 1994, Wagner, 1994; People such as Fennewald, 1996).And observed effect may be (people such as Fennewald, 1995) that this non-specific toxicity causes.Need a kind of novel ligonucleotides inhibition, prove a kind of real antisense effect by the expression that under nontoxic concentration, suppresses VEGF.These oligonucleotide have the specificity that higher binding constant is arranged and/or its target mRNA sequence is increased with respect to existing VEGF antisense oligonucleotide.

About the limited effect of existing VEGF antisense oligonucleotide several possible explanations are arranged.A kind of possibility is that target RNA sequence can be limited in the macromolecular structure, and this structure has hindered the combination of oligonucleotide from the space.For example, rna binding protein and protein translation mixture may hinder the combination of oligonucleotide.In addition, oligonucleotide possibly can't combine with a kind of mRNA of unfavorable conformation.In addition, effectively the position of target sequence is changeable.Effectively target sequence may be positioned on any position of target mRNA transcript, and is that the oligonucleotide of target just is not always effectively (people such as Wanger, 1993 with translation initiation codon or 5 ' non-translational region; People such as Fenster, 1994).Oligonucleotide can cause different biologic activity (people such as Woolf, 1992 too with non-specific interaction between other molecule such as the protein; Stein and Cheng, 1993).And perhaps oligonucleotide itself can adopt on a kind of beyond thought three grades or the quaternary structure position beyond the consideration and combine with DNA.These abnormal combinations may produce some unexpected biology effects (people such as Chaudhary, 1995).

In the process of seeking effective antisense oligonucleotide, also ran into other difficulty.Avidity between oligonucleotide and the target RNA increases along with the increase of fragment length and G-C content.Yet long oligonucleotide is easy to carry out nonspecific the combination with RNA or other oligonucleotide fragment.And the oligonucleotide of high G component easily forms the G-tetramer, thereby reduces antisense in conjunction with the quantity of the oligonucleotide of necessary freedom-spiral status people such as (, 1996) Bioshop.Therefore, oligonucleotide should be relatively short and for its target sequence higher avidity be arranged, even there is higher G content also can not form the G-tetramer.

As previously mentioned, oligonucleotide is a kind of big hydrophilic compounds, and it must pass the hydrophobicity cytolemma could be with the target in tenuigenin or the nucleus in conjunction with (Uhlmann and Peyman, 1990; People such as Milligan, 1993).But because their bigger sizes, wetting ability essence and have negative charge, oligonucleotide can not pass cytolemma effectively.Do not have the cell absorption enhancer in the presence of, oligonucleotide tends in the pronucleus endoplasmic reticulum composition of subject cell accumulation (people such as Fisher, 1993; People such as Guy-Caffey, 1995).In this case, oligonucleotide pass plasma membrane or endoplasmic reticulum transport limited the speed of its internalization and its activity.Therefore, need a kind of new composition and the speed of method to promote that oligonucleotide passes lipid bilayer.

One lipoidis absorption enhancer comprises an energy and the positively charged head group of nucleic acid bonded, and one can with the interactional membrane interaction tail of film component.These compositions can promote oligonucleotide to pass cytolemma by instantaneity disturbance cytolemma.Unfortunately, many cationic lipid preparations, Lipofectin  for example, a kind of cationic lipid DOTMA and fusion lipid DOPE (DOPE) (Life Technologies, company, Gaithersburg, MD) by the mixture of 1: 1 (mass ratio), to the composition and the quantity of many factors such as nucleic acid, the type of target cell, and the sensitivities such as concentration of serum in the cell culture medium.In addition, some preparations itself have cytotoxicity.These pressure factors have seriously limited these compounds and have transported agent as oligonucleotide be used for the treatment of purposes in the animal system.Therefore must establish a kind of improved haulage system that is adapted to oligonucleotide.

In a word, the process of numerous disease increases relevant with caused vasculogenesis of the overexpression of VEGF and vascular permeability.Need to reduce specifically the novel composition of vegf expression and method to be used for these treatment of diseases.Antisense strategy is a kind of attractive approach, because it has the potential selectivity.

Unfortunately, many known VEGF antisense oligonucleotides are only just effective under the deleterious concentration of pair cell, and only show nonspecific effect.And existing antisense oligonucleotide is that chemistry and biology is unsettled, and those more stable showing the unacceptable low-affinity of being low to moderate of its target sequence, and be difficult for passing cytolemma, therefore be difficult to arrive its biology target.At last, the oligonucleotide of high G content easily forms the G-tetramer.

Need a kind of nontoxic, new oligonucleotide composition of its target mRNA sequence being had higher avidity.These compositions should have higher biological stability, comprise the increase of the ability of resisting nuclease degradation.In addition, no matter how its sequence does not all answer polymerization to useful oligonucleotide.Also need a kind of novel composition that can help oligonucleotide to pass cytolemma.

Summary of the invention

The invention provides and be used to slow down composition and the method that increases the diseases associated process with vasculogenesis and vascular permeability.This antisense oligonucleotide composition suppresses to be better than existing antisense oligonucleotide on the celliferous vegf expression in selectivity, and is intended to be used for to this type of treatment of diseases.Selectivity of the present invention comes from the antisense oligonucleotide that can be incorporated on the VEGF mRNA molecule specifically and hinder vegf expression.

The invention provides these oligonucleotide and utilize chemically modified to improve it for the avidity and the specific manufacturing of target mRNA sequence with use their method.Oligonucleotide of the present invention all has higher biological stability and to the avidity of target sequence.These oligonucleotide be hydrophobic environment or under hydrophilic environment insensitive to chemistry all with biological effect.And no matter its sequence how, is difficult for forming polymer.

The invention provides effective and nontoxic VEGF antisense oligonucleotide.Especially, the present invention is used for a kind of new oligonucleotide composition, and this composition is handled cell with the concentration that is lower than 1 μ M and can be caused the cell yield of VEGF to reduce.Antisense oligonucleotide of the present invention is nontoxic and can the interference cell metabolism under these concentration.

The present invention also provides a kind of oligonucleotide that makes to facilitate penetration of composition and method that cytolemma arrives its biology target simultaneously.This is by preparation being provided and using antisense oligonucleotide to use the method for cell absorption enhancer to realize simultaneously.The cell absorption enhancer is nontoxic, and compatible with the VEGF antisense oligonucleotide, can promote that oligonucleotide passes cytolemma effectively.

" oligonucleotide " speech that is used for the object of the invention comprises nucleic acid polymer and the chemical structure similar to the nucleic acid polymer.The equivalent of ribose or ribodesose can be gone in its structure by displacement in oligonucleotide, has with the target sequence specificity and combines required hydrogen bond as long as the composition of bonded base can be kept it.The chemical equiv such as the phosphorothioate bond that can contain similarly, phosphodiester backbone in the oligonucleotide.The base composition that also can comprise in addition, the process chemically modified in the oligonucleotide.Especially, oligonucleotide can be including, but not limited to C5-(proyl or hexin base) uridine or cytidine residue, 6-aza uridine or cytidine residue, and the pyrimidine of modifying through C5 or 6-azepine.

" VEGF " speech comprises the known all proteins that belongs to vascular endothelial growth factor family.VEGF comprises the autoploid of at least four kinds of known human VEGF, it is believed that these four kinds of molecules derive from the difference shearing product with a kind of mRNA, and any homologous protein that similar biological function is arranged.Known protein matter comprises known in the art VEGF206, VEGF185, VEGF165 and the VEGF121 of those codings from the mRNA spliced body.

Antisense oligonucleotide of the present invention prepares in the following method.Length is approximately the nucleotide sequence of 15-30 base, preferred about 19 bases of length, and its sequence is consistent with the mRNA of coding VEGF.The sequence of these VEGF mRNA is known in the art.This RNA sequence can be any one section sequence on proteinic any one the mRNA in the coding VEGF family.Preferredly should be and coding people VEGF206, VEGF185, VEGF165 and VEGF121 mRNA complementary antisense oligonucleotide.Most preferably can with the sequence bonded oligonucleotide (seeing Table 1) that all has on all VEGFmRNA.Anti-VEGF oligonucleotide table 1

I.D.	Kind	Sequence	Modify
				T30615: T30639: T30640: T30641: T30847: T30848: T30849: T30876: T30877: T30878: T30879: T30886: T30887: T30888: T30889: T30890: T30891: T30892: T30893: S96-5296: S96-5297: T30688: T30692: T30807: T30807:	The T30639 ' of antisense mRNA 185-203+ var.of T30615 mRNA seq.204-222 mRNA seq.232-250 var.of T30639 var.of T30639 var.of T30639 mRNA seq.224-242 mRNA seq.406-424 mRNA seq.522-540 mRNA seq.575-593 mRNA seq.171-189 mRNA seq.176-194 mRNA seq.199-217 mRNA seq.195-213 var.of T30639 var.of T30639 var.of T30639 var.of T30639 var.of T30639 var.of T30639 var.of T30615 2-base mispairing has justice ' DNA of T30615 ' that justice ' RNA of T30615 is arranged	5′-g *c *g *c *t *g *a *t *a *g *a *c *a *t *c *c *a *t *g-3′ 5′-g *C *g *C *U *g *a *U *a *g *a *C *a *U *C *C *a *U *g-3＇ 5′-C *g *a *U *U *g *g *a *U *g *g *C *a *g *U *a *g *C *t-3′ 5＇-U *a *C *U *C *C *U *g *g *a *a *g *a *U *g *U *C *C *a-3′ 5′-g *C *g *C *U *g-a *U *a-g-a *C *a-U *C *C *a *U *g-3′ 5′-g *C *g-C *U-g-a *U *a-g-a *C *a-U *C *C *a *U *g-3＇ 5＇-g *C *g *C *U *g-a *U *a *g-a *C *a *U *uC *C *a *U *g-3′ 5＇-g *a *a *g *a *u *U *g *U *C *C *a *C *C *a *g *g *g *U *C-3′ 5′-a *g *g *a *a *g *C *U *C *a *U *C *U *C *U *C *C *U *a-3′ 5′-U *a *C *a *C *g *U *C *uU *g *C *g *g *a *U *C *U *U *g *-3＇ 5′-u *a *a *C *u *C *a *a *g *C *U *g *C *C *u *C *g *c *C *-3′ 5′-C *C *a *U *g *a *a *C *U *U *C *a *C *C *a *C *U *U *c-3′ 5＇-g *a *C *a *U *C *C *a *U *g *a *a *c *t *t *c *a *c *c-3＇ 5＇-g *g *a *U *g *g *C *a *g *U *a *g *C *U *g *C *g *C *U-3′ 5′-g *g *C *a *g *U *a *g *C *U *g *C *g *C *U *g *a *U *a-3＇ 5′-g *C *g *C *t *g *a *t *a *g *a *C *a *t *C *C *a *t *g-3′ 5′-g *c *g *c *U *g *a *U *a *g *a *c *a *U *c *o *a *U *g-3′ 5′-g *c *g *c *t *g *a *U *a *g *a *C *a *U *C *c *a *t *g-3＇ 5′-g *C *g *c *U *g *a *U *a *g *a *C *a *t *C *c *a *U *g-3＇ 5′-g *C *g *C *U-g-a-U *a-g-a-C *a-U *C-C *a *U *g-3′ s′-g *C *g *C *U-g-a-U *a-g-a-C *a-U *c-C *a *U *G-3 ' (AL)-pyrene 5 '-g *C *g *C *U *a *U *g *a *C *a *U *C *C *a *U *g-3′ 5′-g *C *g *C *U *a *C *a *g *a *C *a *U *U *C *a *U *g-3＇ 5′-c *a *t *g *g *a *t *g *t *c *t *a *t *c *a *g *c *g *c-3＇ 5′-c *a *t *g *g *a *t *g *c *c *t *a *t *c *a *g *c *g *c-3′	Total total PT of the total PT.C5-proyl of the total PT.C5-proyl of PT (thiophosphatephosphorothioate) DNA pyrimidine DNA total PT.C5-proyl pyrimidine pyrimidine 4PD key 6PD key 2PD key, the total PT of C5-proyl pyrimidine, the total PT of C5-proyl pyrimidine, the total PT of C5-proyl pyrimidine, the total PT of C5-proyl pyrimidine, the total PT of C5-proyl pyrimidine, the total PT of C5-proyl pyrimidine, the total PT of C5-proyl pyrimidine, the total PT of C5-proyl pyrimidine, the total PT of C5-proyl C (only having), the total PT of c5-proyl U (only having), the total PT of 4 C5-proyl pyrimidines, 6 C5-proyl pyrimidine [AL]-Lip-16PD; The C5-propine, fat chain 8PD; The C5-propine, the total PT of pyrene chain, C5-is the total phosphodiester of the total PT.C5-proyl of alkynyl pyrimidine DNA pyrimidine DNA, the total phosphodiester of DNA, RNA

+ people VECF mRNA sequence source is from people such as Leung., science, 246:1306,1989. initiator codons are positioned at 57 bases.* phosphorothioate ester key.-phosphodiester bond.1C, U represent the base of modified.

A series of complementary or " antisense " oligonucleotide have been prepared.For the purposes of the present invention, " antisense " is meant the sequence complementary oligonucleotide with sequence and mRNA, so they can combine in a specific specificity hydrogen bonded mode with those sequences.Yet as long as oligonucleotide has the activity of anti-VEGF when concentration is lower than 1 μ M, it just might have mispairing or not exclusively correct hydrogen bond mode combination.

Antisense oligonucleotide of the present invention comprises that some improve the modification of its biological stability.The raising of its biological stability is by mix nuclease resistance key such as phosphorothioate bond in different or all nucleotide residues.Oligonucleotide of the present invention also is included in the locational chemically modified of different or all pyrimidines.The base of these modifieds comprises 5-proyl pyrimidine, C5-hexin yl pyrimidines or 6-aza-pyrimidine or in conjunction with the derivative of C5 and 6-aza-pyrimidine, oligonucleotide of the present invention can be further stablized in these modifications.

Comprise at antisense oligonucleotide of the present invention and can increase its modification for the aim sequence binding affinity.Improve binding affinity by in pyrimidine bases, mixing different chemical ingredientss.These oligonucleotide are included in base different or the locational chemically modified of all pyrimidines.These modified bases comprise: C5-proyl pyrimidine, C5-hexin yl pyrimidines or existing C5 have the pyrimidine derivatives of 6-azepine again.

Antisense oligonucleotide can combine with natural mRNA and also can the RNA sequence identical with the sequence with on the VEGFmRNA of chemosynthesis combine.This combination can confirm by several different methods.Describe in wherein a kind of observation bonded method such as the embodiment III.This method comprises mixes the isometric mRNA molecule of antisense oligonucleotide with some chemosynthesis mutually, and this oligonucleotide is annealed in initial heating and cooling step, when observing mixture heating up in the variation of 260nm place light absorption value.Can also utilize other method that combination is detected, for example nuclease protection experiment, oligonucleotide extends experiment, NMR, gel electrophoresis or other technology more well known by persons skilled in the art.

" binding affinity of improvement " or " stability " are meant the oligonucleotide height that the melting temperature(Tm) (Tm) when this oligonucleotide combines with its target sequence is modified than a kind of not process for the purposes of the present invention.Detection method as the melting temperature(Tm) described in the embodiment III is used for this detection.The present invention has adopted those can improve the chemically modified of antisense oligonucleotide/double-helical binding affinity of mRNA target sequence.Usually, the Tm of antisense oligonucleotide is higher than 45 ℃ in described method.Preferred oligonucleotide Tm＞50 ℃.

Specific oligonucleotides of the present invention comprises that the activity through chemically modified is higher than those known VEGF antisense oligonucleotides.The active raising means that the required oligonucleotide concentration of the interior inhibition of body vegf expression is lower.Though the present invention is not limited to the pattern of these modifications, the activity that the present oligonucleotide of paying close attention to increases has at least a part of reason to be because binding affinity and the biological stability that increases.As previously mentioned, these specific chemically modifieds can be used for improving the activity of this oligonucleotide.

The antisense oligonucleotide that the present invention paid close attention to is nontoxic when concentration is lower than about 1 μ M.Measure toxicity with reference to the described method of EXAMPLE V.

Antisense oligonucleotide of the present invention reduces the VEGF output in the cell of handling.In one approach, cell is directly contacted with oligonucleotide composition, make oligonucleotide be dissolved cell and to arrive its target mRNA sequence by interior.Before handling, dissolving or the suspension in a kind of liquid earlier of this oligonucleotide, or make it to mix a kind of solid.The preparation of suitable liquid or solid is known in the art, and available known method is selected.Direct and the cells contacting of the oligonucleotide that is mixed with.In other method, will make this oligonucleotide arrive its target cell by the oligonucleotide location that prescription is made by diffusion, dispersion or similar fashion.The present invention does not require the direct contact target cell of oligonucleotide agent.Invention only requires this Nucleotide to arrive target cell.For example, a kind of oligonucleotide can be imported in the blood, but it may spread out from blood before sacroiliitis cell or the like arriving target tumor cell.In addition, oligonucleotide can be sneaked into a kind of powder and is directly used in skin and is diffused in the following cell.

Handled cell with antisense oligonucleotide of the present invention and under similarity condition, approximately can produce at most 90% VEGF with respect to the cell of unprocessed mistake.And when being lower than about 1 μ M, the oligonucleotide strength of solution can be observed this effect.

A kind of mensuration described in the method such as embodiment VI that VEGF output reduces.But, also can measure the decline of VEGF output with other method, as long as they are the same with method described in the EXAMPLE V responsive.Determine per-cent by measuring by the VEGF of treated cell generation through the amount of handling and untreated cell produces VEGF.Multiply by the 100 output per-cents that are with respect to the cell of handling with the output of the cell of handling again divided by the output of undressed cell.The cell of wherein handling and untreated cell except in substratum, whether exist oligonucleotide agent aspect all be identical in every respect.Therefore the contemporaneity that used cell should have identical type, passage number, phenotype and be in the cell growth in detecting.Cell is grown under identical condition, comprising: substratum (except whether having added the variation of oligonucleotide agent itself), temperature and identical gas condition.Under these conditions, the output of the VEGF of the cell of handling through oligonucleotide of the present invention can reach at most unprocessed mistake cell output about 90%.When the concentration in the antisense oligonucleotide solution during up to 1 μ M, if also will reach similar molecular fraction with solid preparation.

Preferred oligonucleotide mixes some chemically modifieds that can improve its nuclease degradation resistance.The design of chemically modified is meant the modification to naturally occurring chemicals common in oligonucleotide in these embodiments.Particular chemical composition of the present invention comprises phosphorothioate bond.These modifications can be between some or all nucleotide residues.Most preferred oligonucleotide contains 10 thiophosphatephosphorothioates and 8 phosphodiester bonds.Except nucleotide bond, can improve degradation-resistant to nuclease to the chemically modified of base component.More particularly, the modification of pyridine ring is comprised the modification of C5-proyl and hexin base group and/or 6-azepine pyridine.

A kind of method of measuring the nuclease resistance is by detecting the transformation period of oligonucleotide in serum.This is to adopt the known standard method in this area to finish.Be purpose of the present invention, can adopt a kind of chemical ingredients that reduces the nuclease degradation speed of antisense oligonucleotide, if having the have longer serum half-life of the oligonucleotide of this component than this component of nothing.The oligonucleotide serum half-life that contains thiophosphatephosphorothioate substantially exceeds 24 hours.And the transformation period of only containing the oligonucleotide of phosphodiester bond corresponding with it is no more than 3 hours.

Designed oligonucleotide has chemically modified on its pyrimidine ring.Most preferred oligonucleotide includes C5-proyl pyrimidine, C5-hexin yl pyrimidines and/or 6-aza-pyrimidine.These modifications have improved their Tm value, biological stability and activity.Oligonucleotide precursor synthetic that has modification as described in the embodiment I.Adopt the known standard phosphoramidite chemical process in this area from these or other shielded Nucleotide synthetic oligonucleotide.

Specific embodiment is meant that can improve the active cell of oligonucleotide absorbs enhancing composition among the present invention.Usually, these compositions can promote a kind of liquid to pass the transportation of liquid bilayer.In some embodiments, oligomer and a kind of lipophilic molecule is covalently bound.What this can improve oligonucleotide and film combines and penetrates character, as cholesterol, lipid acid or other lipophilic chain.These molecules can be connected with the oligonucleotide chemical by the known standard method in this area.

In other embodiments, can utilize cationic-liposome or Liposomal formulation as the toughener that absorbs.So these reagent are because its adaptability is attractive.The advantage of these embodiments is that same transport agent can be used for the importing of oligonucleotide mixture.Selected Liposomal formulation Cellfectin  in a kind of embodiment especially for use.Comprise polyamino fat absorption enhancer in other the embodiment, comprise SPDC (SpdC).A kind of compound in back has the advantage that plays a role equally effectively in the presence of serum.The preparation method and composition such as the embodiment IV that have the antisense oligonucleotide of cell absorption enhancer are described in VI and the VII.

Specific oligonucleotides of the present invention is the form of salt.This salt form is a kind of oligonucleotide and the atom or the molecule bonded form that have positive charge (positively charged ion).Suitable positively charged ion is including, but not limited to sodium ion, potassium ion, and the ammonium radical ion, spermidine or polyamino fat are as SPDC or the like.

Particular of the present invention also comprises a kind of liposome.Suitable liposome is that this area is known.The specific liposome of the present invention is particularly including Cellfectin .Other composition comprises the SPDC that is mixed with DOPE.Adopt means known in the art to prepare Liposomal formulation.

Particular of the present invention is transported its oligonucleotide composition by sustained release system, including, but not limited to some oligomer release vehicles, for example mixture of polycaprolactone or polycaprolactone and methoxy poly (ethylene glycol).Being used for the method and composition that antisense oligonucleotide of the present invention is incorporated into sustained release system is that this area is known.

Brief description of drawings

Fig. 1. among expression the present invention antisense oligonucleotide synthetic in order to the base of the modification of the present invention of replacing natural base.

Fig. 2. preparation 5-(1-hexin base or proyl)-6-azepine-2 ' deoxyuridine phosphoramidite (referring to Fig. 1)

Fig. 3. the influence that antisense oligonucleotide produces the normal people's keratinocyte VEGF that cultivates.

Fig. 4. use the influence of the antisense oligonucleotide (sequence numbering NO.2) that has or do not have Cellfectin  to the keratinocyte vegf expression.

Fig. 5. use the influence of the antisense oligonucleotide (sequence numbering No.27) that has or do not have Cellfectin  to the keratinocyte vegf expression.

Fig. 6. different cell absorption enhancers is to the active influence of keratinocyte of T30639 (sequence numbering NO.2).

Fig. 7. the cell short burst is exposed to the long term inhibition of oligonucleotide agent of the present invention and vegf expression.

Fig. 8. the cell short burst is exposed to the long term inhibition of oligonucleotide agent of the present invention and vegf expression.

Fig. 9. the cell short burst is exposed to the long term inhibition of oligonucleotide agent of the present invention and vegf expression.

Figure 10. have end modified, VEGF antisense oligonucleotide mosaic have or not absorption enhancer in the presence of to the influence of vegf expression

The explanation of preferred embodiment

Preferred embodiment comprises a kind of ASON, and it can be combined with the common sequence of a kind of multiple VEGF of the mRNA of coding molecule and can suppress in vivo the expression of VEGF. Preferred oligonucleotides contains at the locational phosphorothioate bond of several phosphodiester bonds, and other is in order to improve the chemical modification of oligonucleotides and its target mRNA sequence affinity. Oligonucleotides is made preparation with the Cell uptake reinforcing agent that can improve the ability that they pass cell membrane in preferred composition.

The length range of oligonucleotides of the present invention is from about 17-30 base. Preferred oligonucleotides length is 19 nucleotides. Its sequence selected is based on the principle with the mRNA complementary element of the VEGF gene of encoding. Zone with the oligonucleotides complementation on the mRNA molecule is called target sequence. Preferred ASON can comprise with four kinds of known VEGF mRNA molecules: VEGF206, VEGF185, the upper total target sequence complementation of VEGF165 and VEGF121.

The oligonucleotides of design improves the chemical modification of they and target mRNA binding affinity with some. Preferred oligonucleotides is with C5-propinyl pyrimidine, C5-hexin yl pyrimidines and/or 6-aza-pyrimidine. Temperature when preferred modification raising oligonucleotides and its target sequence dissociate. Synthetic described in the embodiment I with the nucleotide precursor of these modifications. According to standard phosphoramidite chemical method known in the art from protected nucleotides synthetic oligonucleotide.

Preferred oligonucleotides mixes the chemical modification that some increase its nuclease degradation resistance. Although the present invention is not limited to this resistance mechanism, thinks and to disturb the degraded of resisting nuclease with the combination of oligonucleotides by the Binding Capacity pocket place at nuclease through the nucleotides of chemical modification. The oligonucleotides of these preferred nuclease resistances contains phosphorothioate bond between at least some nucleotide residues. Most preferred oligonucleotides comprises 10 thiophosphates and 8 phosphodiester bonds.

In preferred composition, oligonucleotides of the present invention is to prepare or mix with the Cell uptake reinforcing agent that can improve its ability of passing cell membrane. Cell uptake reinforcing agent used in the present invention comprises two oleoyl phosphatidyl ethanolamines, Cellfectin , SPDC etc. Most preferred composition is SPDC and the two oleoyl phosphatidyl ethanolamines that mix at 1: 1 in mass ratio. According to methods known in the art the oligonucleotides of this preparation with 10nM-1 μ M concentration mixed.

Oligonucleotide composition of the present invention is to select according to the activity in their bodies. Preferred composition is in oligonucleotides concentration essentially no cytotoxicity during up to 1 μ M. Be used for method that the standard cell lines toxicity of this detection detects as described in the embodiment I. To prove that also this composition has the ability that reduces cell VEGE output when concentration is lower than 1 μ M.

The present invention comprises that being intended to of having set up the specific embodiments for putting into practice preference pattern of the present invention is set forth. Those skilled in the art should know, and according to spirit of the present disclosure, can carry out multiple modification and variation to specific embodiment in the situation that does not exceed scope of the present invention. All these are modified and all to think and drop in the scope of accompanying claim.

Embodiment

The embodiment I: preparation is used to mix the method for base of the modified of synthetic oligonucleotide

These bases that are used to improve the binding affinity of synthetic oligonucleotide and/or specific modified as shown in Figure 1.What Fig. 2 provided is the synthetic synoptic diagram of preparation 5-(1-hexin base or proyl)-6-azepine-2 ' deoxyuridine phosphoramidite.This synthesizes the antisense oligonucleotide that preparation contains the 6-aza uridine building block is provided.Similar synoptic diagram is also adopted in synthesizing of 6-azacytidine.The preparation 5-synthetic method that (1-hexin base)-6-azepine-2 '-the deoxyuridine phosphoramidite is detailed is as described below.Using the same method can be from 5 '-iodo derivative, 7 preparations, 5 '-proyl derivative.

3 '-5 '-two-oxygen-right-tolyl-5-iodo-6-azepine-2 '-deoxyuridine (7a):

With the chlorine trimethyl silane of 0.5ml join 5-iodo-6-azauracil (5.8g, 33.47mmol) 1,1,1,3,3, the 3-hexamethyldisilane amine (HMDS, 80ml) in the solution, reflux 6 hours.With the reaction mixture cool to room temperature, make the HMDS evaporation in the vacuum.Resistates was drained under high vacuum 4 hours.The silyl derivative that drying is good is dissolved in the methylene dichloride (60ml).In this solution, add 1-chloro-2-deoxidation-3,5-is two-oxygen-right-tolyl-b-D-is red-furan pentose (6,16.3g, 42mmol) and zinc chloride (0.46g 3.35mmol) mixes 24 hours in argon gas.(250ml) dilutes this reaction mixture with methylene dichloride, and uses water saturated NaHCO ₃Solution (100ml) washing dichloromethane solution.With methylene dichloride (4 * 100ml) extracting waters, the organic phase Na of merging ₂SO ₄Dry also evaporation.Resistates silicagel column (4 * 15cm) chromatography purifications.The product methylene dichloride wash-out that contains 0-5% methyl alcohol.The heavy 15g of different body product of drying.Different body of pure b can by with methylene dichloride and methyl alcohol (4: 1, mixture development 200ml) and obtaining.By filtering and evaporation collection solid.Repeat this triturating and can get different body of the pure b of 10.5g.Fusing point 204-205 ℃. ¹H NMR (DMSO-d6): d2.35,2.37 (2s, 6H, 2CH ₃), 2.80 (m, 2H, C ₂' H and C ₂" H), 4.43 (s, 3H, C ₄' H, C5 ' H ₂), 5.55 (brs, 1H, C ₃H), 6.39 (t, J=6.0Hz, 1H, C1 ' H), 7.28 (t, 4H, Tol), 7.85 (t, 4H, Tol), 12.42 (brs, 1H, NH) C ₂₄H ₂₄IN ₃O ₇The ultimate analysis calculated value be: C, 48.58; H, 4.08; N, 7.08, experimental value: C, 48.85; H, 3.80; N, 6.92.

3 ', 5 '-two-oxygen-right-tolyl-5-(1-hexin base)-6-azepine-2 ' deoxyuridine (8a):

3 ', 5 '-two-tolyl-5-iodo-6-azepine-2 '-deoxyuridine (7,3.84 gram, 6.5 mmole) with dried DMF (25ml) coevaporation drying after be dissolved in add among the DMF (30ml) and under the argon gas condition CuI (0.25g, 1.3mmol), triethylamine (1.82ml, 13mmol), the 1-hexin (2.23ml, 19.5mmol) and tetrakis triphenylphosphine palladium (0.75g, 0.65mmol).This reaction mixture at room temperature stirred 18 hours and added the 0.5g tetrakis triphenylphosphine palladium.Evaporating solvent after 48 hours, residue and toluene coevaporation.With silica gel column chromatography column purification residue, product obtains the title compound of 0.9g with the methylene dichloride wash-out that contains the 0-5% ethyl acetate.Fusing point 198-200 ℃. ¹H NMR (DMSO-d6): d 0.87 (t, 3H, CH ₃), 1.45 (m, 4H, 2, CH ₂), 2.37,2.39 (2s, 6H, 2CH ₃), 2.45 (m, 3H, CH ₂And C ₂" H), 2.84 (m, 1H, C ₂' H), 4.45 (s, 3H, C ₄' H, C ₅' H ₂), 5.59 (brs, 1H, C ₃' H), 6.49 (t, J=6.3Hz, 1H, C1 ' H), 7.31 (dd, 4H, Tol), 7.88 (dd, 4H, Tol), 12.40 (br s, 1H, NH).C ₃₀H ₃₃N ₃O ₇The ultimate analysis calculated value: C, 65.80; H, 6.07; N, 7.68, experimental value: C, 65.61; H, 5.73; H, 7.29.

6-azepine-5-(1-hexin base)-2 ' deoxyuridine (9a):

3 ', 5 '-two-oxygen-right-tolyl-5-(1-hexin base)-2 '-deoxyuridine (8a, 0.8g, 1.47mmol) and methyl alcohol (55ml) and sodium methylate (25% methanol solution, 1.28ml) mixture at room temperature stirred 2 hours, add Dowex 50X8 (H+) resin termination reaction.This resin is by removing by filter evaporated filtrate.Residue can be used purification by silica gel column chromatography, produces the title compound of the height suction of 0.38g (84%) with the methylene dichloride wash-out that contains 0-4% methyl alcohol.

¹HNMR(DMSO-d6):d0.88(t,3H,CH ₃),1.47(m,4H,2,CH ₂),2.02(m,1H,C ₂″H),2.33(m,1H,C ₂′H),2.46(m,2H,CH ₂),3.40(m.2H.C ₅′H ₂),3.68(m,1H,C ₄′H,),4.21(br?s,1H,C ₃′H),4.61(t,1H,C ₅′OH),5.17(d,1H,C ₃′OH),6.49(dd,1H,C ₁′H),12.27(br?s,1H,NH).

5 '-oxygen-(4,4 '-two methoxy three acyl groups)-6-azepine-5-(1-hexin base)-2 ' deoxyuridine (10a):

(0.51g, (0.38g is in dried pyridine 1.24mmol) (10 milliliters) solution 1.5mmol) to add 5-(1-hexin base)-6-azepine-2 ' deoxyuridine for 4,4 '-two methoxy three acyl chlorides.Stir after 6 hours, add 0.5g DMT-Cl, this reaction mixture stirs and spends the night.This reaction mixture dilutes with methylene dichloride (100 milliliters), and water (20 milliliters) is washed this organic solution.Water methylene dichloride extracting merges organic phase Na ₂SO ₄Dry also evaporation.Residue and toluene (10 milliliters) coevaporation is with silica gel column chromatography (2 * 10cm) purifying residues.Product obtains 0.45g with the methylene dichloride wash-out that contains 0-1.5% methyl alcohol.

¹H?NMR(DMSO-d ₆):d0.85(t,3H,CH ₃),1.37(m,4H,2,CH ₂),2.08(m,1H,C ₂″H),2.37(m,3H,CH ₂?and?C ₂′H),3.06(m,2H,C ₅′H ₂),3.72(s,6H,2OMe),3.87(m,1H,C ₄′H,),4.20(m,1H,C ₃′H),5.23(d,1H,C ₃′OH),6.35(dd,1H,C ₁′H),6.83(m,4H,DMT),7.16-7.25(m,9H,DMT),12.30(br?s,1H,NH).

5 '-oxygen-(4,4 '-two methoxy three acyl groups)-6-azepine-5-(1-hexin base)-2 ' deoxyuridine-3 '-oxygen-(2-cyanoethyl)-N, N-diisopropylphosphoramidite (11a):

5 '-oxygen-(4,4 '-two methoxy three acyl groups)-6-azepine-5-(1-hexin base)-2 ' deoxidation

Uridine (10a) in methylene dichloride at N, when the N-diisopropylethylamine provides phosphoramidite 11a with known method and 2-cyanoethyl-N, the reaction of N-diisopropylphosphoramidite.

The design of embodiment II oligonucleotide is with synthetic

Selection can with the complementary bonded Antisensedigonucleotsequence sequence of the shearing variant common mRNA target sequence of all VEGF mRNA.Table 1 has provided the example of the sequence of some synthetic oligonucleotide.In order to improve their binding affinities for the mRNA target, these oligonucleotide are synthetic with the pyrimidine that has C5-proyl or C5-hexin base group, as shown in Figure 1 people such as (, 1993) Wagner.Also consider to comprise the base (Fig. 2) of other modifieds such as 6-azepine deoxyuridine and 6-azepine Deoxyribose cytidine, also consider the combination of these modifications.Oligonucleotide T30691 (sequence numbering NO.27) and antisense oligonucleotide T30639 (sequence numbering No.2) complementation, in the experiment below with comparing.It is big or small identical with T30639's (sequence numbering No.2), contains the identical skeleton and the modification of base.

The interactional Tm value of embodiment III antisense oligonucleotide-RNA allos duplex detects

The double-helical temperature of antisense oligonucleotide RNA (Tm) can be used to assess binding affinity.The Tm value is equipped with temperature controlled determination of diode array spectrophotometer (HewlettPackard Model 8452) with one.The RNA target (each 1 μ M) of the equal size of antisense oligonucleotide and synthetic is containing the sodium phosphate pH7.0 of 2mM, mixes in 18mM NaCl and the 1mM EDTA solution.This solution is heated to 90 ℃ in spectrophotometric cell, and 10 minutes, and use more than 10 fens clock times and be cooled to 20 ℃.Balance made duplex form in 10 minutes then.In order to detect double-helical melting temperature(Tm) (Tm), the temperature of cell is heated to 80 ℃ from 20 ℃ at leisure with the speed of 1 ℃/min, is determined at the function of the light absorption value at 260nm place as temperature.The rising of light absorption value signal means double-helically unwinds or is opened into the strand oligomer.Can utilize as the described equation of standard method and obtain the Tm value (Puglisi and Tinoco, 1989) that duplex forms from melting curve.The Tm value is as shown in table 2.The thiophosphatephosphorothioate oligonucleotide that mixes C5-proyl or C5-hexin base modified base causes the Tm value obviously to raise than not modified oligonucleotide.This has shown that antisense oligonucleotide significantly increases the avidity of its target sequence.

Table 2

T30807 (antisense DNA)

	T30615 (unmodified)	T30639 (proyl)	T30688 (alkynyl)	T306s2 (proyl mispairing)
					????T m(℃) AG?at?37℃(kcal/mol) ????Ah(kcallmol) ????AS(eu)	?????43 ????-11.5 ????-136 ????-402	?53 -13.4 -91.6 -252	?????49 ????-11.9 ????-79.8 ????-219	?345 -8.1 -103 -306

T30808 (sense-rna)

	????T30615	????T30639	????T30688	????T30692
					????Tm(℃) AG?at?37℃(kcal/mol) ????AH(kcal/mol) ????AS(eu)	?????42 ????-11.0 ????-128 ????-378	?????57 ????-14.3 ????-88.3 ????-239	?????53.5 ????-13.5 ????-90.7 ????-249	?????43.5 ????-11.3 ????-119 ????-347

The preparation of embodiment IV absorption enhancer:

That the cell of the antisense oligonucleotide of do not have assisting absorbs is very low (people such as Ficher, 1993; People such as Guy-caffey, 1995).In order to improve that the ability that enters cell has adopted multiple commercial absorption enhancer and by the novel polyamino fat of inventor's synthetic preparation.

The Cytotoxic detection of embodiment V:

Cell is inoculated in 96 orifice plates with the density of 500 cells/well.Inoculate after one day, handle cell (each extent of dilution is made four parallel holes) with the oligonucleotide agent of a series of dilutions.Handle after 1 day or measure the influence (Cell Titer96 Aqueous cell proliferation assay, Promega company) of cell viability after 4 days with a kind of on-radiation detection system.Oligonucleotide concentration of the present invention is not observed toxicity when being lower than 1 μ M.

The cytology of embodiment VI oligonucleotide detects

Estimate the activity of counterpart with the different preparations of antisense oligonucleotide, its modified with the human keratinized cell of cultivating.Keratinocyte is an a kind of primary cell system, it can be under normal culture condition secretion of VEGF (people such as Bauaun, 1995; People such as Frank, 1995).Cell is pressed 50-100, and the density of 000 cells/well/0.5mlKGM substratum (Clonetics) is inoculated on 48 orifice plates.With a kind of sensitive, based on (the R﹠amp of Protein Detection system of ELISA detection; D Systerm) measures vegf protein matter level in the cell conditioned medium.Preliminary detection shows that when the NHEK cell is grown 50,000 cells in the 0.5ml substratum produced about 150-200pg VEGF (that is: about 300-400pg/ml in the undressed cell conditioned medium) in 15 hours in the substratum of suggestion.Cell and oligonucleotide agent were cultivated 15 hours together.There are three kinds activity (having under the situation of 10 μ g/mlCellfectin  existence) is arranged in the concentration range of 0.2 μ M in four kinds of anti-VEGF oligonucleotide.And justice (sense) the oligonucleotide pair cell that has of contrast does not influence (result does not provide), and the result as shown in Figure 3.

In order to estimate the effect of antisense, under the situation that adds or do not add the cell absorption enhancer, use oligonucleotide to cell.In initial experiment, the thiophosphatephosphorothioate oligonucleotide that does not have base modification is effective when concentration is lower than 1 μ M, and carrier free is to its active not significantly influence (result does not provide).When concentration during greater than 1 μ M oligonucleotide be easy to the expression (result does not provide) of non-specific inhibition VEGF.These nonspecific effects are known in the art (people such as Stein, 1993; Wagner, 1994).For fear of these nonspecific effects, absorption enhancer is mixed with oligonucleotide.Cellfectin  is a kind of usefulness four palmityl spermidines (Tm-TPS) and two oleoyl phosphatidyl ethanolamine (DOPE) (Tm-TPS/DOPE, press 1: 1.5 LifeTechnologies company of mass ratio) a kind of Liposomal formulation, it is higher than the activity of after testing some other commercialization conveying medium, and toxicity is lower.With the oligonucleotide of the Liposomal formulation of polyamino fat SpdC preparation more effective (people such as Guy-Caffey, 1995; SpdC/DOPE, 1: 1 mass ratio).In typical cell culture experiments, oligonucleotide (10nM-1 μ M) at room temperature is dissolved in the aqueous solution of about 20-40 μ l absorption enhancer, and hatches about 10 minutes.Above-mentioned solution is mixed with the substratum of the warm mistake of 0.5ml and join in the cell.Cell was cultivated 15 hours in the presence of oligonucleotide.Collecting cell supernatant after cultivating, be used for immediately that ELISA detects or in-80 ℃ of storages to be used for detection in the future (discovery) without refrigerated sample and significant difference through the sample VEGF level of freeze thawing.As shown in Figure 4, antisense oligonucleotide T30639 in the presence of Cellfectin  (sequence numbering No.2) activity is stronger, and what effect " justice is arranged " oligonucleotide T30691 (sequence numbering NO.27) of contrast does not have yet under used maximum concentration.As shown in Figure 5.

Fig. 6 has provided and has utilized multiple cation lipid body preparation: SpdC, SPDC (people such as Guy-Caffey, 1995); DC-Chol (Gao and Huang, 1991); CS, CS; DCS, DCS; CF, Cellfectin  use the effect of 0.1 μ M or 0.2 μ M oligonucleotide (sequence numbering No.2).Every kind of cation lipid body preparation is that it is got with fusion lipid DOPE (mass ratio 1: 1) is mixed, stores for future use after the freeze-drying.Earlier liposome is resuspended in 5% glucose (concentration of 1mg/ml) before using, stores in 2 weeks for 4 ℃ and use.By the method for the above, carry out before the cell processing oligonucleotide being mixed with the cation lipid body preparation.

Fig. 7-9 has provided the antisense oligonucleotide T30639 (sequence numbering No.2) with different concns, with and cell cultures result's (seeing Table 1) of phosphodiester-thiophosphatephosphorothioate mosaic T30848 (sequence numbering No.6).What Fig. 7 provided is the effect of 0.1 μ M oligonucleotide, and what Fig. 8 provided is the action effect of 0.2 μ M oligonucleotide, and what Fig. 9 provided is 0.4 μ M oligonucleotide action effect.In each experiment, cell usefulness is added with the substratum of the antisense oligonucleotide of SpdC/DOPE premix and was handled 4 hours.Then substratum is changed into the fresh substratum of not adding.The repressed percentage ratio of expression of figure (graph) 1 provides to be oligonucleotide composition washed off VEGF after 16 hours.Figure (graph) the 2nd washes the result of oligonucleotide after 40 hours off.Figure (graph) the 3rd, oligonucleotide is washed the result after 64 hours off.Detect the amount of VEGF level in the substratum of collecting then.The form that finishes the back cell at about 3 days incubation period is normal, and Fig. 7-9 has provided the long-acting influence of oligonucleotide to vegf expression.That symbol (Δ) expression is the T30848 (sequence numbering NO.6) of 0.1 μ M in these figure (graph); That symbol () is represented is 0.1 μ M T30639 (sequence numbering NO.2).

What Figure 10 provided is the similar result of experiment of doing with the oligonucleotide derivative that has lipophilic group.3 ' end of its molecule of S96-5296 (sequence numbering NO.20) modifies with a C-16 fat base and it contains 8 phosphodiesters and 11 phosphorothioate bonds.It is 3 ' pyrene composition that S96-5297 (sequence numbering NO.21) has identical skeleton and 3 ' terminal modification.These hydrophobicity compositions can improve absorbed degree of oligonucleotide and activity when mixing with Cellfectin , and these phosphodiester bonds can improve the activity of these oligonucleotide.Symbol () expression S96-5296 (sequence numbering NO.20), symbol () expression S96-5296 (sequence numbering NO.20) and 10 μ g/mlCellfectin , symbol (o) expression S96-5297 (sequence numbering NO.21), symbol () expression S96-5297 (sequence numbering NO.21) and 10 μ g/ml Cellfectin , the Cellfectin  of symbol () expression 0.2 μ M T30639 (sequence numbering No.2) and 10 μ g/ml.

The anti-VEGF activity of embodiment VII antisense oligonucleotide

Containing the not generation of the phosphorothioate antisense oligonucleotide pair cell VEGF of carrying base modification does not have tangible influence, not dependency specificity inhibition (data do not provide) of some sequences when surpassing 1 μ M except concentration.These results conform to other research, show: the rub simple sulfur substituted phosphate oligonucleotide of dosage of low sub-micro is not effective inhibition, this oligonucleotide shows the metabolic non-specific effect of pair cell (summary Stein and Cheng, 1993 under high density; Wagner, 1994).But, contain the C5-proyl and modify the oligonucleotide specificity of pyrimidine people such as (, 1993) Wagner thiophosphatephosphorothioate and suppress cell and produce VEGF.See Fig. 3.

The melting temperature(Tm) of the oligonucleotide that these processes are modified exceeds about 15 ℃ than the melting temperature(Tm) of the oligonucleotide of other unmodified.See Table 2.This shows that the avidity of modified oligonucleotide and its target will be higher than those not modified forms.

The ratio of the suitableeest oligonucleotide and Cellfectin : cell depends on the chemical property of each composition in the preparation for the oligonucleotide-absorption portion of cationic-liposome mixture, depend in part on its concentration and relative mass ratio thereof, another part depends on the endocytosis ability of target cell.Oligonucleotide T30639 (sequence numbering NO.2) and Cellfectin  are imposed on the NHEK cell jointly, and the ratio of oligonucleotide and TMTPS is 1: 3 (mass ratio), can reach best activity.In a related experiment, change the concentration of oligonucleotide but keep the ratio of oligonucleotide and cation lipid, mensuration is with respect to the influence of " justice is arranged " contrast T30691 (sequence numbering NO.27) to vegf expression.Oligonucleotide T30639 (sequence numbering NO.2) shows special anti-VEGF activity control oligonucleotide does not then have effect (seeing Fig. 4 and Fig. 5).

SPDC+DOPE or DC-Chol+DOPE (Liposomal formulation, weight ratio 1: 1) preparation is for the influence of oligonucleotide effect: have multiple absorption enhancer to be used to (Behr, 1994 in the exonuclease treatment; People such as Guy-Caffey, 1995, people such as Lewis, 1996).A kind of compound wherein is SPDC conjugate (SpdC) (people such as Guy-Caffey, 1995).This compound is higher than under the concentration of wanting required for the present invention pair cell far away nontoxic, and handle rodent also nontoxicity more than 1 week continuously.Another kind of cationic-liposome DC-Chol (Gao and Huang, 1991) is proved to be and can be used for gene therapy, and also nontoxic relatively in cell system.Is 1: 0.5 with the quality of Liposomal formulation SpdC/DOPE and DC-Chol/DOPE (SpdC or DC-Chol and two oleoyl phosphatldylcholines) than scope, 1: 1,1: 1.5 and 1: 2, wherein 1: 1 ratio with the detection of the anti-VEGF of NHEK cell in the most effective.See Fig. 5.The composition effect of band cationoid reagent is than active high 20%-40% (Fig. 6 of band Cellfectin .)。

The short-term of oligonucleotide agent pair cell is handled just is enough to produce the long term inhibition effect that VEGF is produced.Through ofer short duration be exposed to composition disclosed in this invention after the expression of VEGF descend.For example, the activity of hatching the anti-VEGF that is shown in 4 hours will be spent the night and is exposed to the effect of oligonucleotide.See Fig. 7-9.Surprisingly, this effect continues 3 days at least in whole experimentations.Fig. 7-9.

Other experiment shows that the antisense oligonucleotide that contains thiophosphatephosphorothioate and the chimeric skeleton of phosphodiester is a kind of effective vegf expression inhibition.Especially, contain end modified S96-5296 (sequence numbering NO.20) of the chimeric variant (sequence numbering NO.2) of 10 thiophosphatephosphorothioates and 8 phosphodiester bond T30639 and lipid and S96-5297 (sequence numbering NO.21) have SpdC/DOPE in the presence of show splendid activity (Fig. 7).Be longer than 3 days (Fig. 7) in the inhibition of only cultivating back VEGF in 4 hours.When not having SpdC to exist, this chimeric oligonucleotide skeleton can not influence vegf expression.Therefore the phosphorothioate bond that reduces oligonucleotide can improve effect, and reduces non-specific influence.Absorption is most important to the effect of oligonucleotide.The inhibition of VEGF in the embodiment VIII body.

A. specific purpose.

The expression of vascular endothelial growth factor (VEGF) raises and often participates in and proliferative diabetic retinopathy, the process of the eye angiogenesis that neovascular glaucoma and many other blinding conditions are relevant.Amphiblestroid local asphyxia causes angiogenin protein VEGF synthetic to increase, and this has just caused the hyperplasia of vascular endothelial cell, and has caused a large amount of undesired formation of retina, optic nerve and iris medium vessels.Yet so far to this situation do not have as yet a kind of can received treatment means.Our overall goal by rational design and detection means set up a kind of new, potential curative antisense oligonucleotide vegf expression inhibition is arranged, its purpose is to treat people's the neovascularity hyperplasia relevant with retinal ischemia.Our the up-to-date experiment in vitro result on people's cell culture system shows, we can prepare specific oligonucleotide agent, it can suppress to surpass 50% VEGF cell expressing in sub-micro rubs concentration range, our target for this purpose is that our external discovery is generalized in the relevant neovascularity hyperplasia rat model of VEGF.We are at special purpose:

1. the antisense oligonucleotide " storehouse " that synthesizes a direct antagonism rat VEGF mRNA.Three kinds of main and a kind of accessory VEGF spliceosomes are arranged.Ten kinds of oligonucleotide will be target with the common region of these RNA sequences.They will also contain nuclease resistance skeleton and can improve it and the base of the modified of target mRNA binding affinity.

2. set up rat cell individual layer and spherule model activity and toxicity to detect these antisense oligonucleotides and preparation thereof.Adopted the C6 glioma cell line at this, because it has been widely used in studying the function of VEGF.Whether the oligonucleotide that spherule (cell mass) will be used to estimate us can pass cellular layer.

3. estimate the efficient of oligonucleotide with various cell absorption enhancers.Compared at all cpds and a lot of commercial reagent that Aronex developed.

4. set up a kind of detection method that is amenable to obtain to support the data of antisense mechanism.Designing this experiment in vitro is for verifying whether we can hit only a kind of VEGF phenogen specifically, so that answer whether our oligonucleotide is to work by the pattern of envisioning.This will help the design of future to antisense oligonucleotide.

5. detect most promising antisense oligonucleotide with iris blood vessel hyperplasia model in the body of rat.These researchs will with finish jointly at the Dr.Anthony of Harvard Adamis.

After finishing autotelic research, we wish to obtain the bulk information about effect in the 1-2 kind oligonucleotide body.Also can obtain some about dosage, possible information such as the mechanism of action, cell validity and potential toxicity.Can reduce 20% of the formation of blood vessel and/or vegf expression in vivo as any oligonucleotide, we just think a kind of positive progress and do more detailed research in II phase research process in animal models.

Select oligonucleotide, very high with other RNA bonded risk non-specificly, and the selection of high G content sequence can cause the tetrameric formation of G-do not expected, this will cause in conjunction with in minimizing people such as (, 1996) Bishop of required free spiral form.The another kind of method that have a mind to adopt is the oligonucleotide of crossing with the selective modification that contains C5-proyl pyrimidine, and its modification can cause not having highly effective combination (people such as Wagner, 1993 of overt toxicity; People such as Fenster, 1994).We have found the effect fairly good (seeing initial result) that proyl is modified recently.

Improve the method for oligonucleotide nuclease resistance: the oligonucleotide that contains natural phosphodiester backbone is extremely sensitive to serum and nucleus enzyme.We have determined that the long transformation period of oligonucleotide in serum of a kind of stochastic sequence 17 bases was less than 3 minutes people such as (, 1996) Bishop.Another kind method is to adopt the oligomer have phosphorothioate backbone people such as (, 1991) Stein, and this modification can be brought up to 1 day or longer significantly with the serum half-life of oligonucleotide.The use of phosphorothioate bond is thought can cause some nonspecific effects under high level, but it is said as following discussion, we wish synthetic a kind of oligonucleotide of modifying through specificity, it just can tell under a very low concentration, has therefore reduced the risk of non-specific interaction.Also tested the oligonucleotide of other skeleton, bigger non-specific effect was arranged but all have than thiophosphatephosphorothioate oligonucleotide.

A kind ofly improve the method that the oligonucleotide cell absorbs: the distribution of ubcellular property studies show that, in the acid-treated cell of fluorescence oligonucleoside, it accumulates in the circumnuclear endoplasmic reticulum people such as (, 1995) Guy-Caffey mostly.The oligonucleotide transportation of passing plasma membrane or endoplasmic reticulum seemingly of rate-limiting step in the internalization process.There are two kinds of possible approach can promote oligonucleotide to pass the transportation of film lipid bilayer.In first approach, can and a kind ofly can improve it and cytolemma is affine and the compound covalent coupling of penetrating character with oligomer, for example with cholesterol coupling (people such as Letsinger, 1989).We have proposed a kind of novel suitable modification recently, a kind of lipophilic ferrocene chain (seeing experimental design), and this material has improved the effect of antisense oligonucleotide significantly.In addition, the absorption enhancer that also can adopt other is as cation lipid or Liposomal formulation etc.These reagent are attractive to be because they are multi-functional, promptly can import multiple oligonucleotide simultaneously with same transport agent.Positively charged and a nucleic acid bonded head group are mixed in the design of these cation lipids, and the tail of energy and membrane interaction, and this part is intended to and merges lipid and interact and/or cause the instability of cytolemma.The activity of many cation lipid preparations (as Lipofectin) is subjected to for example influence of concentration of serum etc. in the composition of nucleic acid and quality, cell type and the cell growth medium of multiple factor.Some preparation is cytotoxic in addition.These defectives have seriously limited transportation agent the application in animal system of these compound as therapeutic oligonucleotide, and press for effective absorption enhancer.We have synthesized a kind of new transport agent, and SPDC (SpdC) even having under the situation of serum, also can improve the ability that the oligonucleotide cell absorbs and pass cytolemma people such as (, 1995) Guy-Caffey.To estimate as the part of purpose research the preparation that uses this compound.

Our purpose is to establish a kind of anti-sense oligonucleotide agent that can suppress the blood vessel hyperplasia of ocular cell vegf expression and reduction simultaneously and disease-related.These researchs are subjected to the inspiration of our a nearest discovery, and that is exactly, and through the antisense oligonucleotide of chemically modified very strong inhibition activity are arranged under the dosage that sub-micro rubs.In addition, the outgrowth animal model of VEGF related artery that utilizes Dr.Adamis to set up, we can detect wherein compounds effective in vivo.Preliminary result:

Sum up: the target of our initial a series of successive experiment is to improve active universal principle of antisense oligonucleotide and method in discovery and/or the vegf expression model of verifying some cell bases.Preliminary, our target is: the screening of setting up a kind of quantitative cell levels detects the method for antisense oligonucleotide to the effect of vegf protein matter level.More synthetic have structural modification and make it improve binding affinity, nuclease resistance and specific oligonucleotide target mRNA.Detect the cell internalizing of the oligonucleotide of using with new absorption enhancer (some is synthetic at Aronex) promotion and the preparation that film penetrates.

The design of oligonucleotide: because we expect a kind of final can the test in our compound in human body, the present invention uses directly the antisense oligonucleotide at people VEGF mRNA at the very start.In order to reach the maximum effect expressed of suppressing, we selected can with the common sequences complementary antisense oligonucleotide (table 3) of four kinds of VEGF mRNA.

Antisense oligonucleotide

(initiator codon AUG is at mRNA sequence .57) T30638:mRNA seq.87-105 5 '-a ^*g ^*a ^*g ^*C ^*a ^*g ^*C ^*a ^*a ^*g ^*g ^*C ^*g ^*a ^*g ^*g ^*C ^*T-3T30639:mRNA seq.185-023 5 '-g ^*C ^*g ^*C ^*U ^*g ^*a ^*U ^*a ^*g ^*a ^*C ^*a ^*U ^*C ^*C ^*a ^*U ^*G-3T30640:mRNA seq.204-222 5 '-C ^*g ^*a ^*U ^*U ^*g ^*g ^*a ^*U ^*g ^*g ^*C ^*a ^*g ^*U ^*a ^*g ^*C ^*T-3T30641:mRNA seq.232-250 5 '-U ^*a ^*C ^*U ^*C ^*C ^*U ^*g ^*g ^*a ^*a ^*g ^*a ^*U ^*g ^*U ^*C ^*C ^*A-3

Whole thiophosphatephosphorothioates ( ^*) skeleton has the nuclease resistance.

C5-proyl pyrimidine is to improve the binding affinity to target RNA.

The direct antisense oligonucleotide of table 3. at people VEGF

Consistent with Initial Report people such as (, 1993) Wagner, we find that C5-proyl pyrimidine bases replace the Tm value that the duplex that improved indication interchain avidity forms ,-60 ℃ of oligonucleotide that can be never modified are elevated to above 80 ℃.As a kind of contrast, we have also synthesized " justice is arranged " oligonucleotide T30691 of the identical and identical modification of size simultaneously.

The cell detection of oligonucleotide: the human keratinized cell of adopt cultivating, a kind of primary cell system of secretion of VEGF estimates the activity of the counterpart and the different preparation of antisense oligonucleotide, its modified.Cell is inoculated into by the density of 50,000 cells/well on 46 orifice plates of KGM substratum of 0.5ml (Clonetics).Identify that with enzyme linked immunological absorption (ELISA) method mensuration is secreted into the level (R﹠amp of the VEGF in the growth medium; D Systems).It is linear that this detection is in the 5-1000pg/ml scope.Our detection shows when the NHEK cell is grown in the substratum of recommending, and is seeded in the 0.5ml substratum 100,000 cells and can produces about 150pgVEGF (about 300pg/ml in undressed contrast supernatant) in 15 hours.

A kind of commercial cation lipid body preparation is adopted in the detection of most cell levels, as a kind of in cell a kind of transfection agents of transporter gene finish.(Cellfectin  is available from LifeTechnologies).Find other commercial transfection reagent or poisonous or effect is relatively poor relatively (7 times detect).The elusive effect of a kind of Cellfectin  is when imposing on cell separately, and in contrast, in fact it promote VEGF to produce.Reason the unknown wherein.We begin to adopt SPDC preparation (people such as Guy-Caffey, 1995) recently.

In experiment the earliest, used not have the phosphorothioate antisense oligonucleotide that the C5-proyl is modified, we find that it does not have influence (even under the born of the same parents' extracellular concentration up to 5 μ M, no matter whether having added absorption enhancer) to the VEGF level.On higher level, have seldom appear as non-specific inhibition (data do not provide).When with containing C5-proyl pyrimidine (people such as Wagner, 1993) replace cytosine(Cyt) and thymidine after, the Tm value that records in vivo improves 20 ℃, and this avidity that shows oligonucleotide and its synthetic target RNA is much higher than not modified variant (data do not provide).We are having or not the effect that detects these oligonucleotide under the situation of absorption enhancer, and the ratio of change absorption enhancer and oligonucleotide is in the hope of obtaining the prescription an of the best.We find that different oligonucleotide has different influences to the VEGF level.

Fig. 3. three kinds in four kinds of oligonucleotide have activity in the scope of 0.2 μ M when 10 μ g/ml Cellfectin  are arranged.Contrast has MODN not have effect (not providing).The wherein the most effective oligonucleotide of surveying, T30639 has been used for the optimization research of back.

The suitableeest oligonucleotide and the ratio of absorption enhancer: in the experiment below, we maintain 1: 3 with oligonucleotide (T30639 of antisense and the T30691 contrast that justice is arranged) and the mass ratio of cationic-liposome composition cF, and detect the influence to the VEGF generation.T30639+cF has shown again the anti-VEGF activity of specificity, and control oligonucleotide does not then have effect.Figure 11.

Figure 11. oligonucleotide Cellfectin  (1: 3) preparation is to the influence of vegf expression.

Under high density, show nonspecific effect (not providing).It should be noted that we do not attempt free absorption enhancer and bondage material are separated.When we change a kind of and this is certain to influential during alternative relative proportion.

The influence of oligonucleotide size: in next one experiment, whether we seek can keep the anti-VEGF activity of its specificity under the situation that reduces the oligonucleotide size.Synthesizing of short oligomer is also more cheap.But, detecting with same NHEK, we find that the oligonucleotide of 19 bases is more effective than the derivative of 16 or 14 bases, all oligonucleotide are all used with 10 μ g/mlCellfectin .The ratio that changes Cellfetin  and oligonucleotide can't change its relative reactivity (not providing).

SPDC+DOPE or DC-chol+DOPE (Liposomal formulation, weight ratio 1: 1) preparation is to the influence of oligonucleotide effect: recently, we have begun to develop the surrogate of a kind of Cellfetin , because life-time service Cellfetin  can produce some toxicity by pair cell.Found a kind of absorption enhancer: pair cell is nontoxic on employed level for SPDC conjugate (SpdC) (people such as Guy-Catfey, 1995), and uses on one's body to reach handled rodent and 1 week also do not have the toxicity that can measure.Cation lipid DC-Chol (people such as Gao, 1991) has been approved for the clinical trial of gene therapy, and it has very low toxicity in cell system.Initial data show that this novel Liposomal formulation compares the high 20%-40% of efficient of Cellfetin  in parallel laboratory test.

The short-term of oligonucleotide agent pair cell is handled just is enough to obtain long term inhibition effect that VEGF is produced: in the experiment, we are once with cell and oligonucleotide and the common overnight incubation of absorption enhancer more than all.We seek subsequently, and handling with the oligonucleotide pair cell, whether the time of weak point also can reach identical anti-VEGF activity level.We find it is so really: handle 4 hours flush awaies afterwards, be replaced with the fresh substratum of not adding, can't reduce anti-VEGF activity (Figure 12).And this effect is sustainable to reach 3 days (time of experiment).

Figure 12. handle with four kinds of oligonucleotide+cF, change pure substratum then into.This inhibition almost can continue 3 days.And contrast has MODN not have obvious suppression (result does not provide).

In fact, with respect to contrast (no oligonucleotide), the inhibition effect is more viewed will getting well than in the past.One of them reason may be, in culture experiment, VEGF matter albumen can constantly be synthesized (if not blocked by antisense scant polymer yet) by original already present mRNA, and accumulates in substratum.By after 4 hours, changing substratum, can eliminate the source of " background " VEGF.These experimental datas are very important for experimental system in the body, do not need frequent drug administration because this long-acting antisense effect shows.The checking that this point also need be tested in external and body specially.

Ferrocene link coupled oligonucleotide: our recent findings is a kind of to be the most effective VEGF inhibition in the experiment in vitro of preparation at us of oligonucleotide that metallocenes is modified and absorption enhancer, and in the working concentration scope almost non-toxic property (Figure 13).This oligonucleotide agent that is made into Cellfetin  has the anti-VEGF activity of specificity when 20 μ M concentration.This ferrocene chain has been designed to improve the bonding force (D.Mulvet, Aronex, person-to-person communication) of oligonucleotide to film.In addition, we infer the cellular localization of the active transport system help oligonucleotide that this lipophilic ferrous components may be by utilizing cell and wear the film motion.Surpass the scope of this patent for the further research of modified mechanism, and can be used as the purpose of another research.But we have found the high reactivity of this oligonucleotide of modifying through ferrocene, and prompting is when we also should explore this approach during the detection oligonucleotide in the model in vivo.

Figure 13 .T0639 (antisense) is through the strong Antisense Suppression effect of ferrocene link coupled variant.

As test the detailed description that designs in the joint, adult rat iris blood vessel hyperplasia model provides a method for the activity of detection by quantitative antisense oligonucleotide.In this detection, rat was placed the hypoxemia cabin 1-21 days, with proliferation of vessels quantity in the digital imagery quantitative assay iris.Shown in data (Figure 14 .),, the process of blood vessel hyperplasia is clearly arranged along with the prolongation of incubation time.Retina R NA level also increases but does not reach identical degree (Figure 15).After being subjected to the inspiration of preliminary experimental result and rat model data, we are intended to obtain similar concept nature in the intravital blood vessel hyperplasia model now proves.

C. correlation experience.

Main investigator Nilabh Chaudhary, the Doctor of Science has rich experience in the Celluar and Molecular Biology field.He has obtained biochemical doctorate in 1984 in Canadian London city WesternOntario university.He has obtained a subsidy of DamonRunyon-Walter Winchell cancer foundation again subsequently, to proceed his post-doctoral research in the laboratory of the Dr.Gunter Blobel of Rockefeller university.1986 he be appointed the same breadboard assistant of Howard Hughes Institute for Medical Research.Doctor Chaudhary has added Triplex (being renamed as Aronex recently) beginning the research project of a cytobiology in 1992 as the Research Scientist, and its target is a kind of technology that can improve cell and nucleus to the receptivity of exonuclease treatment of exploitation.He and the combined efforts of an organic chemist troop design, synthesize and test new oligonucleotides-modified and absorption enhancer jointly.Doctor Chauahary is about the common author of the structure-scientific papers such as function mutual relationship of the oligonucleotide that the treatment potentiality are arranged and has designed the approach that improves its cell internalizing and effect.He in design based on the detection architecture of cell, immunochemical technique, proteinic micro quantitative determination technology, nucleic acid purification and molecule clone technology, subcellular organelle separates, membrane protein and fat separate and there is rich experience aspect such as fluorescence microscopy.

Anthony P.Adamis, the doctor of medicine is the co-worker and the consultant of this problem.It is the research assistant of department outside the assistant professor of Harvard medical college ophthalmology system and Boston children's hospital.The dependency of the colleague of doctor Adamis and Ta between the proliferative diabetic retinopathy process of the increase, the blood vessel hyperplasia that confirmed eye VEGF level in 1994 first and the one of the main reasons that causes losing one's sight.By rising at that time in the research of carrying out, he has confirmed stimulating vegf expression to cause anoxybiotic physiological action in the medium vessels hyperplasia.He comprises the extensive studies experience: patient's clinical study, design is based on the exploitation of the rodent disease model of the concept nature proof of cell and the application of molecular biology and clone technology.He has delivered more than 20 piece of article, wherein 10 pieces of fields about blood vessel hyperplasia.

D. experimental design and method.

The method general introduction: the target of this design is to confirm the effect of anti-VEGF antisense compounds in the blood vessel hyperplasia animal model.For this reason, we prepare to carry out a series of experiment in vitro, so that find the antisense agents that is hopeful to be used for experiment in the body most.We will be to begin screening in 10 kinds of candidates' of target the antisense oligonucleotide (19 bases) " library " with rat VEGF mRNA.These oligonucleotide contain C5-proyl pyrimidine, and improving the binding affinity of itself and target mRNA, and key can be given the nuclease resistance between thiophosphatephosphorothioate Nucleotide.

In order to carry out cell detection, we plan to use rat C6 (neurospongioma) cell, and it produces abundant VEGF mRNA and protein under hypoxia condition.Detect on 96 orifice plates, its form is used to screen the activity of various antisenses or control oligonucleotide preparation.They are monitored with the ELISA method for the time course of the influence of excretory VEGF level in the substratum of extracellular.In order to improve the absorption of cell, oligonucleotide and novel absorption enhancer are together used.Measure the different ratios between nucleic acid and the liposome.In addition, therefrom select two kinds of " the best " antisense sequences and be used for and 3 ' the lipophilic ferrocene coupling, this modification can promote that antisense oligonucleotide enters cell.Also hybridize to determine of the influence (with the effect of appropriate contrast compare) of the oligonucleotide (or its preparation) of these two kinds of the bests to VEGF mRNA level with Northern.

When attempting the evidence of antisense mechanism is provided, designed a series of relatively independent experiment in vitro.Be it with one or both VEGFmRNA (in the rat 3 kinds main with a kind of accessory) with the VEGF phenogen specific antisense oligonucleotide of special design respectively be target processing C6 cell.The level relatively that is used to measure every kind of VEGF mRNA with the experiment of RNA enzyme protection.In principle, if this antisense effect truly has sequence-specific, have only the expression of target phenogen to be reduced.Not homotactic oligonucleotide is invalid.

The cytotoxicity of effective antisense compounds detects in two kinds of different clones, and the preparation of its toxic minimum will be used for C6 cell spheroid phantom type, and this model is used for determining whether these oligonucleotide can pass through cellular layer.In the spherule in the successive cellular layer level of VEGF mRNA measure with in-situ hybridization method.The use of absorption enhancer and chain also will detect in this model.Anti-angiogenic proliferative activity with the strongest anti-VEGF oligonucleotide will detect in animal with the iris blood vessel hyperplasia model of rat eye.The albinism rat is put into hypoxemia cabin (reaching for two weeks), and monitor the hyperplasia of iris medium vessels with a kind of non-invasive quantitative digital imaging system.In this model, only can be observed the increase of vasculogenesis under the anoxia condition after 1-2 days.Detected oligonucleotide (or its preparation) is directly introduced in the eyes of rat, the another eyes are as undressed contrast.Processing reaches l after week, measures any influence to vasculogenesis.(vitreum, if possible) change of vegf protein matter and mRNA level detects with ELISA and Northern hybridizing method respectively in the retina.Splash any side effect of appearance.According to initial result, can adopt the multiple doses experiment.Also detect the effect of control oligonucleotide.A series of experimentation on animalies that toxicity the most effective minimum preparation (suppressing＞20% neovascularization) is used to expand then are as the part of these research second stages.

The selection of antisense and control oligonucleotide: for the maximum that reaches vegf expression suppresses, we will synthesize antisense oligonucleotides in 10 common districts that can be incorporated into all VEGF phenogens in rat people such as (, 1990) Conn.Synthetic 10 kinds of oligonucleotide (wherein not having tangible hairpin structure or G-enrichment region) of selecting at random basically are to be used for the screening of the first round.Wherein have 5 kinds to be that rat is specific, other 5 kinds of selections also can with people mRNA bonded.And unclear the evolution go up conservative site as the target spot of antisense or bad, can not predict in conjunction with preferred target spot though most ofs nearest evidence shows for antisense.All synthetic oligonucleotide all contain C5-proyl pyrimidine (to improve its binding affinity to target) and phosphorothioate bond (nuclease resistance) (people such as Wagner, 1993, people such as Fenster, 1994).The oligonucleotide that we have had several " uncorrelated " is with comparing, but if desired, and we will synthesize a kind ofly has the identical size and the control oligonucleotide of based composition with antisense sequences.These oligonucleotide will be synthetic by finishing in the synthetic group of the oligonucleotide of Aronex, purifying (＞95%, use HPLC) and evaluation work.

The oligonucleotide that is used for study on mechanism:, will prepare the phenogen-specific oligonucleotide of several (about 4 kinds according to its effect) 20 bases in order to obtain to support the data of antisense mechanism of action.A kind of oligonucleotide is direct just should not combine with the mRNA of VEGF-120 at a kind of sequence on the VEGF-165mRNA.Similarly, the shearing linker complementation of the oligonucleotide of one 20 base and VEGF-120 (being 10 bases in each exon) just should not also combine with VEGF-165.Synthesize and have the oligonucleotide (two portions sequence oppositely repeats) of tumor-necrosis factor glycoproteins with comparing.These oligonucleotide will be determined by the level relatively of more different mRNA the influence of vegf expression.With the level relatively of every kind of mRNA of RNA enzyme protection measuring (Ambiou, Austin, TX).Probe in this (length is approximately the 150-250 base) has been used rat mRNA sequence specific primers and RT-PCR technology (Perkin Elmer) preparation.

Cell cultures: the biology screening will carried out in the deutero-C6 glioma cell from the rat neurospongioma.Main VEGF phenogen is VEGF-165 (amino acid) and VEGF-120 (each 46%) in this clone, and VEGF-188 only accounts for about 8% (people such as Bacic, 1995).This clone has been widely used in studying the 26S Proteasome Structure and Function of VEGF.To induce the synthetic of VEGF in order stimulating, cell to be placed a hypoxemia cabin (GasPakPlus anaerobism incubator) (BBL.Micobilology System), remove all oxygen molecules with hydrogen and palladium catalyst by hypoxia condition.(people such as Stein, 1995).Typical incubation time scope is 6-8 hour.Perhaps handle culture with the cobalt chloride of 100-300 μ M, cobalt chloride is disturbed protoheme dependency hypoxemia reactive system and is activated the hypoxemia response factor that a kind of VEGF of inducing mRNA transcribes.

The active in-vitro evaluation of antisense oligonucleotide: the C6 cell that grows into individual layer is with containing 5% foetal calf serum and antibiotic Dulbecco substratum is kept.For the oligonucleotide of the anti-VEGF of rough determination, the density of cell by 10,000 or 20,000 cells/well is inoculated on the 96 hole flat boards.Recover to handle cell (in the 0.25ml substratum) with oligonucleotide after 1 day.Test 2 types substratum: conventional C6 substratum that contains serum or Optimen (Life Technologies), this is the few substratum of a kind of serum, by reducing interference in the serum composition through being usually used in improving the efficient of transfection.After different incubation time in the hypoxemia cabin, supernatant transferred to be used for ELISA and detect a new flat board.As a principle, we utilize the form of microscope observing cell to find out unusual change or any tangible toxicity signal when detecting a kind of new preparation.

For RNA analyzes, handle a large amount of cells (＞2 * 10 with a certain amount of preparation ⁶-10 ⁷, shake in the bottle at T75).After oligonucleotide is handled (and through the hypoxemia processing, or the like), collect supernatant once more and detect, and isolation of RNA is analyzed in the following method in order to ELSA.

VEGF ELISA detects: a kind of commercialization detection kit that is applicable to the rodent system is not arranged as yet, so we with known can (RDI-1020 or RDI-4046 be available from Research Diagnostics company, and other is then available from R﹠amp with rat VEGF; D Systerms) fully the antibody of reaction has designed one.Other antiserum(antisera) at VEGF also can be used, so we have selected best combination.ELISA reagent (the enzyme di-is anti-, substrate) is available from Pierce.

RNA extracts, Northern is hybridized, the RNA enzyme protection is tested and hybridization probe: the size of VEGF mRNA mainly is because long non-translational region in 3.8-4 kilobase scope.Analyze for making RNA, with the RNAzol method (Tel-Test company, Friendswood, TX) from handled or untreated cell the total RNA of separation.For being used as probe, adopt C6 RNA and VEGF Auele Specific Primer by associating ThermoScript II-PCR (RT-PCR test kit, PerkinElmer), screen according to the size of the cDNA that derives from different mRNA then, carry out selective amplification with the phenogen Auele Specific Primer again, obtained VEGF specific fragment at last corresponding to common region and phenogen specific probe.With these probing pin clones (Invitrogen) in the PCR II plasmid cloning vector, by these sequences of order-checking conclusive evidence (Sequenase, USB).The RNA polymerase that this PCR II carrier allows radiolabeled rna probe not dependency produces and is used for the experiment of RNA enzyme protection, (test kit is available from Ambion, Austin, TX).Demarcate the amount of RNA with the beta-actin probe.For Northern hybridization, RNA (nearly 20 μ g) is separated on the methane amide gel, transfer on the nylon membrane, utilize the radiolabeled probe to detect again according to the method for standard, demarcate the amount of RNA with beta-actin.In all RNA analyzes, optical imagery instrument (Fuji, optical imagery instrument) be used for radioactivity relatively level quantitatively.

Support the experiment of antisense mechanism of action: the mRNA sequence complementation of antisense oligonucleotide of this research and coding VEGFmRNA.Yet their inhibition effects in living things system not necessarily prove the antisense mechanism of action.In fact, the many oligonucleotide of nearest analysis revealed can non-specific interference cell metabolism, especially when concentration surpasses 1 μ M (summary is seen Stein and Cheng, 1993).As if the evidence about the antisense mechanism of action is difficult to find, and has only some indirect evidences at present.We have designed experiment at present, and (though being indirect) is intended to obtain the evidence of possibility antisense mechanism.Briefly, we have synthesized and have been used for the detection of rat based on cell, suppress " phenogen specificity " antisense oligonucleotide that single VEGF variant is expressed with selectivity.Rat C6 cell (deriving from neurospongioma) can produce three kinds of main type VEGF phenogens.The experiment of RNA enzyme protection has shown in the C6 cell that about 45% VEGF is 120 amino acid whose variants, and 45% is 165 amino acid whose variants, and remaining is 188 amino acid whose variants (people such as Bacic, 1995).Use at the antisense oligonucleotide of one or more VEGF phenogens and handle cell, again with hypoxemia or add can hypoxia-mimicking 200 μ M cobalt chloride stimulate transcribing of mRNA.Handle after 9 hours, the cell levels of VEGF mRNA with the experiment of RNA enzyme protection monitored (Ambion, Austin, TX.).Importantly, determine the level of each VEGF mRNA with us by the phenogen specific probe (length is approximately 100-200 base) that the RT-PCR technology obtains, and use with respect to the level of beta-actin and demarcate.If the antisense mechanism of action is set up, and with single phenogen specific oligonucleotide, then has only expression reduction for other kind of a kind of VEGF.On the other hand, a kind of and all VEGF common region complementary oligonucleotide should reduce the expression of each VEGF.

Synthesizing of ferrocene link coupled oligonucleotide: through after former screenings of taking turns, the oligonucleotide that activity is the highest and side effect is minimum adds a ferrocene chain by 3 ' end, in the hope of further improving the absorption and the activity specific (see PRELIMINARY RESULTS, T30781 (+ferrocene) is not to T30639 (modified)) of oligonucleotide.If effectively, the activity of more this oligonucleotide and a kind of stochastic sequence and contrast that contain same chain.Do we suppose that this ferrocene composition can make active transport or the penetrating system (iron ion of oligonucleotide by utilizing cell?), but this mechanism is studied as yet.

The use of absorption enhancer: in most of the cases, for the ease of entering cell, under the situation that the lipofectamine reagent of being everlasting exists with the oligonucleotide transfered cell.As the transfection agents of gene transportation, now existing many commercial cationic-liposomes, however in our detection (7 kinds of main liposomes of having tested), have only Cellfectin  (Life Technologies) to show consistent effect.Cellfectin  is that a kind of polyamino fat four palmityl spermidines and two oleoyl phosphatidyl ethanolamines (DOPE) are pressed the lipid mixt of 1: 1.5 (wt/wt).We have also used another kind of liposome DC-chol, and this is developed by Leaf Huang (Gao and Huang, 1991), and approved be used for gene therapy clinical trial (Rgene Therapeutics, The Woodlands, TX).In addition, we have have also designed and synthesized a series of cells that can obviously improve oligonucleotide such as (Guy-Caffey people, 1995) and have absorbed, and are not subjected to that serum influences and obvious xicity related novel polyamino fat absorption enhancer not.Wherein the most effective a kind of be SpdC (SPDC).When mouse was gone in intravenous injection, SpdC can promote the transportation of oligonucleotide to many tissues, and when to mouse with the concentration of SpdC up to 100/mg/kg/ days, injected for 1 week continuously and do not see toxicity (data do not provide) when above.

We prepared Liposomal formulation SpdC/DOPE and DC-chol/DOPE (SpdC or DC-chol and two oleoyl phosphatldylcholines) quality than scope from 1: 0.5,1: 1,1: 1.5 to 1: 2.In the anti-VEGF of our NHEK cell early detects, it is 1: 1 for two kinds of the most effective ratios of cationic-liposome.We just be intended to study these preparations C6 clone, C6 spherule and finally in eyeball phantom for the active enhancement of anti-VEGF oligonucleotide.

Definite mechanism of action about these absorption patterns still exists arguement.Cationic-liposome mixed almost always producing some small precipitations with nucleic acid, these precipitations can enter cell effectively.In order to keep consistency, and in order to find a potential principle, we monitor these particulate sizes with a kind of Coulter granular size instrument.

The preparation that is used for injection in the body: we use a not really rigorous noun " preparation " is in order to illustrate at the employed multicomponent mixture of administration process.But when we carry out the body build-in test during stage, it is prudent being designed for and being injected into intraocular optimal formulation, when especially comprising absorption enhancer.Quantity that other parameters that need consider are antisenses and concentration, salt, granular size, pH value and carrier.Dr.Joe Wyse helps us to select and prepares best preparation.

Cytotoxicity detects: the density of cell by 500 cells/well is inoculated on 96 orifice plates.Inoculate after one day, handle cell (4 holes of each extent of dilution) with the oligonucleotide agent of serial dilution.Handle after 1 day or 4 days, monitor the influence (CellTiter96 Aqueous cell proliferation detects, Promega company) of the survival of pair cell with a kind of on-radiation detection system.To the most effective oligonucleotide, on three kinds of different clones, carry out this detection (comprising C6, NHEK and fibroblast).

The active evaluation of antisense in spherule: the C6 cell grows into individual layer (4.5g glucose/L, DMEM, 5%FCS added with antibiotic) usually, can be induced to grow up to about 0.4-0.8mm cell spheroid body or aggregate.Understand our antisense oligonucleotide, make preparation with fat or other antisense oligonucleotide can pass the cellular layer of spherule and still have biological activity highly significant.Utilize the described method of people such as Stein (1995) to prepare spherule.With the Micro-Organism Culture Dish of C6 cell, grew 48 hours from the media transfer of confluent growth to non-adherent property.The spherule that occurs is transferred to rotation shake in the bottle, continued growth 10 days (80 rev/mins) gets spherule of the same size through the liquid getting device suction nozzle precipitation sorting of a 10ml.In 6 weeks of continued growth, every other day change a subculture.In bottle charge into 95% air and 5% CO toward shaking every day ₂, to guarantee to have enough oxygen and pH.

With antisense agents or suitable control treatment spherule, place hypoxemia to reach 1 day then to induce VEGF synthetic, detect the level of VEGF mRNA in the spherule subsequently with in situ hybridization.For this reason, the Paraformaldehyde 96 with 4% is spherule fixedly, and is freezing, is cut into the thick sheet of 10 μ m, makes the VEGF that in situ hybridization detects generation with aforesaid method with the DNA or the rna probe of 35S mark.

These finished sections are redyed with phenodin and cosin dyestuff, and the radioautograph (Guy-Caffey waits the people) through in a few days detects these sections by the bright visual field and dark field illumination.

The distribution of VEGF RNA will reflect the inhibition degree that antisense oligonucleotide reaches.Ideal, the level of VEGF mRNA is all very low in all layers.Most probable, lower at the VEGF on top layer, or this is because medicine does not penetrate cellular layer, or because in the cell of central authorities anoxic more, thereby produce more VEGF.If transportation can only enter the top layer, we will attempt to design new route of administration.

The anti-angiogenic outgrowth activity in vivo evaluation of VEGF antisense oligonucleotide: the relevant iris blood vessel hyperplasia model of the rodent VEGF-that grows up: adult rat generates in iris at low-oxygen environment moderate stimulation neovascularity.The hyperplasia of blood vessel is associated with the rise of VEGF mRNA level in the retina in time.The order of this eyeball incident in monkey iris flush model and people's iris new vessel hyperplasia same as seen, the reason that wherein known ischemic VEGF retina is the iris blood vessel hyperplasia.We are intended to utilize this model to detect the activity of the antisense compounds that can reduce blood vessel hyperplasia.Experimentation on animals comprises that the Northern of processing, surgical operation, photograph, quantitative computer analysis and the VEGF mRNA of animal analyzes and will carry out in the Adamis laboratory.

350-400g is adult, and the albinism Sprague-Dawley of Kingston colony rat (female) placed hypoxemia cabin (1% oxygen) 1-21 days.Before and after cultivating, carry out biology microscope inspection and conventional slit camera imaging.Note the process that iris vasodilation in low-oxygen environment, distortion and vessel density increase.In these albinism animals, can be clear that the blood vessel of iris.The standard photographs scanning input computer program (NIH image, 1.54D software) of iris is also used the area of picture element analysis with the fuzzy quantitative new vessel of mode.The iris blood vessel showed gradual increase in 14 days.The immunostaining of proliferating cell nuclear antigen (PCNA) and factor proves that also endotheli ocytosis starts from the 2nd day; Proof increases blood vessel and represents blood vessel hyperplasia.The isolating retina that is used for Northern hybridization confirms that the VEGF mRNA level of the retina stable state of anoxia condition animal raises.In a word, the growth of adult rat neovascularity in low-oxygen environment moderate stimulation iris.Our purpose is to utilize this model to detect the effect of candidate's anti-VEGF preparation.

Determine of the experiment of the hypoxia condition of interruption to the effect of neovascularization:

Above-mentioned rat model only is used for the successive hypoxemia to be handled.Because if hypoxemia handle can this model of interrupted words can be practical more, for example, for repetitively administered, the degree of condition iris blood vessel hyperplasia after a bit of time that we wish to determine that these animals break away from hypoxemia every day.We estimate that this of short duration shifting out can increase the neovascularity hyperplasia, will have stronger blood vessel hyperplasia reaction (Reynaud and Dorey, 1994) than the animal of depressing at constant oxygen because if handle neonate rat in this way.The anoxybiotic retina can produce reactive oxygen intermediate after the oxygen supply again, and known this intermediate will increase retina VEGF mRNA protein people such as (, 1995) Kuroki.

Rat is placed hypoxemia cabin (10% oxygen) 1,3,5,7,14 and 21 days (3 animals of each time point, 18 altogether).Shifted out 1 hour their every days, and place normal indoor oxygen (oxygen level 21%).After cultivating end, take the iris photo of standard.Put to death animal and the first half of eyeball is used for PCNA dyeing and light microscopy.The 558bp VEGF cDNA random primer labelling probe that the separation retina is used for the 32p mark carries out Northern hybridization.

In 21 days process, the photo that the rise of retina VEGF mRNA and iris neovascularity generate and the data of immunohistochemical methods are relevant.The area of blood vessel can be quantitative from standard photographs, and can compare with the animal that is under the continuous hypoxia condition.From this experiment, we can be expected under the prerequisite that does not influence the hypoxemia effect, can take out number of times that the maximum of animal allows and reach each time time span from the hypoxemia cabin.

The evaluation of antisense action effect in the body: the photo of taking baseline at the 0th day.Eyes of selection anesthetized animal at random are to its injection of VEGF antisense compounds.The another eyes do not deal with.Dosage will be with reference to the result of in vitro study, but administration is no more than 20 μ l.Animal placed unbroken low-oxygen environment 7 days.When testing first, every kind of preparation is handled 6 animal (4 kinds of preparations: only add oligonucleotide; Oligonucleotide+chain, and above two kinds of difference add absorption enhancer again).Purpose is to determine whether really effectively these compounds.If blood vessel hyperplasia has tangible reduction, just use more animal.Following table has been summed up the statistical basis of required animal.In a word, every kind of processing mode will be with about 20 rats.

The quantity of the required rat of table 1.: 90% rat has the new vessel hyperplasia in this model.The level of supposing significance is 0.05 (α=0.05), and power is 0.8 (1-β=0.80), and then the quantity of each experimental group eye can be determined according to the efficient of different angiogenic inhibitors.

The required eyes quantity of control group experimental group

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90????????60????????17

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The hyperplasia of neovascularity suppresses to be defined as in these experiments: compare the area minimizing 20% that experimental group eyes medium vessels forms with the eyes of contrast.The effect of supposing a certain reagent is fine, and then the eyeball ratio of experimental group generation iris blood vessel hyperplasia is just low, has the quantity of the eyes of statistical significance just can reduce greatly in order to acquisition so.

At the 7th day, take a picture and the execution animal.Collect retina as Northern hybridization (independent).With optics imager (Moleular Dyamics) the constant attitude mRNA of VEGF is carried out quantitatively also with 28S ribosome-RNA(rRNA) signal scaling.The iris blood vessel carries out quantitatively and eyeball more treated and contrast.

F. vertebrates handling procedure: all animal processes will be carried out in the children's hospital of Harvard medical college; and strictly observe the regulations that use animal that vision and ophthalmology research association formulated is studied, and the regulations set up of Massachusattes eyes and ear medical faunae protective committee.To use 120 the albinic female Kingston Sprague-Dawley of colony rats altogether.They will be anaesthetized and with No. 30 syringe needle oligonucleotide or the control formulation to intravitreal injection 20 μ l.To impose gentamicin sulphate after the injection.Animal kept under 10% oxygen environment reached for 3 weeks.They suck CO to finish the back in experiment ₂Put to death.All experiments all will meet the regulations of OPRR.

The article Adamis that quotes, A.P., Deng the people, 1994, (molecule eye Science) Arch.Ophthalmol.118:445-450Adamis, A.P., Deng the people, 1993, (biological chemistry and biophysical research communication) Biochem.Biophys.Res.Comm., 193:631-638Adamis, A.P., Deng the people, 1996, people such as (eye Science) Arch.Ophthal.114:66-71Bacic, 1995 (pharmacy news) Pharm.News 2:V, (abst) people such as Bioshop, 1996, (journal of biological chemistry) J.Bio.Chem.271:5698-5703.conn, (3. wait the people, 1990, (PNAS) Proc.Natl.Acad.Sci.USA 87:2628-2632D ' Amore, P.A., 1994, Investigative Ophthai.Vis.Sci.35:3974-3979deYries, C., Deng the people, 1992, (science) Sicence 255:989-991Fenster, S.D., Deng the people, 1994, (biological chemistry) Biochemistry 33:8391-8398Ferrera, N., Deng the people, 1992, people such as (internal secretion summary) Endocrine Reviews 13:18-31Guy-Caffey, 1995, (journal of biological chemistry) J.Biol.Chem.270:31391-31396Gao, X., and Huang, L.1991, (biological chemistry and biophysical research communication) Biochem.Biophys.Res.Comm.179:280-285Miller, people such as J., 1994, (U.S.'s ophthalmology magazine) Am.J.Pathol.145:547-584.Milligan, J.F. wait the people, 1993 (journal of medicinal chemistry) J.Med.Chem.36:1923-1937.Plate, K.H., Deng the people, 1992, (nature) Nature 359:845-848Shima, D.T., Deng the people, 1995, people such as (molecular medicine) Molecular Med.2:64-75Shweiki, 1992, (nature) Nature 362:841-844Stein, C.A. wait the people, 1991, (pharmacological agent) Pharmac.Ther.52:365-384Stein, C.A. and Cheng, Y.C., 1993, people such as (science) Science 261:1004-1012Terman, 1992, (biological chemistry and biophysical research communication) Biochem.Biophys.Res.Comm.187:1579-1586Uhlmann, F and Peyman, A, 1990, chemistry summary (Chem.Rev.) 90:543.Wagner, people such as R.W., 1993,260:1510-1513Wagner, R.W., 1994, (nature) Natre 372:333-335

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Sequence table (1) essential information

(ⅰ) applicant: Chaudhary, Nilabh

Rao,T.Sudhakar

Revankar,Ganapathi?R.

Cossum,Paul?A.

Rando,Robert?F.

Peyman,Anusch

Uhlman,Eugen

(ⅱ) invention exercise question: the Antisense Suppression thing of vascular endothelial growth factor expression

(ⅲ) sequence number: 21

(ⅳ) contact address:

(A) address: Conley, Rose﹠amp; Tayon, P.C.

(B) street: 600 Travis, Suite 1850

(C) city: Houston

(D) state: Texas

(E) country: the U.S.

(F) postcode: 77002-2912

(ⅴ) computer-reader form:

(A) media type: floppy disk

(B) computer: IBM PC compatible

(C) operating system: Windows 95

(D) software: Microsoft Word 7.0a

(ⅵ) nearest request for data:

(A) application quantity:

(B) submission date:

(C) classification:

(ⅶ) date of application formerly:

(A) application quantity:

(B) submission date:

(ⅷ) proxy/proxy information:

(A) name: McDaniel, C.Steven

(B) registration number: 33,962

(C) document/reel number sign indicating number: 1472-07200

(ⅸ) telecom information:

(A) phone: 713/238-8010

The information of (B) fax: 713/238-8008 (2) SEQ ID NO.1 sequence:

(ⅰ) sequence signature:

(A) length: 19 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology configuration: linearity

(E) antisense: be

(ⅳ) antisense: be

(ⅸ) feature:

(A) title/keyword: misc-feature

(B) position: 1-19

(C) out of Memory :/note=" phosphorothioate bond between each residue "

(ⅹ) sequence description: the sequence information of sequence numbering NO.1:GCGCTGATAG ACATCCATG 19 (2) SEQ ID NO.2:

(ⅰ) sequence signature:

(A) length: 19 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology configuration: linearity

(ⅳ) antisense: be

(ⅸ) feature:

(A) title/keyword: misc-feature

(B) position: 1-19

(D) out of Memory :/note=" phosphorothioate bond between each residue, C5-proyl pyrimidine "

(ⅹ) sequence description: sequence numbering NO.2:

GCGCUGAUAG?ACAUCCAUG????19

(2) information of SEQ ID NO.3 sequence:

(ⅰ) sequence signature:

(A) length: 19 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology configuration: linearity

(ⅳ) antisense: be

(ⅸ) feature:

(A) title/keyword: misc-feature

(B) position: 1-19

(D) out of Memory :/note=" phosphorothioate bond between each residue, C5-proyl pyrimidine "

(ⅹ) sequence description: the information of sequence numbering NO.3:CGAUUGGAUG GCAGUAGCCT 19 (2) SEQ ID NO.4 sequences:

(ⅰ) sequence signature:

(A) length: 19 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology configuration: linearity

(ⅳ) antisense: be

(ⅸ) feature:

(A) title/keyword: misc-feature

(B) position: 1-19

(D) out of Memory :/note=" phosphorothioate bond between each residue, C5-proyl pyrimidine "

(ⅹ) sequence description: the information of sequence numbering NO.4:UACUCCUGGA AGAUGUCCA 19 (2) SEQ ID NO.5 sequences:

(ⅰ) sequence signature:

(A) length: 19 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology configuration: linearity

(ⅳ) antisense: be

(ⅸ) feature:

(A) title/keyword: misc-feature

(B) position: 6-7; 9-10; 10-11; 13-14

(D) out of Memory :/note=" phosphorothioate bond between the indication residue "

(ⅹ) sequence description: the information of sequence numbering NO.5:GCGCUGAUAG ACAUCCAUG 19 (2) SEQ ID NO.6 sequences:

(ⅰ) sequence signature:

(A) length: 19 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology configuration: linearity

(ⅳ) antisense: be

(ⅸ) feature:

(A) title/keyword: misc-feature

(B) position: 3-4; 5-6; 6-7; 9-10; 10-11; 13-14

(D) out of Memory :/note=" removing the phosphorothioate bond between the indication residue "

(ⅹ) sequence description: the information of sequence numbering NO.6:GCGCUGAUAG ACAUCCAUUG 19 (2) SEQ ID NO.7 sequences:

(ⅰ) sequence signature:

(A) length: 19 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology configuration: linearity

(ⅳ) antisense: be

(ⅸ) feature:

(A) title/keyword: misc-feature

(B) position: 6-7; 10-11;

(D) out of Memory :/note=" removing the phosphorothioate bond between the indication residue "

(ⅹ) sequence description: the information of sequence numbering NO.7:GCGCUGAUAG ACAUCCAUG 19 (2) SEQ ID NO.8 sequences:

(ⅰ) sequence signature:

(A) length: 19 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology configuration: linearity

(ⅳ) antisense: be

(ⅸ) feature:

(A) title/keyword: misc-feature

(B) position: 1-19

(D) out of Memory :/note=" phosphorothioate bond between all residues, C5-proyl pyrimidine "

(ⅹ) sequence description: the information of sequence numbering NO.8:GAAGAUGUCC ACCAGGGUC 19 (2) SEQ ID NO.9 sequences:

(ⅰ) sequence signature:

(A) length: 19 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology configuration: linearity

(ⅳ) antisense: be

(ⅸ) feature:

(A) title/keyword: misc-feature

(B) position: 1-19

(D) out of Memory :/note=" phosphorothioate bond between the indication residue, C5-proyl pyrimidine "

(ⅹ) sequence description: the information of sequence numbering NO.9:AGGAAGCUCA UCUCUCCUA 19 (2) SEQ ID NO.10 sequences:

(ⅰ) sequence signature:

(A) length: 19 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology configuration: linearity

(ⅳ) antisense: be

(ⅸ) feature:

(A) title/keyword: misc-feature

(B) position: 1-19

(D) out of Memory :/note=" phosphorothioate bond, C5-proyl pyrimidine "

(ⅹ) sequence description: the information of sequence numbering NO.10:UACACGUCUG CGGAUCUUG 19 (2) SEQ ID NO.11 sequences:

(ⅰ) sequence signature:

(A) length: 19 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology configuration: linearity

(ⅳ) antisense: be

(ⅸ) feature:

(A) title/keyword: misc-feature

(B) position: 1-19

(D) out of Memory :/note=" phosphorothioate bond, C5-proyl pyrimidine is fixed "

(ⅹ) sequence description: the information of sequence numbering NO.11:UAACUCAAGC UGCCUCGCC 19 (2) SEQ ID NO.12 sequences:

(ⅱ) sequence signature:

(A) length: 19 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology configuration: linearity

(ⅳ) antisense: be

(ⅸ) feature:

(A) title/keyword: misc-feature

(B) position: 1-19

(D) out of Memory :/note=" phosphorothioate bond, C5-proyl pyrimidine "

(ⅹ) sequence description: the information of sequence numbering NO.12:CCAUGAACUU CACCACUUC 19 (2) SEQ ID NO.13 sequences:

(ⅰ) sequence signature:

(A) length: 19 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology configuration: linearity

(ⅳ) antisense: be

(ⅸ) feature:

(A) title/keyword: misc-feature

(B) position: 1-19

(D) out of Memory :/note=" phosphorothioate bond, C5-proyl pyrimidine "

(ⅹ) sequence description: the information of sequence numbering NO.13:GACAUCCAUG AACUUCACC 19 (2) SEQ ID NO.14 sequences:

(ⅰ) sequence signature:

(A) length: 19 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology configuration: linearity

(ⅳ) antisense: be

(ⅸ) feature:

(A) title/keyword: misc-feature

(B) position: 1-19

(D) out of Memory :/note=" phosphorothioate bond, C5-proyl pyrimidine "

(ⅹ) sequence description: the information of sequence numbering NO.14:GGCUGGCAGU AGCUGCGCU 19 (2) SEQ ID NO.15 sequences:

(ⅰ) sequence signature:

(A) length: 19 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology configuration: linearity

(ⅳ) antisense: be

(ⅸ) feature:

(A) title/keyword: misc-feature

(B) position: 1-19

(D) out of Memory :/note=" phosphorothioate bond, C5-proyl pyrimidine "

(ⅹ) sequence description: the information of sequence numbering NO.15:GGAUGGCAGU AGCUGCGCU 19 (2) SEQ ID NO.16 sequences:

(ⅰ) sequence signature:

(A) length: 19 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology configuration: linearity

(ⅳ) antisense: be

(ⅸ) feature:

(A) title/keyword: misc-feature

(B) position: 1-19

(D) out of Memory :/note=" phosphorothioate bond, C5-proyl cytosine(Cyt) residue "

(ⅹ) sequence description: the information of sequence numbering NO.16:GCGCTGATAG ACATCCATG 19 (2) SEQ ID NO.17 sequences:

(ⅰ) sequence signature:

(A) length: 19 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology configuration: linearity

(ⅳ) antisense: be

(ⅸ) feature:

(A) title/keyword: misc-feature

(B) position: 1-19

(D) out of Memory :/note=" phosphorothioate bond, C5-proyl uridylic residue "

(ⅹ) sequence description: the information of sequence numbering NO.17:GCGCUGAUAG ACAUCCAUG 19 (2) SEQ ID NO.18 sequences:

(ⅰ) sequence signature:

(A) length: 19 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology configuration: linearity

(ⅳ) antisense: be

(ⅸ) feature:

(A) title/keyword: misc-feature

(B) position: 1-19

(D) out of Memory :/note=" phosphorothioate bond, at residue 8,12, the C5-proyl pyrimidine at 14 and 15 places "

(ⅹ) sequence description: the information of sequence numbering NO.18:GCGCTGAUAG ACAUCCATG 19 (2) SEQ ID NO.19 sequences:

(ⅰ) sequence signature:

(A) length: 19 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology configuration: linearity

(ⅳ) antisense: be

(ⅸ) feature:

(A) title/keyword: misc-feature

(B) position: 1-19

(D) out of Memory :/note=" phosphorothioate bond, at residue 2,5, the C5-proyl pyrimidine at 8,12,15 and 18 places "

(ⅹ) sequence description: the information of sequence numbering NO.19:GCGCUGAUAG ACATCCAUG 19 (2) SEQ ID NO.20 sequences:

(ⅰ) sequence signature:

(A) length: 19 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology configuration: linearity

(ⅳ) antisense: be

(ⅸ) feature:

(A) title/keyword: misc-feature

(B) position: 1-19

(D) out of Memory :/note=" 3 ' end is connected in the lipid chain for phosphorothioate bond, C5-proyl pyrimidine "

(ⅹ) sequence description: the information of sequence numbering NO.20:GCGCUGAUAG ACAUCCAUG 19 (2) SEQ ID NO.21 sequences:

(ⅰ) sequence signature:

(A) length: 19 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology configuration: linearity

(ⅳ) antisense: be

(ⅸ) feature:

(A) title/keyword: misc-feature

(B) position: 1-19

(D) out of Memory :/note=" phosphorothioate bond is removed residue 1-5,8-9, and 12-13 is between 14-15 and the 16-19; 3 ' terminal pyrene; C5-proyl pyrimidine "

(ⅹ) sequence description: the information of sequence numbering NO.21:GCGCUGAUAG ACAUCCAUG 19 (2) SEQ ID NO.22 sequences:

(ⅰ) sequence signature:

(A) length: 19 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology configuration: linearity

(ⅳ) antisense: be

(ⅸ) feature:

(A) title/keyword: misc-feature

(B) position: 1-19

(D) out of Memory :/note=" phosphorothioate bond, C5-hexin yl pyrimidines "

(ⅹ) sequence description: the information of sequence numbering NO.22:GCGCUGAUAG ACAUCCAUG 19 (2) SEQ ID NO.23 sequences:

(ⅰ) sequence signature:

(A) length: 19 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology configuration: linearity

(ⅳ) antisense: be

(ⅸ) feature:

(A) title/keyword: misc-feature

(B) position: 1-19

(D) out of Memory :/note=" phosphorothioate bond, C5-proyl pyrimidine "

ⅹ) sequence description: the information of sequence numbering NO.23:GCGCUGACAG ACAUUCAUG 19 (2) SEQ ID NO.24 sequences:

(ⅰ) sequence signature:

(A) length: 19 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology configuration: linearity

(ⅳ) antisense: not

(ⅹ) sequence description: the information of sequence numbering NO.24:CATGGATGTC TATCAGCGC 19 (2) SEQ ID NO.25 sequences:

(ⅰ) sequence signature:

(A) length: 19 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology configuration: linearity

(ⅳ) antisense: not

(ⅹ) sequence description: the information of sequence numbering NO.25:CATGGATGTC TATCAGCGC 19 (2) SEQ ID NO.26 sequences:

(ⅰ) sequence signature:

(A) length: 19 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology configuration: linearity

(ⅳ) antisense: be

(ⅸ) feature:

(A) title/keyword: misc-feature

(B) position: 1-19

(D) out of Memory :/note=" phosphorothioate bond, C5-proyl cytosine(Cyt) residue "

(ⅹ) sequence description: the information of sequence numbering NO.26:AGAGCAGCAA GGCGAGGCT 19 (2) SEQ ID NO.27 sequences:

(ⅰ) sequence signature:

(A) length: 19 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology configuration: linearity

(ⅳ) antisense: be

(ⅸ) feature:

(A) title/keyword: misc-featute

(B) position: 1-19

(D) out of Memory :/note=" phosphorothioate bond, C5-proyl cytosine(Cyt) residue "

(ⅹ) sequence description: sequence numbering NO.27:CAUGGAUGUC UAUCAGCGC 19

Claims (48)

1. one kind is reduced the antisense oligonucleotide that cell VEGE produces in the handled cell, handles under the concentration that is lower than about 1 μ M with this antisense oligonucleotide; The VEGF that the cell of handling produces is no more than about 90% of untreated cell generation.

2. the antisense oligonucleotide of claim 1, wherein said antisense oligonucleotide can combine with the RNA sequence on the mRNA of coding VEGF.

3. the antisense oligonucleotide of claim 1, wherein said antisense oligonucleotide can combine with the RNA sequence on the mRNA of at least two kinds of coding VEGF.

4. the antisense oligonucleotide of claim 1, wherein said antisense oligonucleotide can combine with the RNA sequence on the mRNA of VEGF206.

5. the antisense oligonucleotide of claim 1, wherein said antisense oligonucleotide can combine with the RNA sequence on the mRNA of VEGF185.

6. the antisense oligonucleotide of claim 1, wherein said antisense oligonucleotide can combine with the RNA sequence on the mRNA of VEGF165.

7. the antisense oligonucleotide of claim 1, wherein said antisense oligonucleotide can combine with the RNA sequence on the mRNA of VEGF121.

8. the antisense oligonucleotide of claim 1, it comprises and reduces this antisense oligonucleotide by the chemical ingredients of the speed of nuclease degradation.

9. the antisense oligonucleotide of claim 1, wherein this oligonucleotide comprises thiophosphoric acid ester group and phosphodiester group.

10. the antisense oligonucleotide of claim 1, it comprises the adjacent residues that chemical part a pair of and the nuclease-resistant degraded links to each other.

11. the antisense oligonucleotide of claim 8, this antisense oligonucleotide of wherein said reduction is the thiophosphoric acid ester group by the composition of the speed of nuclease degradation.

12. the antisense oligonucleotide of claim 8, wherein each residue of this oligonucleotide is to link to each other with the thiophosphoric acid ester group.

13. comprising, the antisense oligonucleotide of claim 1, wherein said oligonucleotide be selected from C5-proyl uridine, C5-proyl cytidine, C5-hexin base uridine, C5-hexin base cytidine, the nucleotide residue of choosing in 6-aza uridine and the 6-azacytidine.

14. the antisense oligonucleotide of claim 1, wherein said oligonucleotide contains the thiophosphoric acid ester group and is selected from C5-proyl uridine, C5-proyl cytidine, C5-hexin base uridine, C5-hexin base cytidine, the nucleotide residue in 6-aza uridine and the 6-azepine-cytidine.

15. the antisense oligonucleotide of claim 1, it comprises C5-proyl uridine residue.

16. the antisense oligonucleotide of claim 1, it comprises C5-proyl uridine residue and thiophosphoric acid ester group.

17. the antisense oligonucleotide of claim 1, it comprises C5-proyl cytidine residue.

18. the antisense oligonucleotide in the claim 1, it comprises C5-proyl cytidine residue and thiophosphoric acid ester group.

19. the antisense oligonucleotide of claim 1, it comprises C5-hexin base uridine residue and thiophosphoric acid ester group.

20. the antisense oligonucleotide of claim 1, it comprises C5-hexin base cytidine residue.

21. the antisense oligonucleotide of claim 1, it comprises C5-hexin base cytidine residue and thiophosphoric acid ester group.

22. the antisense oligonucleotide of claim 1, it comprises 6-azepine deoxyuridine residue.

23. the antisense oligonucleotide of claim 1, it comprises 6-azepine deoxyuridine residue and thiophosphoric acid ester group.

24. the antisense oligonucleotide of claim 1, it comprises 6-azepine Deoxyribose cytidine residue.

25. the antisense oligonucleotide of claim 1, it comprises 6-azepine Deoxyribose cytidine residue and thiophosphoric acid ester group.

26. composition that contains a kind of antisense oligonucleotide, when being lower than about 1 μ M, concentration handles cell with this oligonucleotide, the VEGF that handled cell produces reduces, this treated cell produces at most the about 90% of the VEGF that produced by undressed cell, and said composition also contains a kind of cell absorption enhancer.

27. the composition of claim 26, wherein said cell absorption enhancer is a kind of lipotropic component.

28. the composition of claim 27, lipotropic component wherein contains cholesterol.

29. the composition of claim 1, wherein said oligonucleotide also comprises the ionic linkage that forms a kind of salt with positively charged ion.

30. the composition of claim 29, wherein said positively charged ion is a cationic lipid.

31. the composition of claim 30, wherein said cationic lipid are a kind of polyamino fat.

32. the composition of claim 31, wherein said polyamino fat is SPDC.

33. the composition of claim 26, wherein said cell absorption enhancer contains a kind of liposome.

34. the composition of claim 33, wherein said liposome contains Cellfectin .

35. the composition of claim 33, wherein said liposome contain and DOPE blended SPDC.

36. an energy combines with VEGF mRNA and contains the thiophosphoric acid ester group and be selected from C5-proyl uridine, C5-proyl cytidine, C5-hexin base uridine, C5-hexin base cytidine, the antisense oligonucleotide of the nucleotide residue of 6-azepine deoxyuridine and 6-azepine-Deoxyribose cytidine, the duplex melting temperature(Tm) of wherein said antisense oligonucleotide is than identical but not have the antisense oligonucleotide of pyrimidine residue of chemically modified high at least about 5 ℃; This antisense oligonucleotide has reduced the production of the VEGF of cell in the cell that this oligonucleotide that is lower than about 1 μ M with concentration was handled; The cell generation of this processing is no more than by about 90% of the VEGF of untreated cell generation.

37. one kind contains a kind of composition that can combine with VEGF mRNA and have the thiophosphoric acid ester group and be selected from the antisense oligonucleotide of the nucleotide residue in C5-proyl uridine, C5-proyl cytidine, C5-hexin base uridine, C5-hexin base cytidine, 6-azepine deoxyuridine and the 6-azepine Deoxyribose cytidine, wherein the duplex melting temperature(Tm) of this antisense oligonucleotide is than identical but not have the antisense oligonucleotide of pyrimidine residue of chemically modified high at least about 5 ℃; This antisense oligonucleotide has reduced the production of the VEGF of cell in the cell that this oligonucleotide when being lower than about 1 μ M with concentration handled, and the cell of this processing produces and is no more than about 90% of the VEGF that produced by untreated cell; Said composition also contains the compound that polymeric continues release.

38. contain phosphorothioate bond and can with the improvement body of a kind of antisense oligonucleotide of mRNA bonded of coding VEGF in comprise: in antisense oligonucleotide, contain the nucleotide residue that is selected from C5-proyl uridine, C5-proyl cytidine, C5-hexin base uridine, C5-hexin base cytidine, 6-azepine deoxyuridine and the 6-azepine Deoxyribose cytidine; Wherein this improvement body has increased a kind of duplex melting temperature(Tm) of antisense oligonucleotide at least about 5 ℃.

39. minimizing method that cell VEGE produces in cell, its antisense oligonucleotide that comprises with claim 1 contacts a kind of cell, this cell produces the about 90% of the VEGF that is no more than the cell generation that origin handled at most, and this oligonucleotide concentration is no more than 1 μ M.

40. antisense oligonucleotide that is selected from sequence numbering No.2-22.

41. composition that comprises antisense oligonucleotide with sequence numbering No.2.

42. composition that comprises antisense oligonucleotide with sequence numbering No.3.

43. composition that comprises antisense oligonucleotide with sequence numbering No.4.

44. composition that comprises antisense oligonucleotide with sequence numbering No.6.

45. composition that comprises antisense oligonucleotide with sequence numbering No.10.

46. composition that comprises antisense oligonucleotide with sequence numbering No.20.

47. composition that comprises antisense oligonucleotide with sequence numbering No.21.

48. composition that comprises antisense oligonucleotide with sequence numbering No.22.

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