Proc. Natl. Acad. Sci. USAVol. 93, pp. 4851-4856, May 1996Medical Sciences

Oligodeoxynucleotides inhibit retinal neovascularization in amurine model of proliferative retinopathy

(angiogenesis/antisense oligodeoxynucleotide/vascular endothelial growth factor/vascular permeability factor/proliferativeretinopathy)

GREGORY S. ROBINSON*t, ERIC A. PIERCEtt, SUSAN L. ROOK*, ELLIOT FOLEYt,RUTH WEBBt, AND LOIS E. H. SMITHt§*Hybridon, Inc., Worcester, MA 01605; and iDepartment of Ophthalmology, Children's Hospital, Harvard Medical School and V. Kann Rasmussen ResearchCollaborative, Boston, MA 02115

Communicated by James B. Wyngaarden, Washington DC, January 18, 1996 (received for review September 14, 1995)

ABSTRACT Diseases characterized by retinal neovascu-larization are among the principal causes of visual lossworldwide. The hypoxia-stimulated expression of vascularendothelial growth factor (VEGF) has been implicated in theproliferation of new blood vessels. We have investigated theuse of antisense phosphorothioate oligodeoxynucleotidesagainst murine VEGF to inhibit retinal neovascularizationand VEGF synthesis in a murine model of proliferativeretinopathy. Intravitreal injections of two different antisensephosphorothioate oligodeoxynucleotides prior to the onset ofproliferative retinopathy reduced new blood vessel growth amean of 25 and 31% compared with controls. This inhibitionwas dependent on the concentration of antisense phosphoro-thioate oligodeoxynucleotides and resulted in a 40-66% re-duction in the level of VEGF protein, as determined byWestern blot analysis. Control (sense, nonspecific) phospho-rothioate oligodeoxynucleotides did not cause a significantreduction in retinal neovascularization or VEGF proteinlevels. These data further establish a fundamental role forVEGF expression in ischemia-induced proliferative retinopa-thies and a potential therapeutic use for antisense phospho-rothioate oligodeoxynucleotides.

Neovascular diseases of the retina are one of the major causesof blindness in the world, yet the biochemical events respon-sible for this process are only now being understood (1). Visionloss in pathological conditions such as diabetic retinopathy,retinopathy of prematurity, ischemic retinal-vein occlusion,and age-related macular degeneration is characterized by theextensive proliferation of new blood vessels in the retina (2, 3).Current therapies aimed at controlling this aberrant angio-genesis, such as panretinal photocoagulation and cryotherapy,are only partially effective and are destructive to the retina (4,5). Alternative, nondestructive modes of therapy could be ofbenefit to patients with these blindness-causing diseases.

Pathologic states associated with uncontrolled angiogenesis(i.e., ocular neovascularization and tumor growth) have hyp-oxia as a common stimulus (6-10). Over the past 4 decadesresearchers have hypothesized that retinal cells release a"vasoformative factor" in response to hypoxic conditions,causing retinal neovascularization (11, 12). Recently, there hasbeen extensive research on a specific cytokine, vascular endo-thelial growth factor/vascular permeability factor (VEGF/VPF), an attractive candidate for this vasoformative factor.VEGF/VPF appears to play a primary role in angiogenicprocesses. It is an endothelial cell-specific mitogen which isstimulated by hypoxia and required for proliferation in sometumors (13-16). Cultured human retinal cells such as pigmentepithelial cells and pericytes secrete VEGF/VPF and increase

VEGF/VPF gene expression in response to hypoxia (17-21).VEGF/VPF is expressed in the retina prior to the onset ofneovascularization in a mouse model of proliferative retinop-athy (22). Finally, there is a high correlation between VEGF/VPF expression and the retinal neovascularization associatedwith a variety of ocular diseases (10). To further investigate therole of VEGF/VPF in retinal neovascularization, we haveexamined the inhibition of neovascularization using antisensephosphorothioate oligodeoxynucleotides (PS-ODNs) againstmurine VEGF/VPF.

Antisense oligodeoxynucleotide technology provides anovel approach for inhibiting gene expression with targetspecificity as a particular advantage (23-25). Antisense PS-ODNs appear to inhibit protein expression by altering eitherthe splicing, stability, and/or translation of the mRNA throughbinding in a sequence-specific manner to the complementarynucleic acid sequence (26). Modifications in oligodeoxynucle-otide synthesis have created molecules that are stable forseveral days and are relatively nontoxic at effective concen-trations (27, 28). Furthermore, although nonspecific effects ofPS-ODNs have been documented (25), highly selective andsequence-specific inhibition of gene expression has been re-peatedly confirmed (for review, see ref. 29).

In this study, we report sequence-specific PS-ODN inhibi-tion of retinal neovascularization in a murine model of pro-liferative retinopathy. This model provides a reproducible,quantifiable system to study retinal neovascularization (21).The reduction in retinal neovascularization following intra-vitreal injection of a 21-nt antisense PS-ODN targeted againstmurine VEGF/VPF depended on the injected dose of PS-ODN. A reduction in the levels of VEGF/VPF protein inresponse to the antisense PS-ODNs was also observed. Thesestudies confirm a role for VEGF/VPF in proliferative reti-nopathy; they further suggest that antisense PS-ODNs mayprovide a viable therapy for diseases characterized by retinalneovascularization.

MATERIALS AND METHODSSynthesis of PS-ODNs. Antisense PS-ODNs were synthe-

sized on a Biosearch 8700 automated synthesizer using eitherthe phosphoamidite or H-phosphonate procedures. PS-ODNswere purified by low-pressure reverse-phase chromatography,low-pressure ion-exchange chromatography, and dialysis (3times) against sterile, pyrogen-free water. The sequences ofthese PS-ODNs were as follows: Vm (5'-CAGCCTGGCT-

Abbreviations: VEGF, vascular endothelial growth factor; VPF, vas-cular permeability factor; P, postnatal day.tG.S.R. and E.A.P. contributed equally to the research described inthis paper.§To whom reprint requests should be addressed at: Department ofOphthalmology-Fegan 4, Children's Hospital, 300 Longwood Ave-nue, Boston, MA 02115.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

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CACCGCCTTGG-3'), complementary to bases 648-668 ofthe murine VEGF/VPF sequence (30); M3 (5'-TCGCGCTC-CCTCTCTCCGGC-3'), complementary to bases 37-56 of themurine VEGF/VPF sequence; Vm-sense (sense control, 5'-CCAAGGCGGTGAGCCAGGCTG-3'), equivalent to bases648-668 of the murine VEGF/VPF sequence; V2 (controlnoncomplementary to mRNA, 5'-TCCGAAACCATGAAC-TTTCTG-3') equivalent to bases 78-98 of the murine VEGF/VPF sequence. The purity of the sequences was analyzed bycapillary gel electrophoresis and ion-exchange high- pressureliquid chromatography. Endotoxin levels in the final PS-ODNpreparation were examined using a limulus amebocyte lysateassay (31) and found to be <2 endotoxin units/ml.RNase H Cleavage Assay and RNA Secondary Structure.

RNase H catalyzed cleavage was performed in a cell-freesystem. The murine VEGF/VPF cDNA (980 nt) was clonedinto the EcoRI restriction site of pBluescript II (SK-). MurineVEGF/VPF mRNA was transcribed in vitro using a Mega-Script kit (Ambion, Austin, TX). RNA (10 ,lI, 15 pmol) wasphosphorylated through incubation with 5 ,ul y2P ATP, lxAll-phor-one buffer (Pharmacia), and 25 units of T4 polynu-cleotide kinase for 60 min at 37°C in a reaction volume of 25,ul. The labeled mRNA was purified on a G-50 Sephadex spincolumn.An equal volume of the labeled mRNA was incubated with

1 ,uM of each of the PS-ODNs in 10 ,ul of RNase H buffer (40mM Tris-Cl, pH 7.6/4 mM MgCl2/1 mM DTT) and 1 unit ofRNase H for 15 min at 25°C. Formamide stop buffer (10 ,lI)was added to stop the reaction, and the cleavage products wereseparated on a 6% polyacrylamide/7M urea/TBE gel (100 nMTris/90 mM boric acid/i mM EDTA) and exposed to x-rayfilm (Kodak).The computer generated secondary structure of the murine

VEGF/VPF mRNA was determined using FOLDRNA andSQUIGGLES software (Genetics Computer Group) on a SunMicrosystems workstation.

Immunoprecipitation. NB41 murine neuroblastoma cellsthat endogeneously express VEGF/VPF were grown in com-plete DMEM containing 10% fetal bovine serum, 2 mMglutamine, and 100 units per 100 ,tg of penicillin/streptomycinto a confluency of 90%. Cells were treated with 1 ,tM ofPS-ODN in the presence of 2.5 ,tg/ml DOTAP (BoehringerMannheim). Forty-six hours after initial PS-ODN addition, thecells were incubated in methionine-free medium for 4 hr in thepresence of 150-200 ,uCi 35S-Translabel (ICN).

Trichloroacetic acid-precipitable counts from labeled me-dium (± 10%) were immunoprecipitated overnight at 4°C inthe presence of a polyclonal rabbit anti-human VEGF/VPFantibody, which crossreacts with the murine VEGF/VPFprotein (Kevin Claffey; personal communication). The anti-body-VEGF/VPF complex was removed from the immuno-precipitation solution using protein A-Sepharose. The proteinA-Sepharose was washed three times, and VEGF/VPF wasanalyzed by SDS/PAGE under reducing conditions.Murine Model of Proliferative Retinopathy. All procedures

involving animals were conducted in accordance with theAssociation for Research in Vision and Ophthalmology(ARVO) Statement for the Use of Animals in Ophthalmic andVision Research. Postnatal day 7 mice (P7; C57BL/6J, Chil-dren's Hospital Breeding Facilities, Boston; Taconic Farms)were exposed for 5 days to hyperoxic conditions (75 ± 2%oxygen) in a sealed incubator, as described (32). After 5 days,P12 mice were returned to room air. Maximal retinal neovas-cularization was observed 5 days after return to room air atP17.PS-ODN Treatment in Vivo. PS-ODNs were injected into the

vitreous of Avertin anesthetized using a 32-gauge Hamiltonneedle and syringe (Hamilton) to deliver 0.5 plI of PS-ODNsolution diluted in balanced salt solution (Alcon Laboratories,Fort Worth, TX). Repeat injections were done through a

previously unmanipulated section of the limbus. This proce-dure limited the number of injections possible in neonatal eyes(n = 2). The amount of solution actually remaining in thevitreous cavity was <0.5 ,ul in some animals due to a smallamount of leakage at the injection site. Thus, the concentra-tions ofPS-ODN listed below are only approximate. Mice wereinjected twice in one eye with antisense PS-ODN and in theother eye with control (sense or random) PS-ODN to give afinal estimated concentration after each injection of PS-ODNin the vitreous of 50 AM. Injections were done immediatelyupon removal from oxygen on P12, and were repeated on P14.Animals were sacrificed at P17 by injection with a lethal doseof Avertin followed by cardiac perfusion with 4% paraformal-dehyde (Sigma) in PBS.

Quantification of Retinal Neovascularization. Followingsacrifice, as described above, the eyes were enucleated, fixed in4% paraformaldehyde, and embedded in paraffin. Serial 6-,tmsections of the whole eyes were cut sagittally parallel to theoptic nerve, and stained with hematoxylin and periodic acid/Schiff stain according to a standardized protocol (32). Theextent of neovascularization in the treated and control eyes wasdetermined by counting neovascular cell nuclei extendingthrough the internal limiting membrane into the vitreous. Allcounting was done using a masked protocol. For each eye, 10intact sections of equal length, each 30 ,um apart, wereevaluated. The mean number of neovascular nuclei per sectionper eye was then determined. The extent of neovascularizationresulting in antisense-treated eyes was then compared withthat found in control eyes using Student's t test or the WilcoxSigned Rank test. All statistical calculations were performedusing SIGMASTAT for Windows (Jandel, San Rafael, CA). Eyesfound to have intraocular inflammation, infection, or retinaldetachment were excluded from analysis. This accounted for10% of all eyes. All other eyes were included, even those eyes

where leakage of injection material may have occurred.Western Blot Analysis. For Western blot analyses, mice were

injected with antisense PS-ODN in one eye and controlPS-ODN in the other eye as described. Mice were sacrificed atage P17, and the retinas isolated and frozen in liquid nitrogen.For each analysis, retinas from the antisense or control in-jected eyes of three or four animals were pooled. Total retinalprotein was prepared by homogenizing pooled retinas in SDSsample buffer and analyzing 50 ,ug of total retinal protein foreach time point by SDS/PAGE (33). Partially purified recom-binant mouse VEGF/VPF (rmVEGF/VPF; Kevin Claffey,personal communication) was included on each gel. Followingelectrophoresis, protein was transferred to Immobilon-P (Mil-lipore) membranes, and VEGF/VPF protein was detectedusing affinity-purified anti-VEGF/VPF antibodies accordingto established methods with enhanced chemiluminescent re-agents (Amersham) (34-36). Only exposures that did notsaturate the film were used for quantification. The results wereanalyzed using IMAGEQUANT software (Molecular Dynamics).PS-ODN Uptake. Tissue samples from animals subjected to

hyperoxia as described and injected with Vm PS-ODN at P12were sacrificed at P14. PS-ODN uptake in these tissues wasdetermined using a described procedure (37) and modified bythe authors (unpublished results). Briefly, the tissues (retinaland nonretinal) were homogenized in 1 ml of lysis solution (10mM Tris, pH 7.5/10 mM NaCl/3 mM MgCl2/1% NonidetP-40/1% SDS) and centrifuged at 4°C for 10 min at 3000 RPM.The supernatant was extracted with phenol/chloroform (1:1)and then with chloroform/isoamyl alcohol (24:1). The aqueousphase was filtered through a 0.2-,um nylon membrane (Bio-trans; ICN) using a slot blot apparatus (Schleicher & Schuell).Vm PS-ODN was detected through hybridization with a 32plabeled complementary (sense) phosphodiester oligode-oxynucleotide. The results were analyzed using IMAGEQUANTsoftware (Molecular Dynamics).

Proc. Natl. Acad. Sci. USA 93 (1996)

Proc. Natl. Acad. Sci. USA 93 (1996) 4853

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FIG. 1. Identification of antisense PS-ODN against murineVEGF/VPF. (A) A computer generated map of the predicted sec-ondary structure of murine VEGF/VPF mRNA is shown. The po-tential hybridization sites of Vm and M3 are indicated. (B) RNase Hcleavage assay. 32P-labeled murine VEGF/VPF mRNA was preparedand analyzed as described. Arrows mark the positions of the cleavageproducts generated by incubation with M3 (125 nt) and Vm (800 nt).(-)ODN, RNase H cleavage in the absence of any added PS-ODN.Markers: y-32P-labeled qbX-174 HincII digest.

RESULTS

Cell-Free Analysis of Antisense PS-ODNs Against MurineVEGF/VPF. Inhibition of gene expression has been observedusing oligomers complementary to the 5'-untranslated region,the translational start site, the translational stop site, or the3'-untranslated region (26). There is also evidence that RNAsecondary structure may play a role in making the targetaccessible to the oligodeoxynucleotide (38, 39). Areas that donot cooperate in intramolecular hybridization (i.e., loopedstructures) may be targets for antisense inhibition. AntisensePS-ODNs were selected by means of computer modelingtechniques and a novel RNase H cleavage assay (Bruce Frank,

personal communication). We have tested 33 antisense PS-ODNs targeting regions of murine VEGF/VPF that includestem-loop structures of varying size. Two PS-ODNs targetingsequences in either the 5'-untranslated region (M3) or thetranslational stop site of murine VEGF/VPF (Vm) were themost effective at inhibiting VEGF/VPF expression. A com-puter generated diagram of murine VEGF/VPF RNA sec-ondary structure suggests that there are stem-loop structuresat the sites of hybridization for both M3 and Vm (Fig.L4).The PS-ODN cleavage of transcribed RNA in vitro was

tested using an RNase H cleavage assay (Fig. iB). The additionof Vm or M3 PS-ODN to this assay resulted in the expectedcleavage pattern based on the comparative size of the frag-ments with molecular weight markers on a denaturing poly-acrylamide gel. Control PS-ODNs (Vm-sense, V2) did notresult in RNase H cleavage.

Antisense PS-ODNs Inhibit Murine VEGF/VPF ProteinExpression in Cultured Neuroblastoma Cells. NB41 murineneuroblastoma cells were used to test PS-ODN inhibition ofVEGF/VPF expression. PS-ODN caused a reduction in thelevel of murine VEGF/VPF protein (Fig.2). Untreated cells orcells treated with control PS-ODNs did not result in a reduc-tion of VEGF/VPF protein. M3 PS-ODN was also able toinhibit the expression of VEGF/VPF in NB41 cells (data notshown).

Antisense PS-ODNs Reduce Proliferative Retinopathy inthe Mouse. We examined PS-ODN inhibition of VEGF/VPFgene expression in a murine model of proliferative retinopathy.In this model, exposure of neonatal mice to 75% oxygen for 5days (P7-12) results in cessation of normal retinal blood vesseldevelopment and obliteration of the posterior retinal vascu-lature. Return of animals to room air is believed to result inrelative hypoxia of the nonperfused areas of the retina. Retinalneovascularization then occurs in 100% of animals (22).Thirty-five animals were injected on P12 and P14 in a pairedfashion, with one eye receiving the antisense PS-ODN (Vm orM3) and the contralateral eye receiving a noncomplementarycontrol PS-ODN (V2). As seen in Fig. 3A, injection of Vm orM3 antisense PS-ODNs caused a significant decrease in retinalneovascularization compared with control injections with thenoncomplementary PS-ODN V2. Injection with Vm or M3decreased neovascularization by 25% (P = 0.0002) or 31% (P= 0.0212), respectively. Fig. 3B shows the neovascular nucleicounts in the antisense-treated and control eyes for all animalsinjected. A decrease in neovascularization was observed in 20of 25 (80%) of the animals following injection with Vm, and8 of 10 (80%) following injection with M3. It can be seen thatindividual animals demonstrated up to a 75% decrease inneovascularization following injection with antisense PS-ODN. Saline solution or a random PS-ODN, synthesized withthe availability of any of the four deoxynucleotides at each

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FIG. 3. Inhibition of VEGF/VPF with antisense PS-ODN suppresses retinal neovascularization in vivo. (A) Mice were injected intravitreallywith antisense (M3 or Vm) or sense control (V2) PS-ODN. Retinal neovascularization was determined from neovascular cell nuclei counts ofantisense and control PS-ODN injected eyes. Error bars indicate standard error for all animals in each group. Statistical differences betweenantisense and control injected eyes are indicated. (B) Average number of neovascular cell nuclei per 6-Am histological section per eye wasdetermined as described. Eyes from the same animal are connected by solid lines. Arrows mark the mean of each group.

position in the sequence, showed no significant effect onneovascularization (n = 16; data not shown).The Reduction of Retinal Neovascularization Is Dependent

on the Dose of Antisense PS-ODN. To determine if theinhibition of retinal neovascularization observed with anti-sense PS-ODN Vm was dose dependent, 22 animals wereinjected intravitreally with 50 ,tM or 5 piM of either antisense(Vm) or noncomplementary (V2) PS-ODN in a paired fash-ion. Results show that inhibition of proliferative retinopathy isdependent on the concentration of the antisense PS-ODNinjected (Fig. 4). Injection with 5 ,uM of Vm PS-ODN did notresult in a statistically significant decrease in neovascularnuclei counts compared with V2 control PS-ODN. In contrast,50 ,uM of Vm PS-ODN decreased neovascular counts by 34%(P = 0.0156), consistent with prior experiments.

Fig. 5 shows light micrographs of representative retinalsections from an animal treated with the antisense PS-ODNVm in one eye and the control V2 in the other. The eye injectedwith control PS-ODN (A) demonstrates more preretinal neo-vascularization than the antisense-treated eye (B) (arrows). Noretinal toxicity was observed by light microscopy at the dosesof PS-ODN used for these experiments.

Antisense PS-ODN Treatment Reduces VEGF/VPF Pro-tein. VEGF/VPF protein levels in the retina following injec-tion of PS-ODN were examined to determine whether theobserved decrease in retinal neovascularization was correlatedwith a reduction in VEGF/VPF expression. Animals wereinjected with either antisense (Vm or M3) or control (V2 or

Vm-sense) PS-ODN in a paired fashion. VEGF/VPF proteinexpression was evaluated by Western blot analysis. VEGF/VPF protein expression is decreased -44% in response totreatment with Vm compared with treatment with V2 (Fig. 6,lanes 3 and 4). Similarly, injection of M3 decreased VEGF/VPF protein levels =40% compared with control V2 (Fig. 6,lanes 5 and 6). To verify the inhibition of VEGF/VPF proteinexpression by antisense PS-ODNs, an additional control (Vm-sense) was used. As shown in Fig. 6 (lanes 7 and 8), injectionwith antisense PS-ODN Vm decreased retinal VEGF/VPFprotein levels -66% when compared with injection of controlVm-sense PS-ODN. The anti-VEGF/VPF antibody used in

these experiments detects murine VEGF/VPF, whereas non-immune serum demonstrates nonspecific binding only (Fig. 6,lanes 1 and 2).The uptake and distribution of PS-ODNs in tissues of

treated animals is an important factor in animal experiments.We found a 19-fold greater uptake of Vm PS-ODN in theretinas of mice (P14) injected at P12 as compared with thePS-ODN uptake in the nonretinal tissues of these eyes (datanot shown). Thus, Vm PS-ODN is present in the tissue of theeye where VEGF expression is induced and neovascularizationoccurs.

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Proc. Natl. Acad. Sci. USA 93 (1996)

Proc. Natl. Acad. Sci. USA 93 (1996) 4855

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FIG. 5. Antisense PS-ODN reduce histologically evident retinal neovascularization. Retinal neovascularization was induced as described. Oneeye of each mouse was injected with antisense PS-ODN (Vm, B) and the other eye with noncomplementary PS-ODN (V2, A). The figure showstypical findings from corresponding retinal locations from both eyes of the same mouse. An area of retinal neovascularization with vascular cellsinternal to the internal limiting membrane is indicated with arrows.

DISCUSSIONWe investigated the role of VEGF/VPF in retinal neovascu-larization and evaluated antisense PS-ODNs directed againstmurine VEGF/VPF as potential therapeutic agents for retinalvascular diseases. In vitro selection of specific oligodeoxynucle-otides and studies aimed at inhibiting VEGF/VPF expressionin cultured cells show that antisense PS-ODNs Vm and M3 areeffective inhibitors. Vm PS-ODN hybridizes to a sequencesurrounding the translational termination codon that can forma stem-loop structure. This structure may provide an optimalsite for antisense oligodeoxynucleotide hybridization to thetarget sequence (40). Inhibition of VEGF/VPF expression byVm PS-ODN may be the result of incomplete translation of theVEGF/VPF protein, resulting in degradation by cellular pro-teases, or enzymatic cleavage of the mRNA by RNase H. Bycontrast, M3 targets a sequence in the 5'-untranslated regionof the mRNA (41, 42). This sequence can also form astem-loop structure, again providing an advantageous site forPS-ODN binding and inhibition of VEGF/VPF expression.

Intravitreal injection of Vm and M3 PS-ODN in neonatalmice prior to onset of proliferative retinopathy showed a

statistically significant reduction in the level of retinal neovas-cularization. Incomplete inhibition of neovascularization byPS-ODN directed against VEGF/VPF might imply that otherangiogenic factors are involved in the induction of retinalneovascularization. Alternatively, further optimization of an-tisense oligodeoxynucleotides (sequence, chemistry, uptake)and treatments (dose, schedule) may provide increased inhi-bition. In addition, other mechanisms of delivery such as slowrelease polymers and systemic administration need to beinvestigated more thoroughly. Finally, it is worth noting that100% inhibition of VEGF/VPF expression may not be desir-

VEGF -,(23 kDa)

able in treatment of retinal vascular diseases because VEGF/VPF may be required for blood vessel maintenance, and thatcontrolled angiogenesis to relieve hypoxia may be the goal inmany proliferative retinopathic diseases (43).Three pieces of evidence suggest that Vm and M3 antisense

PS-ODN inhibition of retinal neovascularization was sequencespecific. First, injection of a control (sense) PS-ODN revealedno significant decrease in neovascularization of the retinacompared with injection of saline. Second, the reduction in thelevel of proliferative retinopathy following antisense PS-ODNtreatment showed a trend toward dose dependence. Third, inanimals treated with Vm and M3, there was a 40-66%decrease in the level of VEGF protein, when compared withanimals treated with control PS-ODN (V2 or Vm-sense).However, we cannot rule out the possibility that an aptamereffect may be playing a minor role in the reduction of retinalneovascularization.

Inhibition of retinal neovascularization by sequence-specificPS-ODN directed against VEGF/VPF demonstrates thatVEGF/VPF is required for the development of ischemia-induced retinal angiogenesis. Taken together with the findingsthat VEGF/VPF is elevated in the ocular fluids of patientswith proliferative retinopathies (10, 44), our data imply thatVEGF/VPF is one of the causative factors responsible forneovascularization in ischemic retinal diseases. In our murinesystem, we see retinal uptake of PS-ODN by day 2 followingintravitreal injection. PS-ODN are internalized by most cellsand we have not yet determined which cells of the retina arespecifically responsible for the uptake of the PS-ODN. Glialcells or other cells expressing an abundance ofVEGF/VPF arepotential targets in this model of ischemia-induced retinopathy(22, 45).

FIG. 6. Antisense PS-ODN reduces retinal0 VEGF/VPF protein expression. Mice were injected

0 intravitreally with the antisense or control PS-ODN,C& S' e, indicated, and VEGF/VPF detected by Western blot

4', cwN e ffi $> z, eco analysis. The experiment was repeated three timest -+ x~. z seand a representative blot is shown. Lane 1: 1 ,ug of

partially purified recombinant murine VEGF/VPFwas probed with anti-VEGF/VPF polyclonal anti-body (23 kDa, arrow). Lane 2: Total retinal proteinfrom a control treated animal probed with nonim-_ 2 ' 3~*~'I.Mr mune rabbit serum. Lanes 3 and 4: Total retinalprotein from antisense (Vm, lane 3) or noncomple-

*'l* 1k -Lanes 5 and 6: Total retinal protein from antisense(M3, lane 5) and noncomplementary (V2, lane 6)

_ PS-ODN-treated eyes. Lanes 7 and 8: Total retinalprotein antisense (Vm, lane 7) and nonspecific (Vm

2 3 4 5 6 7 8 sense, lane 8) PS-ODN-treated eyes.

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Angiogenesis is a complex multi-step process involvingdifferent factors. Intervention at any step could have a ther-apeutic advantage. Recent work has targeted the inhibition ofVEGF/VPF action and has included chimeric molecules thatcontain extracellular domains of the VEGF/VPF receptorsFlk-1 and Flt-1 (46), anti-VEGF/VPF monoclonal antibodies(14), and the expression of a dominant negative VEGF/VPFreceptor (Flk-1) mutant (47). We have enlarged this field toinclude antisense oligodeoxynucleotides. Oligodeoxynucle-otide technology may provide improved therapeutic agents toprevent or reduce neovascular disease.

We thank Richard Sullivan and Terrance Meehan for technicalassistance, Dr. Lloyd Paul Aiello for helpful discussion in the prepa-ration of this manuscript, and members of the research staff atHybridon for critical reading of this manuscript. We are grateful to Dr.Kevin Claffey for his gift of partially purified rmVEGF/VPF and toDr. Janice Nagy for her gift of anti-VEGF/VPF antibody. Thisresearch was supported in part by grants from the National EyeInstitute (EY00343 to E.A.P. and EY08670 to L.E.H.S.) and from theV. Kann Rasmussen Foundation (E.A.P. and L.E.H.S.).

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