CA2235685A1 - Hyaluronic acid as dna carrier for gene therapy and vegf antisense dna to treat abnormal retinal vascularization - Google Patents

Abstract

The invention provides methods and compositions for gene therapy, including antisense therapy. In one embodiment, the compositions comprise hyaluronic acid to promote uptake of nucleic acid by the target cells. The invention is illustrated with reference to treatment of retinal diseases caused by neovascularisation.

Description

CA 02235685 l998-04-23 WO 97/15330 PCT/AU~)61'~C~q EIYALURONIC ACID AS DNA CARRIER FOR GENE TEIERAPY AND VEGF ANTISENSE DNA TO
TREAT ABNORMAL RETINAL VASCULARIZATION
Thi~ invention relate~ to u~e of hyaluronic acid to target active agent~ which ablate the function oi-tar~eted gene~ in the control or treatment of di~ea~e. In 5 one embodiment, thiQ invention relateQ to a method and c~ ition for treating ocular ai~ease~, in ~articular retinal ~isea~e involving neova~culari~ation of the choroia and/or retina. It makeQ uQe of the ~ha~ocytic characteri~tic of ~ecific cell~ in the eye to ~rovide an l0 effective ~o~ o~ delivering an active agent to the target, for either Qhort term or long term treatment oi-neova~culari~ation. The methoa~ and com~ositions of the invention are u~eful for cielivering DNA, RNA, anti-~en~e nucleotiaeQ, peptiaeQ or other thera~eutic agentQ to 15 ~ha~ocytic cell~ or ~urro~;~g cell~.

~R~RO~ND OF THE lNV~_. lON
A) ~yal~ . ;c ACi~ a~ an Ad;~v~L or Tar~et~ Agent Hyaluronic acid (HA) is a large, complex oligoQA~chA~iae con~i~ting of u~ to 50 000 ~air~ of the 20 ba~ic ~;~Acc~ide glucuronic acia-~(1-3) N-acetylgluco~-amine ~ 4). It ia founa in vivo as a major component of the extracellular matrix. It~ tertiary ~tructure i~ a ranaom coil of about 50 nm in aiameter.
HA has the ~bility to bina a large amount of 25 water, which in vivo make~ it a vi~cou~ hydratea gel w:Lth vi~coela~tic pro~ertie~. It i~ founa in thi3 form in 1:he mammalian eye, both in the vitreous ana in the extracellular matrix.
HA has been u~ea in the treatment of certain O 30 ~i~eases and conaitions of the r -n boay both systemically ana to~ically, because of its ability to target an active agent to sites where the aisease or conaition is localisea (International Patent Publications No. WO 9l/04058 and No.
wO 93/16733). It has been shown that HA forms aepots, for 35 example at the injurea carotid artery (relative to WO 97/15330 PCT/AU95'~C~

uninjured contralateral arteries) and in colorectal tumour~
growing in experimental a~imals, ana is ret~ in the skin of such animals. In all these cases, the sites of the ~e~osit~ are areas of high HA rece~tor expression, indicating that HA targets specifically to ti~sues that are ex~re~sing hi~h level~ of these rece~tors, ~articularly to tissues unaergoing unusual ~roliferation and migration, incluaing ti~ues res~on~;~ to injury, inflammation, develo~ --~, and tumorigenesis.
The characteristic of HA which i~ im~ortant to its action a~ a ~otential adjuvant is its ability ~imultaneously to bind to other molecules and to bind to cell membranes. Cell surface rece~tor~ specific for HA
have been iaentified, incluaing the hi~tocom~atibility antigen CD44, the receptor for hyaluronic acid~ tea motility (p~MM), intercellular adhesion factor (ICAM), ana ~ome homolo~ous ~roteins in the CD44 family. The b;~;~
of virus to the cell ~embrane facilitatea by HA would allow the usual endocytotic mech~n;sms of viral u~take to be more effective.

B) n;~ of the Eye A variety of ocular diseases such as macular de~eneration and diabetic retino~athy are characterisea by neovasculari~ation of the choroid and/or retina. This ~rocess is the major cause of bl;~e~s in patients ~uffering from these condition~.

Prior Art Trea ~ t~
In age-related macular degeneration (ARMD), the formation and h~ ~rhaging of a subretinal neovascular membrane (SRNVM) results in ra~id and substantial 1088 of central vision. Various treatments are available, but all are unreliable. ~aser photocoa~ulation is the most acce~table ty~e of treatment, but it still suffers from the di~advantages that dama~e by the laser rays causes aense, ~ -n~ scotoma (Sch~ch~t, 1994; Ibanez et al, 1995 and WO 97/15330 PCT/AU~C/'~

Hudson et al, 1995) resulting in temporary 1088 of vision, ana inability to ~ ev~..L pro~re~sion of the conaition in the long term becA~e of recurrence of the neovascular m~hrane.
Thu~ thi~ treatment proviaes an advantage only in term~ of ~reventing profound visual 1088.
Similarly, sur~ical ~ v~l of the SRNVM or of subretinal blooa~ or re-positioning of the fovea by rotating the retina have largely been unsuccessful, due to ~ost-surgical com~lications and to ; n; or temporary imp ov - t in vision. These inva~ive forms of treatment and the corre~on~;ng complications therefore far outwei~h the advanta~es gainea~ ana are limitea in usefulnes~.
~~ ; n; ~tration of interferon a2a, which has some anti-angiogenic activity (Fung, 1991; Guyer et al, 1992 ana Engler et al, 1994) ana trans~lantation of retinal ~igment epithelial (RP~) cell~ (Algvere et al, 1994) have also ~roved to be of limited usefulne~, and initial ~romising results obtA; n~ with small groups of patients have not been confirmed in larger trial~.
In adaition to la~er photocoagulation which, as describea above, suffers from various di~advantages, the other main method of treating aiabetic retinopathy is the control of blooa glucose and blood ~ressure. ~he efficacy of ~uch form~ of treatment i~ limited by the motivation and com~liance of the patient involved.
About 30% of the ~o~ulation above age 75 su~fers from macular de~eneration, and about 3 in 1000 indivi~
suffer from aiabetic retino~athy. As each of these number~
will increa~e due to the aging of the ~o~ulation, and the increa~e in incidence of diabetes, there is a need fo:r a more effective manner of treating these and other ocuLar diseases -~~;~ted by neovasculari~ation.

D~ech~ni~m of Neovascularization Va~cular endothelial cell growth factor (VEGF) is a dimeric, di~ul~hide-bridged ~lycoprotein which i~ W~311-PCT/AU9GI'~ C I~C 1 known to be synthesised and secreted by a ~ariety of normal a8 well as tumour cells. Recent ob~ervations indicate that VEGF is frequently aetected in the neovascular retinal membrane3 of ~atients with aiabetes (Malecaze et al~ 1994), and in the ocular fluid from ~atient~ with either diabetic retinopathy or with central retinal ~ein occlusion (Aiello et ~1, 1994). More recently, it wa~ found that VEGF
expre~sion was induced in condition~ such a~ central vein occlusion, retinal detachment and intraocular tumours. In a rabbit model, levels of V~GF mRNA were elevated in the hy~oxic xe~ion of the retina ~ollowing induction of retinal vein occlusion. (Pe'er et al, 1995). Stimulation of VEGF
expres~ion by hypoxia has al~o been observed in other An; ~ ierce et al, 1995; Niller et al, 1994), and in vitro in all types of cell cultures (Simorre-Pinatel et al, 1994p Hata et al, 1995 and Thiema et al, 1995).

C) Anti-Sense DNA d Gene '~he ~ in TreA~ - of n;r_ A
The su~pression of expression of genes encoding ~roteins which ~~;Ate undesirable activity has been achievea in a variety of ~ituations by the introduction or ~n s~tu ~roduction of 'anti-sense' DNA se~nc~ in the target cells. These anti-sense se~nce~ are DNA ~e~e~
which, when transcribed, result in synthesis of RNA whose sequence i~ Ant;rA~allel to the se~uence encoding the protein. Such anti-sense ~e~uences have been testea in a ~ e~ o~ ~iral aiseases. Alternatively, anti-sense oligoaeo~nucleotides can be introduced into target cells;
such short se~n~s are not ~ clves tran~cribed, but inhibit transcription and/or subsequent tran~lation of the correspo~;~g ~ense DNA Requence in the target cell.
~ntil recently it wa~ widely thought that the m;~; ~equence length nece~sary in order to effect anti-~en~e inhibition of gene expression was 12 to 14 nucleotides (Wagner, 1994). However, it ha~ now been Rhown that the s~ecificity of bin~ins to the target -WO 97/15330 PCT/AUg61C~~ 1 sequence can be ~ufficiently ~nh~n~ed by use of modif~ed oligonucleotiaes com~rising C-5 pro~yne ~yrimiaines ana ~hos~horothioate internucleotide linkages that seql~en~ as ~hort as 7 or 8 nucleotide~ are effective in ~rovidin~
gene-selective, ;r -tched sensitive, r;~o~clease H-de~ nt inhibition, in which fl~nk; ng 8eql~nc~ of the target RNA are important in aet~ ;n;~g s~ecificity (wa~ner et al, 1996).
~owever, ~uccessful use of anti-~ense nucleotides to counter expression of a gene in ~ivo i~ limite~ by factors such a~ the need for s~ecific sup~ression of mutant gene expresnion (Milan, 1993; McInnes and Ba~com, 1992), or the need for hi~h concent~ations of the anti-sense nucleotiaes (~h~A~ and Ivinson, 1993).
To date, this form of thera~y has largely involvea use of anti-sense se~ence ~c~e~ in li~osomes, or airect a~lication of antisense cDNA or oligonucleotiaes to the ~ite of di~ea~e. Thus attempts to increase u~take of anti-sense sequences into the tar~et cell by e~r~ulating the~e ~equences in li~osomes have been largely unsucces3ful. It is also difficult to tar~et liposomes efficiently, and uptake is even lower than with viru~es.
The targeting may also be achievea by virus-~';Ated DNA tran~fer, using viruses such as the Sendai viru~ en~; virus is an RNA virus which has been 3hown to deliver DNA and ~roteins into cell~ with more than 95%
efriciency (F~ et al, 1987). In this gene tran3fer system, DNA nuclear ~rotein com~lex in li~osomes i~
directly introducea into the cyto~lasm of the cell by the fu~ion activity of Sendai viru~. The DNA i~ deliverea ra~idly into the nucleus with nuclear ~rotein. Sendai viru~ ted gene transfer occurs by fusion o~ the virus with the cell membrane, ana by~asses the endocytic pathway.
Recently, highly efficient delivery of anti-sense or ;~ DNA into target cells by Sendai virus ha~ been observea. Both the anti-sense and plasmia DNAs ret~; ne~

W097/1~330 PCT/AU~6/OOC~1 their activity not only in culture but also in vivo (~n~
et al, 1987). ~owever, the use of this virus is limited by the fact that there are no ~uitable constructs available at ~resent to use as vectors. In addition, the transferre~
DNA can only be ex~reasea for a limited ~eriod of time ~ince the gene transfer i~ ted by fusion.
Retroviruses have been widely used for somatic tissue gene thera~y (Boris-~awrie and ~emin, 1993). They can tar~et and infect a wide variety of host cells with hi~h ef~iciency, and the transgene DNA integrates into the host ~enome. Theoretically, the integration of t~e DNA
will ~rovide ~ermanent ~ro~uction of the transgene which could result in ~ermanent rescue of the cells. However, retroviruses cannot infect non-dividin~ cells (,~ and Gunzbur~, 1993). Furthermore, the retrovirus ~articles are unstable in vivo, which makes it difficult to achieve hi~h virus titre with inoculation. In addition, there are ~i~ni~icant concerns regaraing the onco~enicity of the ~nte~rated viruses. The inability of retroviruse~ to infect non-dividing cells means that they cannot be ~elected as c~n~ tes for ~ene transfer in the eye, as the most im~ortant tar~et cells ~uch a~ ~hotorece~tors and RPE
cells are non-dividing cells.
The usefulne~s of her~es simplex virus vectors has been limited by their ~oor efficiency of infection (Culver et al, 1992). Two ty~es of vectors have been developed, namely the re~lication aefective recomb;~t~
and the ~lasmia-derivea ~rlicons- The latter requires a hel~er virus. Althou~h the toxic ~enes can be removea from the herpes simplex virus with difficulty, the constructs ~ - -;~ c~totoxic (Johnson et al, 1992). In ad~ition, the lon~ term ex~ression of the seguences inserted has been unsucces~ful to date, and there are ~roblems with the re~ulation and stability of the constructs. The application of mo~ifiea herpes sim~lex viruses to the eye in ~ene thera~y ~oses major concerns because of their ~atho~enicity. ~er~es zoster virus infection causes PCT~AU9C~'~lC 6 ~erious infections in the eye, frequently resultin~ i~n bl;~ requiring corneal trans~lantation.
Adenoviruses have been wiaely used for gene transfer in both non-dividing and proliferatin~ cells.
They can acc~ te DNA u~ to 7 . 5 kb, and provide efficient tran~fection and high viral titre. The main advantage of using these rather than retroviruse~ is lthe ability to infect a wiae range of non-diviaing target cells (Kozarsky an~ Wilson, 1993). Replication-defective adenoviruQeQ are considered to be relatively safe, in that these viru~es are c - - ~athogens in l- - ~, usually causing relatively benign conaitions such as colds. 'rhe vectors carry tumour genes with a deletion mutation, lowerin~ the pos~ibility of bec i~ oncogenic (Siegfrie~, 1993). In the first experimental gene therapy trial approvea by the ~S National Institutes of Health R~ ~inant DNA A~vi~ory Committee, ~_ 'inant aaenoviruse~ were used to treat indiv;~nAl~ suffering from cystic ~ibrosis.
However, the main aisadvantage of adenoviru~es i~
their transient ~ene expression. This is a result of the lack of integration of the trans~ene into the cellular genome. Furthe - e, few attempts at gene delivery to non-dividing cell~ have been succes ful. The first succe~~sful gene transfer into the brain, which consists of non-dividing cells, was reported in 1993 usin~ adeno~iruse~ (Le Gal La Salle et al, 1993).
These results indicate that gene therapy is a theoretically viable approach in the treatment of diseases, but that the technical aifficulties of efficient targeting and u~take neea to be overcome by using viruses which adhere to and are taken up by the target cells. This proces~ i~ inefficient, and the use of viruses may entail an undesirable level of risk of iatrogenic disease.
Positive result~ have, however, been published that teach that regulation of biolo~ical proces~e~ by gene therapy i~
feasible.

PCT/AU9G~ 61 There i~ therefore a need for i~proved methods of targeting gene therapy for the treatment of disea~e, and for suitable compositions comprising hyaluronic acid for use in ~uch treatment.

D) Ge~e ~h- ~,,~ an~ ~.l~_ n~
In Australian Patent Ap~lication No. 75168/94 (Hybridon Inc), it was shown that in vitro expres~ion of murine VEGF could be inhibited in COS-l or NB41 cell~ by incubation with 19- to 21-mer anti-sense oligonucleotide~
ba~ea OIl murine V13:GF. A 21-mer anti~ense nucleotide targeted against the translational ~top site wa~ shown to be the effective sequence. There i~ no discloRure or ~ug~e~tion of ~ecific targeting of se~l~ces to any tissue in the eye, or of treatment of any ocular conditions other than aiabetic retinopathy.
In ~.S. Patent No. 5,324,654, a method of ~timulating proliferation of non-malignant cells is disclo~ed. The method comprisQs the in vitro treatment of cells with an anti-~en~e nucleotiae corres~o~;~ to the retinoblastoma (Rb) ge~e to inhibit expre~sion of the Rb ~ene pro~uct, resulting in suppres~ion of the expres~ion of proteins which inhibit cell growth. In this way, ~roliferation of cells is encouraged. The ~roliferated cells can then be re-impl~te~ if ~esired, and the cells may be genetically engineered to replace a ~ecific gene ~rior to re-im~lantation. Howe~er, there i~ no reference to u~e of this anti-~en~e sequence to treat conaition~ of the eye. The in~ention of ~S-5324654 is directed to establ;~ cell lines ca~able of long-term ~roliferation and to treatment of conditions such as ~uscular aystrophy and diabetes, caused by failure to express a gene.
The targeting of a ~pecific gene to a specific cell has not been attempted, and no one ocular type ha~
been sin~led out. Specific targeting u~ing adenovirus alone is expected to be difficult, a~ the viru~ ha~ the ability to transfect a large ~ariety of cell types.

-CA 02235685 l998-04-23 PCT/A~G~ q WO97/lS330 g For treatment of ocular disease~, in which other ~ite~ in the body are largely or entirely unaffectea, it i~
highly aesirable to aeliver the thera~eutic agent 3electively to the target tissue in the eye. For antii-~ense DNA, it i~ e~sential that the DNA be actually takeninto the~e target cells.
The advances in gene therapy referred to above have led to further stuaie~ of the delivery and expre~sion of transgenes into target cells, such a~ the ~-galactosiaase transgene into the retina (Bennett et al, 1994, Li et al, 1994 ana t~ ~r et al, 1994) using recombinant adenovirus as a aelivery system. The retinal pigment epithelium (RP~) is a non-renewable ~ingle cell layer in the eye, situate~ between the neural retina ana the choroia. The cells of the RPE are phagocytic neuroe~ithelial cell~ which form the outer most layer of the retina. The phagocytic pro~ertie~ of the~e cells have long been known, and have been reviewed (Bok and Young, 1979). High levels of transgene expre~sion within 3 days in the RP~ layer ana within two weeks in the ~hotoreceptor cells of the neural retina in young animal were ob~ervea.
The expres~ion of the re~orter gene was followea u~ to 9 weeks. In older ~ , neither subretinal nor intravitreal injections ;~ce~ the expression of the ~-~alacto~iaase trans~ene in the photorece~tor cells (Li etal, 1994).
Australian Patent A~plication No. 61444/94 ~hows that replication-defective recombinant aaenovirus is taken u~ by various tis~ues in the eye following injection into the anterior chamber, the vitreous ? ~, or the retrobulbar s~ace, and that the re~orter gene ~-galactosidase is expressea. ~owever, this do~l - t does - not show that such forms of viruses 3ucces~ully incor~orate the active agent into the target cell or area.
Nor is there any aisclosure or sug~estion t~hat V~GF can be usea to heal any ocular condition.

_ , WO 97/lS330 PCT/AU~)GI~

One s~ecific obstacle to success of using anti-~ense nucleotides a~ a form of thera~y for the eye is the inability of the nucleotide to enter the target cell~, and the limited stability of the oli~onucleotiaes which have been modified, e~. ~hosphorothioate oligonucleotidea (Helene 1991). These factors greatly restrict the success of ~ene theraRy in vivo, particularly in the long term. In the treatment of retinal diseases, the ability to delay ~rogres~ion o~ the conditions by about 12 - th~ would greatly increase the value and e~fectiveness of long term therapy.
Cytotoxicity has been observed in association with u~e of adenoviruses as a transport vector ~or retinal gene thera~y. This cytotoxicity has been ~hown to be do~e-15 de~enaeIl.t (M~l - ~, 1994) and iposes another di~ficulty in using such a vector. In order to decrease the aose of a ~iven vector but retain its transfer e~ficiency, an adjuvant may be usea. Adjuvants such a~ lipofectin have been ~hown to increase the uptake of ~naked~ DNA by cells.
Even though HA has been widely used in eye surgery as a re~pl AC' - t for vitreous humour lost durin~
the ~urgical procedure, we are not aware of any suggestion in the art that HA promotes u~ptake of any pharmaceutical agent into any cells or tissues in the eye. Similarly, although HA has been suggested to promote ~enetration of ~h~ tical a~ents such as antibiotics or anti-c~ncer a~ents, ~8 set out in Au~tralian Patent A~lication No.
52274/93 by Nor~pharmco, this specification doeQ not sug~est that HA ~promotes uptake of any agent, let alone DNA or viruses, by individual cells of any ty~e. In ~articular, this s~ecification does not teach the use of HA via intra-oc~ ~ injection.
We have now found that the phagocytic nature of the RP~ cells will increase the u~take of molecules such as oligonucleotide~ and viruse~, following injection into the vitreous s~pace in vivo. TheQe RPE cellQ show increased u~take o~ virus compared to other cell type~. Our f;n~;ng~

, CA 02235685 l998-04-23 PCT/AU~C/O0 C~q enable the induction of both long-term and short-term ~ nh; h~ tion of V~GF ex~ression in retinal or choroid e~i~h~l;~l cell~, and hence inhibition of neova~c--lA~isation o~ the retina or the development oi SRNVM.
-S~MM~RY OF THE lwv~..llON
Accoraing to one a~ect, the invention proviae~ acompo~ition com~ri~ing a nucleic acid and a hyaluronic: acid or a derivative thereof, to~ether with a ~harmaceutically-acceptable carrier.
The nucleic acia may be a DNA or RNA, and/or maybe a nucleotide ~equence which is in the anti-~en~e orientation to a tar~et ~equence. The target ~equence is a nucleic acid sequence which is im~licated in the causation or exacerbation of a ~athological condition. This tar~et nucleic acid sequence ma,y be a genomic DNA, a cDNA, a me~senger RNA or an oligonucleotide. Where the target nucleic acid sequence is a genomic DNA, it may be pre~ent in a co~;~g region, or in a regulatory region, such a~ a ~romoter seque~ce.
Alternatively, the nucleic acid may be ~resent in a vector com~rising a nucleic acid ~equence to be tran~ferred into a tar~et cell. Again the nucleic acid sequence may be geno~ic DNA, cDNA, me~en~er RNA, or a,n oligonucleotide. However, in thi~ case the nucleic a~id may either be a sense sequence to be provided to a target cell in order to exert a function, or may be an anti-~ense ~equence to be ~rovided to inhibit the ~unctioning o~ a nucleic acid ~resent in the target cell.
The vector comprising the DNA to be transferred may be a virus, ~uch a~ an adenovirus, an adeno-a~sociated - virus, a herpe~ virus or a retrovirus. The use o~ all o~
these cla~es of viru~ a~ vectors for gene thera~y ha~ been extensively canvassed in the art. Alternatively the vector may be a liposome.

CA 0223~68~ l998-04-23 ~CT/AU 9 ~ / O O .
~FCE~YE~ t ~ J.'~, For the purposes of this specification the term ~comprising~ is to be understood to mean "including but not limited to".
The invention also provides a method of treatment of a pathological condition in a subject in need of such treatment, comprising the step of administering an effective dose of a composition according to the invention to said subject.
It will be clearly understood that the dose and route of administration will depend upon the condition to be treated, and the attending physician or veterinarian will readily be able to determine suitable doses and routes. It is contemplated that the compositions of the invention may be administered parenterally, for example by intravenous or subcutaneous injection, topically, for example adsorbed on gels or sponges, or directly into the tissue to be treated, for example by intra-ocular or intra-tumoral injection The subject to be treated may be a human, or may be an animal, particularly domestic or companion mammals such as cattle, horse, sheep, goats, cats and dogs.
In the compositions of the invention the nucleic acid or vector may simply be mixed with the hyaluronic acid, or may optionally be physically or chemically coupled to hyaluronic acid.
In a preferred embodiment this aspect of the invention provides compositions and methods for treatment of a retinal disease mediated by abnormal vascularization, in which the nucleic acid is an anti-sense nucleic acid sequence corresponding to at least a part of the sequence encoding vascular endothelial growth factor (VEGF), and is administered together with a hyaluronic acid as described below.
Many forms of HA are suitable for use for the purposes of the invention. In particular, both low and high molecular weight forms of HA may be used. The only re~uirement is that the HA be of a degree of purity and H:\~.uisd\l~eep\specis\hydl-pc~-00664.doc 9/01/98 ~MEND~D SHEET
EA~AU

CA 0223~68~ l998-04-23 ~CT/AU ~ ~ / O O ~
R E C E I ~ q~ J

sterility to be suitable for pharmaceutical use; pre~erably the HA is also pyrogen-free. High molecular weight preparation of HA may require dilution prior to use In particular, commercially-available HA products suitable for use in the invention are those supplied by Hyal Pharmaceutical Corporation, Mississauga, whi~h is a 2%
solution of HA having a mean average molecular weight of about 225,000; sodium hyaluronate produced by Life Core~
Biomedical, Inc.; Pro Visc (Alcon Laboratories); and "HEALON" (Pharmacia As, Uppsala). It will be clearly understood that for the purposes of this specification, the term derivatives of HA encompasses homologues, analogues, complexes, esters and fragments and sub-units of HA.
Derivatives of HA which may be used in the invention include pharmaceutically-acceptable salts thereo~, or fragments or subunits of HA. The person skilled in the art will readily be able to determine whether a given preparation of ~A, or a particular derivative, complex etc. of HA, is suitable for use in the invention.
According to a second aspect, the invention relates to a composition for treatment of a retinal disease mediated by abnormal vascularisation, comprising an anti-sense nucleic acid sequence corresponding to at least a part of the sequence encoding vascular endothelial growth factor (VEGF), and optionally further comprising one or more adjuvants such as hyaluronic acid or a dendrimer compound for increasing cellular uptake, together with a pharmaceutically acceptable carrier. The use of dendrimer compounds to transport genetic material into target cells is disclosed in International Patent Application No. WO 95/24221 by Dendritech Inc et al.

H:\Luis~\lCeep\specis\hy~l-pct-00664.doc 9/01/98 ~MENDED SHEEr 3~J~ ~

CA 02235685 l998-04-23 PCT/AU~C~

The V~GF i~ mo~t ~referably h m~n retinal pigment e~îthelial (RPE) or choroidal endo~h~l;~l V~GF.
In se~arate - ~o~;ment~, this a~ect of the invention i~ directed to treatment for ~uch retinal aisea3e in the ~hort term (u~ to about two - h~ ), the long-term (u~ to about one year), and indefinitely (for the life of the ~atient). In the fir~t embodiment, for short-term treatment the invention ~rovides one or more anti-~en~e oligonucleotide~ having 100% com~l.~ ~ t~ity to a corres~onA;~ region of the VEGF gene. The oligonucleotide should have 16 to 50 nucleotides, ~referably 16 to 22, and more preferably 16 to 19 nucleotide~. Modified oligonucleotides of the kind described by W-gne~ et al (1996) may be u~ed, and enable the lower limit of gequence length to be re~ce~ to 7 nucleotiaes.
For long-term inhibition, the invention ~rovide~
a recombinant viru~ com~rising VEGF DNA in the anti-~ense direction. Thi~ V~GF DNA is a lon~ ~equence, which for the ~urpo~e~ of thi~ ~ecification i~ to be under~tood to re~rese~t a VEGF ~equence of ~reater than 20 nucleotide~ in length, preferably greater than 50 nucleotide~, ranging u~
to the full length sequence of VEGF. In this ~ ~o~; - L, the recombinant virus is accumulated in RPF cells, and prs~ce~ anti-sense VEGF in situ, thereby inhibiting VEGF
expression in the RPE cell.
For indefinite inhibition, the invention ~rovide~
a virus com~ri~ing V~GF DNA in the anti-sense direction in which t~e viru~ is one capable of integrating the anti-sense ~equence into the genome of the tar~et cell.
Preferably the virus is an adeno-associated or ~imilar virus. A~ in the embo~; - t directed to long-term treatment, thi~ V~GF DNA is of at least 20 nucleotides, ~referably greater than 50 nucleotide~. The a~eno-as~ociated or ~imilar viru~ facilitate~ integration of anti-~ense VEGF DNA into the RPE cell ~enome, thu~ enabling expres~ion of anti-~ense VEGF for a~ long as the cell ~ functional.

W O 97/15330 PCT/AU~GI!UO6Cq Eye ~;Pe~es ~- ;ch may be treated u~ing the compositions an~ methods of the invention include, but are not limited to, a~e-relatea macular de~eneration (ARMD) and diabetic retino~athy. Other ocular conditions and tissues in which neovascularisation occurs, for exam~le branch or central retinal vein occlu~ion, retino~athy of ~rematurity (al~o known as retrolental fibroplasia), rubeoQis iriai~ or corneal neovascularisation, may also be treatea by the invention.
In another a pect, the invention ~roviaes a method of ~revention or amelioration o~ a retinal disease mediated by abnormal neovascularisation, compri~ing the ~te~ of A-- in; ~tering an effective amount of an anti-sense nucleic acid ~equence directed again~t VEGF into the e~re~
thereby to ;~h;h~t neovascularisation.
The anti-~ense sequence may be carriea in a re~lication-defective rec~ h~n~t viru~, as a vector o~-vehicle. The vector ~referably com~rises re~lication-~e~ective a~enovirus carrying ~romoters such as the res~iratory syncytial viru~ (RSV~, cytr ~alovirus (CW~), aaenoviru~ major late protein (M~P), VAl pol III or ~-actin promoters. The vector may al~o com~ri~e a polyaaenylation signal ~equence ~uch as the SV40 si~nal ~equence. In a ~articularly preferred embodiment, the vector i~ ~Ad.RSV, ~Ad.M~P, or ~Ad.VAl. In a more particularly ~referred embodiment the vector is Ad.RSV.aVEGF or Ad.VAl.aVEGF.
In a preferred ~mhoaiment, } -n VEGF is ~ubcloned into the ~ector, in order to create the restriction ~ites necessary for insertion, to form an adenovirus ~1~ i~ carryin~ VEGF or ~artial sequenceQ
thereof in an anti-~en~e airection, which can then be linearizea by restriction enzyme di~e~tion. The linearized ~ pl~ ;~ can then be co-transfected with a linearize~
replication defective adenovirus, in a ~uitable permis~ive host cell ~uch a~ the kidney 293 cell line.
The composition~ of the invention may be delivered into the eye by intra-vitreal or sub-retinal CA 02235685 l998-04-23 WO 97/15330 PCT/AU~ 61 injection, preferably in an appro~riate vehicle or carrier.
Such method~ of ~ tration an~ vehicle~ or carrier~ for such injection are known in the art. Alternatively, ex vivo delivery of the compo~itions of the invention may be achieved by removal of RPE cells from the patient to be treated, culturing the cells and ~ubjecting them to infection in vitro with a re~lication-defective adenoviru~
or an adeno-associated viru a~ defined above. RP~ cells carrying the viru~ are then injectea into the Qub-retinal layer o* the eye of the patient.
While the invention is ~pecifically describe~
with re~erence to con~itions of the eye, the ~er~on skilled in the ~rt will be aware that there are many other patholo~ical conditions in which VEGF is of im~ortance.
Such a person will under3tan~ that the antisen~e oligonucleotide~ and the recombinant viruse~ of the invention are applicable to treatment of ~uch other condition~. Similarly the ~kille~ ~er~on will under~tan~
that while the invention is specifically illu~trated with reference to VEGF the methods des~ribed herein are applicable to use with other proteins.

BRIEF D~SCRIPTION OF ~HB FIGURES
Figure la ~hows the result~ of GeneScan analy~i of persistence of anti-sense oli~onucleotides in vi~o in the retina followin~ a sin~le intra-vitreal injection.
Figure lb show~ a confocal microscopic image of the retina of a RCS-rdy' rat at different tim~s following injection of CATSCF.
Figure 2 i~ a gra~hical representation of the number of phagosomes in the RP~ layers of Long-~vans rats.
Do~es were as follows: Low 6.6 ~g, medium 66 ~g and high 132 ~g of CATSC anti-sense oligonucleotide. Each column shows the mean an~ stA~d deviation of the number of ~hagosomes in five r~ y ~elected areas in the rat retina~.

PCT~AU~C~C~Cq Figure 3 is a graphical re~re~entation of the number of ~hago~omeM in the RPE layer~ of RCS-rdy+ ra1;~.
~x~er; -ntAl animals were injected with 66 ~g of ~en~e t oli~onucleotides (Sl) and 66 ~g of anti~en3e oligonucleotide (CATSC).
- Figure 4 shows the effect of increa~ing the titre of adeno~iral vector on the ~e~ of cell~ ex~ressing the aaeno~iral tran~gene. In all case~, the incubation pQriod wa~ 16 hours. RPE7 denote~ Human retinal pigment e~ithelial cells from a 7 year old donor; F2000C ~enot:es F2000 fibroblastic cell~. The C ~uffix on the F2000 key indicate~ that the count~ for the F2000 cell ex~re~ion have been corrected for ~irect com~arison with the RPE7 cells.
Figure 5 ~how~ the effect of increa~in~ the time of incubation with the adenoviral ~ector on the number of cells ex~ressing the adeno~iral transgene. In all ca~les, the co~e~t~ation of the adeno~iral ~ector wa~ 2 x lO6 p.f.u./ml. The C ~uffix on the F2000 key inaicates that the counts for the F2000 cell ex~re~ ion ha~e been corrected for direct com~ari~on with the RPE7 cell~.
Figure 6 i~ a graphical re~re~entation of the effect of Hyaluronic Acid (HA) on the number of RPE7 cells expres~ing an adenoviral tran~gene for a 4ixed ~iral titre.
The three bar~ indicate the e-ffect of 0.001% HA, 0.005% HA
an~ no HA (control). The error bar indicates one ~n~d deviation.
Figure 7 i a ~raphical re~resentation of the effect of Hyaluronic Acid (HA) on the number of F2000 cells expres~ing an adenoviral tran~gene for a fixed viral titre.
The three bars indicate the effect of 0.001~~HA, 0.005%HA
and no HA (control). The error bar indicate~ one st~n~d deviation.
Figure 8 shows the immunofluore~cent s~A;nin~ of HA rece~tors in RPE7 and F2000 fibrobla~t~ 8a. CD44 ~tA;n;ng on RPE7; 8b. ICAN stAining on RPE7; 8c. RHAM~
~tA;n;n~ on RPE7; 8~. CD44 stAin;ng on F2000 fibroblasts;

8e. ICAM s~;~;ng on F2000 fibroblasts; 8f. RHAMM st~;n;~
on F2000 fibrobla~ts.
Figure 9 ~hows micro~ ~hs of chorioca~illary endothelial cells isolated from ~orcine eye, illustratin~
their characteristic a~pearance (top ~anel), ~resence of Factor VIII-related anti~en (middle ~anel)~ and ability to take u~ acetylated low-density lipoprotein into the cyto~la~m (bottom panel).
Fi~ure 10 shows the effects of a variety of hyaluronic acid ~re~arations on tube formation by chorioca~illary endothelial cells.
Figure 11 shows the alkaline ~hosphatase st~;n;ng Of CD44 antigen in retinal ~igment epithelium cell~. In each ca~e the e~ithelium i3 at the bottom of the ~icture with choroid above.
. ~nbleAch~ ~igment e~ithelium layer B . Pigment e~ithelium layer bl~Ach~A to 1- -ve ~1 ~n; ~ ~ranule8.
C. Ble~he~ ~igment e~ithelium ~t~i~e~ with alkaline phos~hatase-labelled anti-CD44 antibody.
Fi~ure 12 ~hows the results of DNA PCR and RT-~CR
analysis of transfection of a retinal ~igment e~it cell line with VEGFl65.
Figure 13 shows the effect of VEGFl65 produced by tran~fected RPE cells on tube formation by chorioca~illary enaothelial cells.

DE~TT~T~n DESCRIPTION OF THE lNV~-N-llON
The invention will now be described by way of reference only to the following non-limiting exam~les. In some of these exam~les, the feasibility of the methods utilised in the invention is ~- ~qtrated using anti-sense oli~onucleotides compl~-~nt~y to cathe~sin S (CATSC).

CA 02235685 l998-04-23 PCT/AU~~'~C~t~

Example 1 Ac~ l~tion of Antisense Oli~onucleot:iaes in the RPE Cell Layer Human retinal ~igment e~ithelial cell~ were culture~ and on the third ~assage were used for in vitro ex~eriments. Confluent culture~ were ;~c~Atea with bovine rod outer ~ (ROS) to mimic the in vivo situation. A
fluore~~cein-labelled anti-sen~e oligonucleotide com~ ~y to ~ -~ cathe~sin S (CATSCF) was ~A~e~ to the medium of the~e cell~ an~ after 7 day~ of incubation, the cellQ were harvested. The presence of f luore~cein-labelled oli~onucleotides within the RPE cells was detQcted by fluorocytometry ~FACS). A GeneScan DNA analy~er wa use~ to a~se~~ the ~resence and ~tability of the oligonucleotiaes in the cell~. The fluore~cence of culture~ RP~ cells wa~ increased by about 100-fold, demonstrating the presence of the anti-sense oligonucleotide~ within the RPE cells. These result are ~~i~ea in Table 1.

Table 1 Fluorocytometer measurements of human RPE cell~
~c~ te~ with or without com~l~ - L A ~Y CATSCF

RPE I ROS 5,94 RPE + ROS I CATSC 8.50 RPE I ROS I CATSCF 461.50 It was not known if the f luorescence was emitt:ea by the full length CATSC or by degradea olisJonucleotiae~~.
ing GeneScan, it was ~r ~ ~trated that the fluorescence wa~ largely due to a l9-mer oligonucleotide~ which appeared at a ~osition similar to that of CATSCF. ~sing a ~~imilar ~rocedure, it wa~3 observea that CATSC oligonucleotide~~ were ~~till intact after 7 aay8 of incubation.

, WO 97/15330 PCT/AU!)G~ q Example 2 Cellular Distribution of Oli~onucleotiaes in Retinal Cell~ and StabilitY of Oligonucleotides Following Injection ~nto ~YeA
One nmole of CATSCF was injected into the vitreou~ humour of 6-week old non-~igmented RCS-rdy' rat~, ana the movement of the oligonucleotide~ were followed by confocal fluoromicrosco~y. Fluorescein (1 nmole) was also injected as a control. A~; ~ 1~ were euth~ ed 2 hours, 3 days and 7, 14 and 28 and 56 days after injection.
Following eu~hAn~ia~ the injected eyes were enucleated, frozen, sectioned and immediately used for confocal microQco~y without fixation.
Two hours after intravitreal injection of CATSCF
the ~enetration of the oli~onucleotides were observed in the ganglion cell layer at 2 hour~ and also in the ~hotorece~tor and ~igment e~ithelial layers at 3 day~.
However, 7 aays following injection, only the RPE layer had significant amounts of CATSCF. At 14, 28 and 56 days, a 20 _luore~cent Aignal was ~;~;~e~ in the RPE layer, and no ~i~nal was ob~erved in any other cell type~. The~e resultn ~how that a large ~ro~ortion of CATSCF was taken u~ by the phagocytic RPE cells.
Following intravitreal injection as described above, eyes were ~issected, the retina was l.- -,ved, and the DNA extracted. The purified DNA was subjected to GeneScan analysis. ~he ~resence of undegraded fluorescein-labelled oli~onucleotide was ~r ~ ~trated in the rat retinas after 7, 14, 28 and 56 day~ of injection, as ~hown in Figure la.
The intensity of the signal had ~ignificantly ~; ;n; ~he~ by 56 days.
Confocal microsco~ic analysis wa~ ~erforme~
followin~ a single injection of 10 nmol CATSCF into non-pi~mented ~CS-rdy+ rats. Retinas were eY~ ;~e~ at interval~ after injection, and the results are shown in Figure lb, in which g re~resents the ganglion cell layer, i the inner nuclear layer, o the outer nuclear layer, and r WO 97/15330 PCT/AU~Ci~ ~ 561 the retinal ~igment e~i~h~l~l layer. The ~An~l~ show retinas 2 hours (B), 3 day~ (A), 7 day~ (C), 28 days ~D) and 56 day~ (E) after injection of 10 nmol CATSCF, an~
3 daya (F) after injection of FITC as a control.
These results demon~trate that following intravitreal injection, oligo~ucleotide~ accumulate in the ~P~ cells. The oligo~cl~otides are pre~ent in the RP~
layer u~ to 56 days, An~d ~ -;n in a biologically active ~orm durin~ thi~ ~eriod o~ time.

Example 3 Biolo~ical Acti~itY o~ Anti-Sen~e Oli~onucleotide~
Female ~ixty day-old ~igmented rats of the Lon~-Evans strain were obtA;~e~ from Charle~ River Bree~in~
Laboratorie~, W;l ;n~ton~ MA.
Sixty day old non-~igmented RCS-rdy+ rat~ were obt~;n~ from our colony. The A~; - 1 ~ were acclimati~ed to a 12 hr light/ 12 hr dark light;ng cycle, with an average illuminance of 5 lux for at lea~t 10 aays ~rior to ex~er; - tAtion.
~n; -1~ were anae~theti~ed by intra~eritonea:L
injection of ~odium ~ntobA~h~tal (50 mg/kg body weight).
Intravitreal injection~ through the ~ars ~lana were made using a 32 gauge needle. The left eye~ ~erved as controls, and the ri~ht eyes were injectea with 3~1 of 150 mM ~odium chloride (~aline), or with 3 ~1 of ~ali~e co~tA;n;ng ~.6, 66 or 132 ~g of CATSC respectively, an anti-~en3e oligonucleotide described earlier (Rakoczy et al, 1994) or 66 ~ of ~ense oligonucleotide Sl, 100% com~l~ -ntAry to CATSC. Injected animals were allowed to recover from anaesthesia, and at one week ~o~t-injection were sacrificed by an overdo~e of sodium ~ntohA~bital and uQed for morphological ~Y~m;n~tion. All An; -lC were killed within half an hour at the same time of the day, a~proximately 4 hour~ after li~ht onset. Two to three animals were used 3 5 for each dose.

WO 97/15330 PCT/AU~)6/OCC6 1 Following enucleation, whole eye~ were immerse~
in 2.5% ~lutar~qlA~yde an~ 1% paraformaldehyde in 0.125M
sodium cacodylate buffer, pH 7.35. The co~n9A and lens were dis~ected free and the e~_~ trimmed for orientation ~urpo~es. The tisque wa~ fixed overnight at 4~C and then ~ost-fixed for 1 hour in 1% osmium tetroxide at room tem~erature. After ethanol dehydration, the tis~ue was embeAA~ in epoxy resin. Retinal ~ections were prepared for trAn ;~3ion electron micro~co~y a~ described previou~ly (Re~neAy et al, 1994).
Hi~tolo~ical data were obtA; ~ by light microscopy. Semi-thin 1 ~m sections were cut using a L~B
2088 ~ltratome (LKB-Produkter, Swe~en) with a diamond knife and st~; ~eA with toluidine blue. The number of ~hagosomes that ac~ ted in the RPE cell~ of eaah s~ecimen injected with ~aline, low (6.6 ~g), medium (66 ~g) or hi~h 132 ~g) dose of CATSC and 66 ~g of Sl ~ense oligonucleoti~e wa determined. From each eye, fi~e sets of count~ were made at 40 fold magnification ana the stA~A~d ~eviation was calculated. Each set con~istea of the total number of ~hagoso~es in 250 ~m length of RPE from 6 different randomly ~elected area~. The ~umber of phagosomes that ac~ l~ted in the RPE of the control eye~, low medium and high do3e~ of CATSC were analysed and graphically re~re~ented. C _q~isons were made using the analysis of variance following the general linear model~ procedure of the SASR (version 6) statistical ~acka~e (SAS In~titute Inc., ~SA).
The results show that we successfully tested an anti-sense oligonucleotide (CATSC) in two strains of rats.
The number of pha~o~ome-like inclu~ion bodieç ~resent in control Long-~vans and RCS rdy I rats was not ~ignificantly different, 35.8l11.6 and 47.29~14.8 (mean ~ SD), respectively, The intravitreal injection was non-traumatic. Light microscopic e~r-;~rtion of the retina~ of the saline injected eyes revealed no damage to the outer layers of the retina, and there was no increa3e in the CA 02235685 l998-04-23 ~ o~ ~hagosome-like inclusion bodies in the RPE layer when com~ared to the control non-injected An; -~. ~ong-Evans rats were usea to identifr the minimum amount of CATSC required to induce biological changes in the RPE
layer. In the control eyes and in those injected with low do~e (6.6 ~g) of CATSC, the number o~ pha~osome-like inclusions within the RP~ cells were 35.8111.6 and 35.0+7.4 respectively. In animal~ injected with higher doses (66~g and 132 ~g), the ~e~ of ~hagosome-like inclusions were 96.2+13.6 and 141.0134.7, respectively, and the difference was statistically ~iqnificA~t when c _-~ed to the control and low do~e sam~le~ (Figure 2).
RCS-rdy+ rats injected with 66 ~g of CATSC al~o demon~trated a statistically significant increase in the number of ~hagosome-like inclusion bodies, ie 204.20+39.3 when com~ared to the 47.20+14.8 in controls. In contrast, the injection of 66 ~g o~ ~ense oligonucleotide (Sl) dia not increase the ~ ~ of ~hagosome~ (Figure 3) ~resent in the RPE Layer, (34.4~12.54).
The inclusion~ found in RPEs of CATSC-injected ~ong-~vans and RCS-rdy+ animals were s~herical in ~ha~e, and clearly di~tin~;~h~hle from the very dark, small elliptical mel~n; n granules ~reQent in ~ong EvanY rat~. In the ~resence of 66 ~g of CATSC, the tips of the outer segments ~howed si~ns of di~organisation and there were ~ome vacuole~ ~re~ent in the outer ~cJ 9A~ layer. However these chan~e~ were not ob~erved in Sl ~en~e oligonucleotide-injected An; ~
Electron microscopic eYA ; ~tion of the RPE layer of a CAT SC-injected eye revealea no ~ignificant changes in the morphology of RPE cell~. ~el~;~ granule~ a~eared smaller and le~s concentrated due to regional difference~.
Individual mitoch~ial ~rofile~ were ~maller in the treated grou~ than in the controls, althou~h the number was greater in the treated than in the untreatea animals.
Electron microsco~ic e-lr;~tion confirmed that the ~tructures o~ the undigested material wa~ ~imilar to that WO 97/15330 PCT/AU~)G/~1~66q of phagosome~. The numerou~ ~hagosomes ~een in the RPE
layer of rats treated with CATSC were ~aranuclear, and ContA; ne~ mainly compacted pho~pholi~id membranes, resembling undigestea ~hotoreceptor outer 8e~ ~nt ( pos ) and conf; ;n~ their ~hotorece~tor origin. There were no other morphological change~ ob~ervea in the POS layer, except for the disor~ani3ed a~earance of the apices in treated ~n; ~1~

Exam~le 4 Gene Transfer to the RPE Cell Layer The nature and dynamics of gene transfer u~ing an adenoviral vector were examined. The effect~ o$ adjuvants on the u~kake o~ the a~enoviru~ wa~ al~o studied.
-n RP~ cultures (HRPE7) were obtA;ne~ from a 7-year old CA~cA~ian donor and prepared as described in Rakoczy e~ al (1992). -n F2000 fibrobla~t cells were cultured, harvested and pooled in Minimal ~agles Medium (MEM, Multicel TM Trace Biosciences, Au~tralia), with 10%
FBS (MultiserTM, Trace Bio~cience~) and contA;n;ng 125~1 gentamicin (Delta We~t, Bentley, Australia) ~er 100 ml medium. One ml aliquot3 of the ~ooled cell suspen3ion were placed into each well of a 24 well plate, to ensure equal ~ee~;ng of well~. ~XPeriments were carried out with cells at confluence, and at lea~t two parallel set~ of each exper; - tAl points were obtA;n~.

Expre~~~ion of Adenoviral ~ranE~çrene Replication-deficient ~e~ovirus 5 carrying a RSV
~romoter and ~-Galactosida~e gene (Ad.RSV.~gal) (Stratford-Perricaudet, 1992) wa~ cultured and purified as de~cribed by Graham and Prevac, 1991. Ad.RSV.~gal was added to each well as a 1 ml aliquot, in NEM, at a concentration of 4X106 ~.f.u./ml. for the time-basea trials, gi~ing a final concentration of 2X106 p.f.u./ml.
For the titre-based trials, conc~nt~ations of 8x103, 4x104, 8x104, 2.4x105, 4x105 ~.f.u./ml were ~A~ to the wells in a 1 ml aliquot, m~k;ng the total volume 2 ml in each well WO 97/15330 PCT/AlJ-36~1C~q (the ~inal viral conGent~ation i~ half of that aaded). All of the trials examining the effect of increasing viral titre~ involved ;n~hAtion of the culture with the ~iral ~uspen~ion for a fixed period of 16 hour~.
~xperiment~ were terminated by L.- ving the - meaium ~rom each well and fixin~ the cell3 with 0.5 ml o.f 0.5% glutaraldehyde. The glutar~ yae waR remo~ed after 5 minute~ and the cells w-Qh~ once with Pho~phate Buffered Saline (PBS). Following this, 0.5 ml of X-gal ~tain tFor 1 ml of ~olution (co~cent~ation in final ~olution): 25~1 X-Gal (0.5mg/ml, BioRad, Hercules, California), 44~1 HEPES
buffer (44mM), lOO,Ul K4Fe(CN)6(3m~) 1oo~ll K3Fe(CN)6(3m~, 100~1 NaCl(15mM), 100~1 MgCl2(1.3mM), sterile di~tilled water to make 1 ml (531~1)] was a~ded to each well and ;~c~Ated overnight (about 16 hour~) at room tem~erature.

Cell Co~ln~; n~
An Olympu~ TO41 ~hase contra~t micro~co~e (Olym~u~ Optical Co ~td, Tokyo, Japan) at a agn;fication of 200x was u~ed. Counting wa~ carried out by a ~in~le ob~erver. A ~econ~ ob~erver then blind counted 25% of t~he ~ample~ a~ a countercheck. A counting graticule in the micro~co~e wa~ usea to define the region for counting when averaging was u~ea.
All cell~ stA;n;ng ~o~itively with the X-Gal ~tain were counted. At low expre~ion of tran~gene (< a~roximately 2000 cell~/well), the entire ~late was counted. When the cell count waR higher, averaging was u~ed. Cells were countea in fi~e ~tAn~A~dized region~ and their a~erage waR u~ed to calculate the total count for each well.
In the trial~ comparing the rate of expre~ion in HRP~7 and F2000 fibrobla~ts, the figure for the number o~
F2000 cell3 expreasing the gene was correcte~. This correction reflect~ the different total cell number of each cell type in a confluent culture in a 24 well plate. The count for HRPE7 i~ 3x105 per well and for F2000, it is CA 02235685 l998-04-23 WO97/15330 PCT/AUgG,~6C1 2x105 ~er well. The gra~hical figures (Fisures 4 and 5) al~o co~;n corrected counts to allow direct ~ _~ison.
Where there i no c~ on between cell ty~es, no alteration of the raw count is carried out.
In the titre-ba~ed trials, the profiles of ex~ression were markealy aifferent in terms of rate o~
increase and ab~olute ex~ression. For HRPE7 cell~, the expression rate a~peared to have an ex~onential form, while in F2000 fibrobla3ts the ~rofile was more linear. There was a wi~n;ng ga~ in expression throughout the trial comparing titre. At higher viral titre, HRPE7 expre~sion was an order o~ ma~nituae greater than F2000 cells. For the conditions and titres triea in this experiment there was an overall and constant increase in the number of cells expressing with increasing vector titre (Figure 4).
In the study of the effect of incubation time on the ~rofile of transgene ex~ression, the concentration of AdV.RSV.~gal was ke~t constant at 2X106 p.f.u./ml. The profile~ of ex~ression of transgene in the two cell types were markedly aifferent, both in terms of rate of increase and ~n;tude of nl~m~er of cells expressing the ~ene.
There was also a notable ~elay between the ~harp increaAe in number of HRPE7 and F2000 fibrobla~t~ expres~in~ the ~ene. For HRPE7 cells, the u~turn in ex~ression rate occurre~ at 4 hours while in F2000 fibrobla~t~, it occurred at 24 hours. There is a "window'~ ~erioa between 4 and 24 hours where the HRPE7 ex~ression is an or~er of magnituae ~reater than that of F2000 cells (Fi~ure 5).

Exam~le 5 Effect of HA as an Aa~uvant on the ~take and expression of the ~-gal Gene using a Viral Vector HRPE7 and F2000 cells were aliquoted into 24 well ~lates. The cells were incubated as described in Example 4, and allowed to reach 95% of confluence.
Solution~ of 0.001% to 0.005% buffered sodium hyaluronate ~HA) (1% Hyaluronic acid from rooster comb; HEALON, WO 97/1533U PCT/AU9f/OOG~q ph~ ~cia AB, ~ala, Sweden) were ~repared with MEN. A
dose of 10 ~1 of viral solution at a conr~ntration of 4 x 106 ~.f.u. was added to 10 ml of each of the diluted HA
solutions and lO ml of MEM for the control, A~d incubated for 30 minutes at 25~C with intermittent ~entle Rh~k;n~
To separate wells of the 24 well ~late, 1 ml of each of the test and control ~olutions was ~. There were four parallel ~am~les for each test concentration and for the control, which were counted and averaged.
The viral/HA solutionR were incubated with the cell cultures for 16 hourR. Each experiment was t~ ;r~tea according to the procedure given in Example 4.

Table 2 Experiment l: Ex~ression in HRPE 7 Cells 1 2 3 4 Mean (0.001%) (0.005%) 7/Cont The mean nu_ber of HRPE7 cells expressing the transgene in each well for adenovirus alone was 14 705 (SDl2228). For a~eno~irus with 0.001% HA the mean ~l~mhe~
of expressing cells was 19 168 per well (SD 1561) and for 0.005% HA the mean was 20592 (SD 2143) (Figure 6). This shows an increa~e in ~ of cells expre~Ring the transgene of 30.4% for 0.001% HA and of 40.0% with 0.005%
HA.
AR a88egged by Student's t test, the ~robability of the significance of the increase in number of HRPE7 cells expressin~ the gene, when 0.005% HA is usea, com~ared with the control, is 0.0097, which shows a level of significance of ~<0.01. The significance reflects the CA 0223~68~ l998-04-23 PCT/AUsG,~66q lar~e difference between the means (20592 (te~t) v 14705 (control)) and the se~aratio~ of the means by more than two st~n~d deviations.
The t test ~robability of the significance of the increase in he~ of RP~7 cells ex~ressing the gene, when O.001% H~ i~ used compared with the control, is 0.02931, which shows a level of significance of ~0.05. ~he r~c~
~ignificance reflects the smaller difference between the means (19168 (test) v 14705 (control)).

Table 3 Ex~eriment 2: Expression in F2000 Cells.

1 2 3 4 Mean F 2000/~A 4358 4620 4195 NA 4391 ( 0 . 001%) (0.005%) Cont The ~rotocols for exa~;n;~ the effect of HA on the exprQ~sion of a transgene in F2000 fibroblasts were t~e same as that for HRPE7. The numbers of cells ex~ressing transgenes were si~nificantly less than for HRPE7, which i8 consistent with the results ~ trate~ in Example 4. The mean number of cells expressing in each well for adenovirus alone wa~ 3780 (SD~100). For adenovirus with 0.001% HA, the mean number of ex~res~ing cells was 4391 ~er well (SD+214) an~ for 0.005% HA the mean was 4378 (SD355)(Fig.
7.). This shows an increase of 15.8% for 0.001% HA and of 15.5% with 0.005% HA in the number of cells expressing the aaenoviral transgene.
The two-tailed Student's t test was used ~o aQsess th.e significance of the difference between the means for each set of experimental data. For each experiment, the means~ the StAn~A~a error of the aifferences of the CA 02235685 l998-04-23 WO 97/15330 PCT/AU9~ 61 means an~ the p value for the t test are ~iven. In both ex~eriment~, HA gave very significantly increasea u~take (P < 0.05).
The t test probability of the significance of the increase in number of cells ex~ressing transgene for the - F2000 fibrobla~ts with 0.005% HA, compare~ with the control, is 0.0044, which ~hows a level of significance of ~<0.01. The high si~nif;c~n~e here reflects the large aifference between the mean~ (4391(teQt) v 3790 (control)) an~ the small variation within the two ~amples. The stAn~a deviation is 214(test) and lll(control).
The t test probability of the significance oE
the increase in number of cells ex~ressing transgene for the F2000 fibroblasts with 0.001% HA, c _-~ea with the control, i~ 0.0195, which show~ a level of ~ignificance of p<0.05. There is a greater variation in the raw figures, and the stAn~a aeviation i~ higher than for the 0.005%
sam~le (355 v 214), which accounts for the hi~her p value.
Preliminary trials of chon~oitin sulphate an~
li~ofect~ ;ne as aajuvants were also carried out in order to a~se~ the likely efficacy. These agent~ ha~ no significant effect on gene expression in HRPE7 cell~.
The followin~ aoses of adjuvant~ were also us~ea:-Table 4 HA Con~nt~ation ~mount o~ viral 0,05%0.01%0.005% 0.001% Control Control ~olut~on 5 ~l 176a 318 319 316 279 282 10 ~1 305 906 802 645 623 609 30 25 ~ 714~16821822 1478 1184 50 ~1 _a 277226923328 2250 1822 The figures re~resent the effect of HA
concentration on the u~take an~ expression of ~-gal transgene. Increasing virus concentration resulted in an WO 97/1~330 PCT/AU961~0661 increase in the ~ o~ ~-~al expressing cells. The numbers represent the h~ of RPF cells st~;~;~ ~ositive for ~-gal following 16 hours incubation of viru~ in the ~resence of HA in a 24 well ~late (cc 2xlO" ~fu/ml).

~ The viscosity of the~e solutions ~recluded adequate ~i3~ersion of the HA and made them ~ery difficult to manipulate.
b It was not clear why this fi~ure fell so far outside of the ~ormal distribution of the other results.
~0 Example 6 Effect of HA Molecular Weight on the ~ptake and ~x~ression of the ~gal Gene ~in~ a Viral Vector ~ e~ovirus with a ~-galactosida~e marker gene and a RSV promoter (AdV.RSV.~gal) wa~ cultured in cell~ of the R293 ~ryonic 1 -~ kianey cell line. Su~ernatant wa~
collected, and the concentration of virus was determined by ~erial ailution with 4 re~licate~ of each dilution. The concentration of the viru~ wa~ calculated to be 5 x 108 pfu/~l. The virus was sus~ended in ~RM medium with 10% fetal bovine serum (FBS) and 125~1/lOOml g~t~;cin.
~ ~ Retinal Pi~ent Epithelial Cells (HRPE) were from a 20 year old donor and cultured in meaium aR
aescribea above. They were aliquoted into 24 well ~lates from the same stock and allowed to reach confluence.
Fourth pas~age cells were used.
The following HA preparation~ were tested:
1. Hyal (MW approx. 300 000)

2. Provisc (MW a~prox. 1 900 000)

3. Healon GV (MW a~prox. 5 000 000) Each of the ~reparations was diluted to a solution of O .002% in MEM without FBS.
The virus solution as above wa~ mixed in a 1:1 ratio with the adjuvant olution giving a final viral concentration of 2.5 x 108pfu and an HA concentration of 0.001%. The two ~olutions were incubated in this mixture WO 97/15330 PCT/AU9Ci~ C ~q for 30 minutes at room temperature with gentle ~hAk;n~, The control solution consistea of a mixture of the viru~
with MEM without FBS with no HA present.
To each of the 24 well cells 1 ml of the viral/HA
mixture was added. Tnc~ tion was for 24 hours in a C02 incubator (5% CO2) at 37~C. The experiment was t~rm;n~ted by ~ -vin~ the viral/~A mixture and ~A~in~ 0.5ml of 0.5%
~lutaraldehyde for 5 minutes to each well. The well was ~- ~h~ once with PBS ana reacted with X ~al stain.
An Olympus T041 phase contrast microscope (Olympus Optical Co. Ltd. Tokyo, Ja~an) at a aDn;fication of lOOX was u~ed throu~hout. Counting was carried out by a single observer and ch~c~e~ against a second blind observer who counted a quarter of the samples. A counting ~raticule in the microsco~e was used to define the region for countin~. All cells st~;n;ng ~ositively blue with the X-~al stain were counted as ~ositive. Cell~ were counted in five 3t~ ~dized regions and their average was used to calculate the total count for each well. The results ana ~tatistical analysis are presentea in Tables 5 to 9.

Tabie 5 (count i~ of ~ only) Control ~Ql Pro~nc ~llon GV
' - of cell~ expr~ion 2043 2486 2424 2756 -g~ 1 25 Stati~tic~
Anova: Single Factor Between all grou~

Table 6 S~aRY
G~ ~.. ",,~ Cou~t Sum Averas~e Varian¢e Control 3 61292043 15769 Hyal 3 74582486 4225 Pro~isc 3 72712423.667 36677.33 Healon GV 3 82682756 3692~

CA 02235685 l998-04-23 PCT/AU9G~ C1 - 3~ -Table 7 ANOVA
Source of~ va~iation SS d~ MS F
Between Group777567.0 3259189 11.07653 Within Groups187198 7 ~23399 83 Total 964765.7 ll Anova: Single Factor Between Adjuvant~

Table 8 ,~MM~2Y
Grou~sCount Sum A~erasreVari,-r~ce Hyal 3 7458 2486 4225 Provisc 3 7271 2423.667 36677.33 Healon GV 3 8268 2756 36928 Table 9 ANOV~

0~ SS df MS FP-value Fcr~ t Va~;~
Between187230.9 293615.443.610.094 5.14 Grou~s Within155660.7 625943.44 Grou~

Total34Z891 _ 6 8 There was an increase in transgene expres~ion in 25 all o~ the HA-containing samples relative to the control (P < 0,003). The percentage increa~e was 21.7%, 18.6% and 34.8% ~or Hyal, Provisc ana Healon GV HA preparation respectively. There is no ~ignificant difference between the effect o~ different molecular weight~ of hyaluronic acid (p = 0.09).

SUBSTITUTE SHEET (RULE 26) WO 97115330 PCT/AU~GI~ ~C1 The~e re~ult~ demonstrate that hyaluronic aciLd increa~e~ viral ~ector uptake, ~ trating an adjuvant ef~ect. In aclclition it wa~ ~hown that the adjuvant effect is independent of the molecular weight of hyaluronic acia between MW 300 000-5, 000 O00.

~xample 7 Demon~tration of HA Receptor~ on the cell membrane of HRPE7 and F2000.
Polyclonal R~MM (Receptor for 8yaluronan M~; Atea Motility) antiboaie~ were kindly pro~idea by Dr E Turley, Manitoba In~titute of Cell Biology, C~nA~.
The antibody was u~ed at a ailution of 1:75. MonoC
InterCellular A &e~ion Molecule 1 (ICAM-l) antiboclies (Boehringer-~~- nnhei ) were used at a concentration of

4~g/ml and monoclonal homing receptor CD44 antibody (CI)44) was u~ed at a concentration of 4~g/ml (Boehrin~er M~nh~;
Bio~-;ca, Ge- -~y). Monoclonal anti-l -~ IgG antibocly and rat non-immNne ~erum were kinaly ~rovidea by Dr N RA;ne~, Lions Eye In~titute, Perth, Au~tralia. They were u~ed at a concent~ation o~ 4~g/ml and a dilution of 1:75 respectively. Anti-Mouse IgG (Fab specific)-FITC
conju~ate ~eC~n~A~y antiboay was u~ed at a 1:64 dilution and anti-Rabbit I~G (whole molecule)-FITC conjugate C~nA~y antiboay wa~ u~ed at a 1:100 dilution (Sigma ochemical~, St Loui~, Miq~ouri).
HRPE7 and F2000 fibrobla~t cell~ were cultured in Lab Tek 8-well sliae chr ~_rs (Nunc Inc. Naper~ille, Illinois). Cell culture~ were fixed with methanol at -20~C
for 10 minutes before immunofluore~cent st~;n;ng. All primary antiboay ~olutions were incubatea for 1 hour. The primary antibodies usea for each of the two cell types were monoclonal anti ICAM-l, anti-CD44 a~ test and monoclonail anti-T- ~n I~G as control, and polyclonal anti-R~MM with a non-i ? rabbit ~erum a~ control. Following the ~o~l of the primary antibo~y, each well waq wa3hed three time~
with PBS ana the ~eco~y antibody wa~ applied for 1 hour.
The ~econdary antibody to the monoclonal antibodies wa~

CA 02235685 l998-04-23 WO97/15330 PCT/AU9C/~D~C1 antimouse I~G and the A~olyclonal wa anti-rabbit IgG. The A~ecQn~A~~y antibodies were a~lied to ti~sue without primary antibody a3 a further control. Finally, on . ~cv~l of the ~ec~A~~y antibody, each well was w~he~ a further three time~ before the well chambers were l.- -ved and the slides mounted with o Fluore Mounting Medium (ICN Biomedical~
Inc, Aurora, Ohio).
I~ noh; ~tochemical st~; n; n~ for CD44 u~ing a monoclonal antibody ~ - ~trated ~ositive stA;~;n~ for both HRPE7 cell~ and F2000 fibroblast , a~ shown in Figure~ 8a and 8b res~ectively. The A~tA;n;~g had a di~tribution consi~tent with the cell ~urface, a~ the stA;n~y ~attern wa~ the Rame a~ the cellular outline of cultured tis~ue.
A monoclonal human anti-IgG wa~ u~ed a~ control, and was negative for both HRPE7 and F2000 fibroblasts. A
second control, usin~ s~co~~y fluore~cent antibody with no primary antibody was also negative for both cell ty~es.
Im o~;~tochemical stA;~;~ uA~ing a monoclonal antibody for ICAM-l demonstrated positive ~tA;~;n~ for both HRPE7, and F2000 fibroblaA~ts, as ~hown in Figures 8c and 8d res~ectively. The ~t~;~;~ had a similar distribution to that of CD44, but the ~ignal was ~lightly weaker. The aame controls as for CD44 were used for ICA~A-l stA;~;~g, and were al~o negative.
S~;~;ng for ~MM receA~tor~ using a rabbit ~olyclonal antibody wa~ positive for both A~RPE7 and F2000 ribroblasts, aA~ ~own in Fi~ureA~ 8e and 8f res~ectively.
The di~tribution of ~t~;n;n~, howe~er, was markedly ~ifferent in the two cell ty~es. In HRPE cells the ~t~;~;ng pattern was ~redom;n~ntly nuclear, with a very faint cyto~lasmic outline (Figure 8e). ~he distribution of ~t~;~;~g in F2000 fibroblaA~t~ was ~imilar to that of CD44 and ICA~ with no A~ignificant nuclear si~nal ob~ervable over the cytopla~mic or cell outline A~attern.
The control A~erum waAq a rabbit non-immune A~erum, which was negative for HRPE7 but gave a verAy weak A~ignal in F2000 fibrobla~t~. In both ca~e~, the ~ecoAn~y PCT/AU~ q ~luorescent antibody alone did not lead to a ~ositive ~ignal from either cell type.

Exam~le 8 The ~ffects of Hyaluronic Acid Preparations of Dif ferent Nolecular Weight on Tube Formation Reagents Hank~ r~ced salt ~olution (Hank'~ BSS) without calcium or magne~ium, medium Hams F12, -;n;
essential medium with ~arles salts (EM~N), foetal calf serum (FCS), ~enicillin-streptomycin, amphetericin B, and trypsin-~DTA were obt~;n~ from Australian Biosearch (Perth, Western Au~tralia). Collagena3e A, endothelial cell growth su~plement (~CGF), mouse anti-l -n monoclonal antibody against factor VIII-relatea anti~en, an~ anti-mouse Ig-fluorescein were acquired ~rom Boehringer ~~-nnhe;m Australia Pty. ~ta. (Perth, Western Australia). Gelatin, heparin, a~corbic acia were ~urchasea from Sigma Chemical Cc _ ~ny (Sydney, Australia), acetylatea low-density li~o~rotein (DiI-ac-~DL, 1,1'-aioctaaecyl-3,3,3',3'-tetramethyl-; n~oc~ ~bo-cyanine ~erchlorate) from Biomedical Technolo~ies, Inc. (Sto~ghtor~, Ma~hl~etts), Natrigel from Collaborative Research (Beafora, Mas~ch~etts), recombinant human va~c~ ~ endothelial cell growth factor (VEGF) from Pepro Tech EC ~td. (Rocky Hill, New Jersey), Pro~isc (MW 1.9 x 106) from Alcon Laboratorie~, Healon (MW 2.5 x 106) and Healon GV (MW 5.0 x 106) from Pharmacia.

I~olation and Culture of Porcine Choriocapillary ~ndothelial Cell~
Porcine eye~ were obtained from a local abattoir 2-4 hour~ after death of the An; ~l ~ . The choriocapillary endothelial cells (CECs) were isolated as ~reviously de~cribed (Morse et al, 1990, Sakomoto et al, 1995).
Briefly, Hank's b~l~n~e~ salt ~olution (Hank'~ BSS) without calcium or magnesium, but with 0.1 % collagenase A was used to release endothelial cells at 37~C for 1 hour. After CA 02235685 l998-04-23 WA ~h; ~ twice in Hank~ 8 BSS, the cell~ were ~lated in 1 %
gelatin-coated 75-cm2 cell culture fla~ks in 5% CO2, 95%
air at 37~C. The growth medium consisted of H~ F12 ~lus 10% fetal calf serum (FCS), 100 ~ ~enicillin-100 ~g stre~tomycin/ml, 2.5 ~g/ml am~hotericin B, 37.5 ~g/ml endothelial cell growth su~lement (ECGS), he~arin 100 ~g/ml, and ascorbic acid 25 ~g/ml. After 24 or 48 hours of ~lating the ca~illary segments, the colonies of endothelial cell ~howing a cobblestone ap~earance flattened and spread. On the thir~ or fourth ~ay, the non-endothelial colonie~ were recognised and were circled with a ~e -~t marker ~en on the to~ of 75-cm2 flasks. A
glass ~i~ette which had been drawn through a flame to ~roduce a bead ti~ wa~ u~ed to remove and crush any non-endothelial colonies within the circles (Folkman et al.,1979). This technique was carried out under a ~hase contrast micro~co~e (x10 pha~e objective) in a laminar flow hood. The medium was changed twice to remove floating cells. This ~rocedure was re~eated three to five times to enrich the ~rimary cell~ for endothel; A1 cells before they became confluent. The cells were identified a~ vascular en~o~h~l;~l cell~ by ty~ical cobble~tone mor~hology, ~re~ence of factor VIII-related antigen (.SA~ to et al, 1995), and ~ositive s~A;n;~g (uptake) with DiI-ac-~DL
(Folkman et al, 1979).

~e Eff~ectB of Nyaluronic Acid on l!ube Formation The tube ~ormation as~ay was performed a~
~reviou31y described (Haralabopoulo , et al, 1994.
Briefly, Matrigel (16.1 mg protein/ml) was pre~ared from the Engelbreth-Holm Swarm tumour wa~ u~ed to coat 24 well cluster ~late~ (250 ~l/well) as reCOmmen~e~ by the product sheet. After ~olymeri~ation of the Matrigel at 37~C for 30 minute~ in CO2 incubator, 0.5 ml medium co~t~;~;~g 10 or 20 ~g/ml of hyaluronic acid ~reparations of different molecular weight~ (ProVisc, MW 1.9 x 106; Hyal MW 2.5 x 106 and HealonGV MW 5.0 x 106 re~ectively) in MEM with 10% FCS

WO 97/15330 PCT/AU~/Ca~OE~

wa~ A~e~ to the Matrigel coatea wells. 10% FCS in O 5 ml MEM wa~ u~ed for c~- _~~ison of a relati~e unit of the tube area. The CEC~ (pa~sage 3-7) were lifted from fla~ks by 0.25% try~in-0.02% ~DTA, sus~enaed in 5% MF~, and ~e~ to the coated well~ (50,000 cell~/well in 0.5 ml medium). To evaluate the areas of tube-like ~tructure~ on the gel, ~hotogra~hs were taken with a ~ha~e-contra~t microsco~e after six hour~. Five to ~even fields (xlO
objective) were cho~en randomly in each well for quantitative stuay.

Cllorioca~illary Endothelial Cells Primary culture~ of ca~illary enao~h~~ celln have a characteri~tic a~earance that distingli~hes them from other cell type In addition they were characterize~
by 8~;n;~ for factor VIII-related antigen, and a~aying for the ability to phagocytize DiI-ac-LDL. More than 95%
of the CECs ~howed a positi~e reaction to factor VIII-relatea antigen. Almo~t e~ery cell 3howea u~take of DiI-ac-LD~ into the cyto~lasm, a~ shown in Figure 9. T'hi~
inaicate~ that at lea~t 95% of the cell~ were chorioca~illary enaothelial cells (CEC cell~).

Q~Ant; fication of ~rube Fo~mation and StatiE~tical Ana}ysi~t The tube area~ from du~licate well~ were measured u~ing a Com~uter Imaging Analyzer System (Profe~sional Image Proces~in~ for Window~, Matrox Ins~ector). The ~lide ~hotographs were ~c~n~e~ into a com~uter and the h~c~ou~d adjusted to obtain the best contrast between the tubes and ~atri~el. Tube formation wa~ then quantifiea ~y mea~uring the total tube area of each ~hotogra~h. The results were ex~res~ed as the mean and the st~n~d error of the ~ercentage of tube area in the ~re~ence of 7.5% FCS alone (the final co~c~t~ation) and were analyze~ by Student's t-test for at least two ex~eriment~.

PCT/AU9'/JQ~ I

~ube Formation After 1 hour of being ~eeaea on the toy o~
Matri~el, the CEC~ hecr-- attA~he~. Within 2-3 hour~ the OE C3 ra~idly mi~ ated into a reticular network of aligne~
cell~. After 3 hours the CBCs ~tarted to flatten and ~orm ca~illa~y-like ntrUcture~ on the ~ur~ace of Matrigel-. By 6 hour~ ca~illary-like ~tructures he~: - ap~aren~, ~howing an ana~omo~ing network like ve~el tube~. Tube ~ormation in control and experimental sam~les cont~;n;n~ di~ferent ~re~aration~ of hyaluronic acid at a biolo~ically active concentration waQ a~se~ed after ~ix hour~, ana the re~ults are ~. ~ized in Figure 10 and Table~ 10 to 13.

Table 10 Pro Vi~ Pro Vi~ Healon ~
15 (5 ~ml) (10 ~g~ml) (5 ~ml) (10 ~q/ml) 60.3 139.91 119.37 118.33 49~64 122.96 134.01 111.22 90.03 47.38 39.96 47.48 41O78 36.1 37.12 68.9 59~04 99.4 142.32 126.36 12g.2 106.05 72.63 117.69 Table 11 GV ~ GV Control (5 ~g~ml) (10 ~g~ml) 25 115.22 86.16 155.09 111.57 62.57 118.71 114.42 78.88 85.08 105.06 145.59 43.34 22.64 136.12 94.91 30 104.08 102.94 , CA 02235685 l998-04-23 PCT/AU~6fJ_~
WO97/lS330 Table 12 Ano~a: Single Eactor S~aRY
G o~ Count SIm Average Variance Col~m~ 1 6 42g.99 71.67 1062.86 Column 2 6 551~8 91~97 1724.36 Column 3 ~ 545.41 90.90 2226.80 Column 4 6 589~98 98.33 1035.70 Column 5 6 572.99 95.50 1295.7~
Column 6 5 509.32 101.86 1351.08 Co~umn 7 6 600~07 100.01 1370.50 ~able 13 AN~VA

Source SS ~ MS F P-val~e Fcrit o~
Variation Between3655.25 6609.21 0.42 0.86 2.38 Groups Within48984.10 341440.71 Groups Total52639.35 40 There was no statistically significant aifference in CEC tube ~ormation between the control ana hyaluronic acia-cont~;~;n~ samples, aemonstrating that hyaluronic acid o~ molecular weight between MW 300,000-5,000,00C aoe not induce neo~ascularisation in the absence of another agen.t.

Sl~d S 11 1 ~JTE SHEET (RULE 26~

WO 97/15330 PCT/AU~Gi~ 6q ~xamPle 9 D~ tration Or the Presence of CD44 HA
Receptor in the 1. qn Retina Pre~aration of r~ ~n Retina A 1 ~n ~ye Bank donor eye was dissectea followin~ the removal of the cornea. After discarding the anterior segmLent the ~itreous was carefully ~- -vad, leavin~ behind some ~art~ of the neural retina and the com~lete layer of pigment epithelium att~ch~ to the choroid. The eye ca~ was filled with 2~5~o glutaraldehyde for fixation. Sections of the fixed tissue were subjected to paraffin embe~; ng . Paraffin blocks were cut and sections were trans~errea on to glass histoch~-;cal sliaes, dewaxed i.n xylane and ethanol, and w~h~ in distilled water and Tris-buffered saline ~H 7.2 (TBS).

Alkaline Phos~hata~e St~;";"~ of Sections R~ ~1 of -l~n;~ ~ranules was achie~ed by incubating the eye sections in 50 ~1 0.25% potassium ~erm~n~n~te for 45 minutes followed by 50 ~1 1.0% oxalic acid for 5 minute8, Bl~Ch; ng was carried out following incubation with serum 50 ~l/section of 10% normal horse ~erum/TBS (CommLonwealth Serum Laboratories, Perth, Australia) for 30 miDLutes. Sections were then w ~e~ twice in TBS, 5 minutes ~er wash and incubated in 50 ~1 mouse anti-CD44 monoclonal antibody (Boehrin~er ~nnhe;
Biochemica, M~nnh~;, ¢~ -~y) or 50 ~1 of mouse anti-81-11 monoclonal antibody (non-immune co~trol) for 60 minutes.
After incubation sections were a~ain washed twice in TBS,

5 minutes ~er wash, incubated in 50 ~1 of 1/250 horse anti-mou~e IgG (H+~) conjugated to alkaline phos~hatase conju~ated to alkaline ~hosphatase (secondary antibody) for polyclonal antibodies for 60 minutes and washed twice in TBS, 5 mi1~utes per wash. Sections were incubated in 50 ~1 FAST RED (Sigma Aldrich, St louis, ~SA) for 20 minutes, washed twice in TBS, 5 minutes ~er wash and counterst~;ne~
in Meyer~ Haemotoxylin for 10 minutes, followed by 5 minutes in ta~ water. Sections were allowed to dry, then CA 02235685 l998-04-23 PCT/AU9 -'011 ~6q mounted using glycerol ;elly.
Bl~ch;~ of --1~;~ wa~ carried out succes~fully without cau ing dama~e to the tissue sections, as ~hown in Figure 10. St~in;ng of glutar~l~hyde-fixed 1 ~n eye ~ections with the mouse control monoclonal antibody and FAST RED resulted in clear 8t~; n; ng of the RPE layer in unbl~c~oA tissue (Figure 10A) and following bleAch; n~
after incubation with 10% ~o ~1 horse serum (Fi~ure 10B).
A strong ~ink si~nal demonstrating the ~ecific ~re~ence of CD44 HA receptor~ in the retinal ~i~ment epithelium but not in the choroid wa~ ob~er~ed in tissue st~ineA with anti-CD44 monoclonal antibody (Figure 10C). As in choroid there was no si~nal detected in the neural retina.
The~e result~ A -_ ~trate that the retinal ~igment e~ithelial cells ~referentially expres~ HA
receptor~, thun facilitating an enh~n~o~ u~take of HA
com~lexes.

Example 10 ~p and Down Regulation o~ CatheP~in D
~xPression in NIH 3T3 Cells A 1620 b~ U;nATTI fragment of human cathe~sin D
was subcloned into ~UR~ -neo vector in both ~en~e and anti-~en~e directio~. Positi~e clones were selectea, and the orientation of the fr~ t~ was confirmed by ~coRI
restriction enzyme analysis. For the transfections of NIH 3T3 cells the clones carrying cathepsin D in the an~i-sense and sen~e direction~ were on caesium chloriae aen~3ity çrraaient~, NIH 3T3 cells were seeaea on to 6-well tissue~
culture plates at a con~nt~ation of 2 X 105 in 2 ml DNE~
su~lemented with 10% fetal bovine serum (FBS). The cell~
were incubated o~ernight at 37~C until they became 70%
- confluent. Having ro~cheA confluency, the cell~ were washed twice with serum and antibiotic-free medium.
Li~ofection reagent (10 ~1) (GIBCO-BRL) ailuted in 100 ~1 of OPTI-MEM (GIBCO-BR~) were ~ently iY~ and incubated at room temperature for 15 minute~. Following incubation, an CA 02235685 lsss-04-23 additional 800 ~l of OPTI-MEM wa~ A~e~ to the mixture.
This ailuted mixture wa~ gently overlaid onto the wa~hea NIH 3T3 cells. The cells were ;~c~h~tea for 16-20 hr~
before the transfection -~; A was ~ aa ana re~laced with 5 DMEM 5U~l:. - t~ with 10% FBS. After a further 48 hrs incubation the cells were try~sini~ed and subculturea at 1:5 in -';~ c~tA;~;~g 10% FBS and Geneticin 418 (GIBCO-BRL) at 1 ng/ml co~ent~ation. Successfully tra~sfected cell~ ~elected with Geneticin 418 were -;~tA;~ in meaia ~u~lemented with FBS and Geneticin 418 as describea above.
Confluent transformea cultures were frozen for storage and ~ubcultured for further analy~is. The ~re~ence of cathe~in D in the tran~formed NIH 3T3 cell~ was aetected with ~olyclonal antibody against cathe~sin D, u3ing a conventional cytsch ;cal tech~;que ana an alkaline ~hos~hata~e-labelled ~econd antibody.
The presence of cathe~in D frA~ -nt of the vector wa~ Qtratea with XindIII ~igestion. Positive clone~ ~howea the ~resence of a 1620 kb fragment. The orientation was establishea by ECO RI restriction enzyme dige~tion, which ~ave two fra~ - ~ at 5.7 and 5.9 kb in the ca~e of the anti-~ense orientation an~ 4.3 an~ 7.3 kb in the case of the sense orientation. All NIH 3T3 cells ~urvivin~ Geneticin 418 selection carried cathe~in D
clones, which are antibiotic resistant. The trans~ormea control NIH 3T3 cells aia not survive the selection ~rocedure. The ; ocytoch~ ;~try re~ults sug~est that NIH 3T3 cells carryin~ cathepsin D in the sense direction u~-regulated cathe~sin D ~roduction, while those carrying cathe~sin D in the anti-sen~e direction down re~ulated cathepsin D ~roduction.

Exam~le 11 Production of a VEGF165-Expressing RPE Cell Line Cell Cultu~e The 1- ~ RPE cell line 407A (Davi~ et al, 1995), was ~;~t~;~e~ at 37~C in a humidifiea envi c --t WO 971$5330 PCT~AU5~6 contA;n;n~ 5% CO2. The culture medium consigted of M;n; -1 E~ential Medium (MEM, Trace Bio~cience~, Sydney, NSW, Au~tralia) ~up~l.s t~ with 10% FCS (Trace Biosciences, Sydney, NSW, AuQtralia) and 100 I~/ml Penicillin/100 ~g/ml Stre~tomycin (P/S) (ICN ph~ ~ce~ticals Inc, Co~ta Mesa, CA, ~SA). Cell~ were pas~agea 1 in 5 with 0.25% tryp~in (Trace Biosciences, Sydney, NSW, Australia)/0.05% EDTA (BDH
Chemical~ Australia Pty Lta, Ril3yth, VIC, Au~tralia) approximately e~ery 5 day~.

lO Cloning of V:Z~GFl6S into the ~ ,L~sion Vector Mouse VEGFl65 in Bluescri~t RS was obt~; n~ from Dr Geor~ Breier, Max Planck Institut, G~ - ~ (Breier e~t al, 1992). VEGFl65 wa in~ertea into the Bam HI ~ite of pH~APr-l-neo (Figure 1) (Gl~nn;n~ et al, 1987). Thi~
cloning wa~ ~erformed ~ia pGem 7Z~(I) ( ,- -~, M~ o~, WI, ~SA) ~or the aaaition of a Bam HI ~ite to the 3' end o~
VEGFl65. A sense VEGF-pH ~Pr-l-neo clone was identified by Eco RI aigestion (Promega, Madison, WI, ~SA).
VEGF-pH~APr-l-neo DNA was pre~ared using the Qiagen Pla~mid Midi Rit (Qiagen GmbH, Hilden, Germany). The extraction was carriea out as de~cr~bed in the manufacturer's ~rotocol, an~ the resulting pellet was resuspended in 500 ~1 TE buffer (10 mM Tris HCL, ~H 8.0, 1 mM EDTA).

~ransfection of RP~ Cell ~ine VEGF-~H~APr-l-neo DNA was transfected into 407A
cells usin~ Lipofectin (Gibco BRL, Gaithersburg, MD, USA) a~ described in the manufacturer~s instructions. Briefly, 2 mg of VEGF-pH~APr-l-neo DNA in 100 ~1 OPTI-MEN (Gibco BRL, Gaithersbur~, MD, ~SA) was ;~e~ with 5 ~1 Lipofectin reagent in 100 ~1 OPTI-MEN. The mixture wa~ allowed to - stand at room temperature for 15 minutes, then made up to 1 ml with OPTI-MEM, and overlaid on to 60% confluent 407A
cells. The cells were incubated at 37~C overni~ht in a humidified envi ~ and 5% CO2, then 4 ml MEM with 10% FCS and P/S wa~ adde~. The cells were re-incubated for WO 97/15330 PCT/AU9GI!UQ 6~1 24 hours before 1 mg/ml Geneticin (Gibco BRL, Gaither~burg, MD, ~SA) was included in the cell culture medium. After one week a ~erie~ of discrete colonies wa~ ~elected, and grown in 1 mg/ml Geneticin until established. The co~c~nt~ation of Geneticin wa~ then decrea~ed to 300 ~g/ml cell culture medium.
A control cell line con~isting of 407A cells transfectea with pH~APr-l-neo only (407A-~H~APr-l-neo) wa~
al~o ~roduced using ~i~ofectin. Both cell lines were _~;nt~;n~ in MEM contA;n;n~ 10~~ FCS, P/S and 300 ~g/ml Geneticin.

Selectio~ of Primers ~or DNA and R~ PCR
Primer~ were selected to allow specific amplification of tran~fected mou~e VEGFl65, without bach~u~d am~lification of 1 n VEGFl65 from the 1 ~n 407A cell line. The ~e~uences of mou~e VEGFl65 and human V~GF,65 as listed on the ~nR~nk databa~e were c~ _~ed u~ing the IBI Pu~tell Analy~is Software (IBI ~td, Cambridge, ~ngland). l9mer regions which were les~ than 70% homolo~ous with human VEGFl65 were ~elected from mou~e VEGFl65. Primer ~equences were: 'v~,~Ol", 115-134 b~ on mouse VEGF65, 5'-AGG AGA GCA GAA GTC CCA T; 'v~-~rl1~2", 300-318 b~ on mou~e VEGFl65 5'-CGT QG AGA GCA ACA TCA C.
Analysi~ o~ ~rimer ~e~e~e~ by the Ba~ic Local Alignm~nt Search Tool (BLAST, National Centre for Biotechnology Information, Bethe~da, ~D, ~SA) ~ - ~trated homology to mouse VEGF ~orm~ only.

DNA PCR
Cell~ were harve3ted u~ing 0.25% tryp in/0.05%
~DTA. Sa~ple~ of 2 x 106 cells were collected and washed with PBS, then incubated for 3 hour~, 37~C, in the pre~ence o~ 100 ng~ml ~roteina~e X (Boehringer M~nnheim, MAnnheim, Germany) and 0.5% w/v Sodium Dodecyl Sulphate (SDS) ~BDH
Chemical~ Au~tralia Pty Ltd, Kil~ythr VIC, Au~tralia). DNA
wa~ isolate~ by ~henol/chloroform extraction and ~odium CA 02235685 l998-04-23 WO 97/1~;330 PCT/AU9~/~5~1 acetate/ethanol preci~itation. DNA ~ellets were resus~enaed in 100 ~1 T~ buffer.
All PCR reagent~, includin~ ~ltra Pure Water, were ob~A;~e~ from Biotech International ~td. (Bentley, WA, 5 Au~tralia). The PCR reaction mixture consiste~ of 5 ~1 5X PolymeriQation Buffer, 25 mM MgCl2, 1~ Tth PluS DNA
Polymera~e, 50 ng VEGFMOl, 50 ng VEGFMO2 and ~ltra Pure Water to 25 ~ 1 of each DNA ~ample wan u~ed for PCR.
For each ~erie~ o4 PCR reaction~ carried out, a positive control ~o~tA;~in~ 20 ng VEGF-~H~APr-l-neo DNA, and a negative control co~A;n;~g ~ltra Pure Water in the pla,-e of DN~, were included. PCR reactions were carried out UQing a Perkin Elmer GeneAmp PCR System 2400 ~h~ ~cycler (Perkin-Elmer Corporation, Norwalk, CT, ~SA). Cycle~ u~ed were 1 cycle of 92~C for 5 minutes, 55~C for 1 minute~ 74~C
for 1 minute; 35 cycle~ of 92~C for 1 minute, 55~C for 1 minute, 74~C for 1 minute; 1 cycle of 92~C for 1 minute, 55~C for 1 minute, 74~C for 10 minute~. The PCR products were electro~horesed on a 2% agarose gel, and ~i~ualisea by ethidium b ~ tA;~;~g.

~everse Transcri~tion PCR (RT PCR) RNA waQ extracted using RNAzolB (Biotecx ~aboratorie~ Inc., Hou~ton, Texas, ~SA). The ~rocedure wa~
carried out a3 describe~ in the manufacturer'Q ~rotocol, with RNA bein~ extractea airectly from confluent 25 cm3 fla~ks of cell~ (4 x 106 cell~ ~er fla~k). The re~ulting ~elletQ were resu~enae~ in 50 ~1 Diethyl PyrocA~ho~te (DEPC) (BDH ~t~, Poole, Dorset, ~ngland) treate~ water.
RT PCR was performed U5 ing the GeneAmp Th~ -~table rTth Reverse TranscriptaQe RNA PCR git (Perkin-Elmer Cor~oration, Norwalk, CT, ~SA). Reverse transcription and PCR reaction~ were carried out a~
ae~cribea in the manufacturer's instructions. 200 ng R~A
~ wa~ usea for each reaction. Water u~ed for all reaction~
was ~ltra Pure Water. The RT PCR ~ositive control cont~;~ 20 ng of VEGF-~H~APr-l-neo DNA. The negati~e CA 02235685 l998-04-23 WO97/15330 PCT/AU~ C1 control recei~ed Ultra Pure Water in the ~lace of RNA.
Control~ $or DNA cQn~ ;nAtion were pro~c~ by the a~a;tion of r~th DNA Polymerase after completion of the Rever~e ~ranscription ste~. RT PCR ~roducts were ~recipitatea using sodium acetate/ethanol. Samples were washea in 70~ ethanol and resus~ended in T~ buffer to l/5 the PCR reaction volume. PCR products were electrophore~ed on a 2% agarose qel and visualisea by ethidium bromide st A ; n ; n~ , Production of a V:EGF~65-lZxpres~ing Retinal Pigment Epitheli~ll Cell l~ine V~GFl6s was successfully clonea into the Bam ~I
site of pH~APr-l-neo. The identity of the clone was confirmed using a Bam HI digest which yielded two fr~
of l0.0 kb, corresponA; ng to pH~APr-l-neo, ana 656 b~, COrre8pO~; ng to mouse VEGFl65. ~co RI digestion of the VEGF-~A~-l-neo clone proA~c~ two fragments of 5.7 kb ana 5.0 kb, confirmin~ that VEGF was in the sense orientation.
VEGF-~ neo was transfectea into the 407A
cell line using Li~ofectin. The ~resence of mou~e VEGF 65 DNA in the transfected 407A cell line was confirmed using DNA PCR. DNA was extracted from VEGF-~H~Apr-l-neo transfected 407A colonie~, along with DNA from the control 407A-~H~pr-l-neo cell line. PCR of the VEGF-r~r~-l-neo transfected 407A DNA resultea in the production of a 200 bp DNA frA~ - t in every colony te~te~. This fragment was the ~ize preaicted from the ~osition of the primers on the mouse V~GFl65 gene, and agreed with the frA~ -nt size pro~ce~ from the VEGF-positive control. One establishea colony of transfectea cells was chosen for the ~- q;n~e~ of the experiments (407A-VEGF). No signal wa~ detected on PCR
of 407A-pH~Apr-l-neo. The results are illu~tratea in Fi~ure llA. DNA from untransfectea 407A cells also produced PCR si~nal, conf; ;n~ that the primer~ being usea were ~pecif ic to mouse VEGFl65.

PCTtAU9C ', l756 wo 97/15330 RT PCR was used to verify the ~roduction of mouse VEGFl65 _RNA by 407A-VEGF. On RT PCR of 407A-VEGF total RNA, a fra~ment of 200 b~ was ~roduce~, corres~on~;ng t:o the fr~ t size ~redicted from the ~osition of the mouse VEGFlC5 ~rimer~. No ~i~nal was received from 407A-pH~A~r-l-neo total RNA. Both RNA sam~les were shown to be free of contr linating DNA by omission of the cDNA synthesis ste~
auring RT PCR. The result~ are ~hown in Figure llB. RT
PCR using untransfected 407A RNA did not produce any signal.

~ube Formation A~~J3ay The a~say was performed aa described in Exam~le 8. CEC adhered to the Matrigel su~ort within 1 hour of se~;ng. After 2 to 3 hours of culture, the C~C
had mi~rated ra~ialy to form a reticular network of ali~nea cells, and subsequently began to form ca~illary-like ~tructures on the urface of Matrigel. By 24 hours the CEC
had the a~earance of an anastomo~ing network, which i~
ty~ical of vascular tubules. The quantitative analysi~ of tube formation, obt~;n~ from com~uter images, is summarised in Figure 13.
The most extensi~e ca~illary network was ~een in C~C cultured in the ~resence of 100 ng/ml human reco_binant VEGF (Figure 13B). The amount of capillary tube formation ;~c~ by the 407A-VFGF conditioned medium was 8;milAr to that from the human reco_binant V~GF. In contrast the le~el of tube formation from conaitioned medium of the control 407A-~H~APr-l-neo-cell line was significantly less, and was comparable to the control culture~ co~t~;n;ng Ham's F12 me~ium with 5% FCS ana P/S only.
There was a 100% increase in the amount of tube formation ;n~l~ce~ by 407A-VEGF conditioned ~ when com~ared to 407A-pH~APr-l-neo. Thi~ ~ifference wa~ found to be si~nificant (P = 0.009, Student's t-test). The aif~erence between the control culture ana the culture co~t~;n;ng 100n~/ml ~ ~n recombinant VEGF wa~ also found CA 0223~68~ l998-04-23 WO97/15330 PCT/AU~ G~61 to be ~i~nificant (P = 0.002, Student'q t-test).

Exam~le 12 Cloninq ana Characterisation of -~ RPE
Vascular Enaothelîal Growth Factor (RPE-V~GF) ~ -~ RPE cell~, available in our laboratory, are ~rown in ti3sue culture. To u~regulate V~GF expresqion, cell cultures are treated in hy~oxic con~itions. The upre~ulation of V~GF expression i~ monitored with ; o~;~toch~i~try. The mRNA iq extracted from 107 RPE
cells, ana a cDNA library carrying all gene~ ex~resse~ in the RPE~choroid is establishea using method~ known in the art.
VEGF is a hi~hly conserved molecule which i5 highly homologous between aifferent s~ecie~. A murine VEGF
cDNA clone, available in our laboratory, is use~ to screen the l ~ RP~ cDNA library in order to identify the full len~th humAn RPE-VEGF clone. Poqitive clones are analy~ed by re~triction enzyme analysis and finally by DNA
sequencing. Full length RPE-VEGF clones are analysed to elucidate their genomic structure (initiation ~e~nce~, start and sto~ co~onq, ~utative exonq etc. ) .
Clones carrying the full length RPE-V~GF are analysea for the expression of V~GF ~rotein with in ~itro tran-qlation. The identified clones are usea to aerive the anti-sen~e molecule ~or insertion into the vehicle sy~tem, ana for the selection of the anti-sense oligonucleotiaes.

Exam~le 13 ph~ ~ceutical Agent for the Short-Term Inhibition of VEGF ~x~ression ~nti-sense DNA technology enables the sequence-specific inhibition of target molecule~ without affectin~the non-tar~eted functions of the cell. As describea above, we have ~ trated both in vitro and i~ ~ivo that anti-sense DNA can be usea effectively to inhibit the anti-sense oligonucleotide into the vitreous.

PCT/AU~ C ~C~
WO 97/1~5330 A ~anel of 16 to 19-mer oli~onucleotides, 100%
complementary to ~arts of the VEGF gene, is ~elected from the 5~ and 3~ ends of the DNA ~equence. Sense and ~crambled ~equences are al~o u~ed a~ control.
Phos~horothioate-~rotected oligonucleotides are ~ynthesized on a DNA synthesizer and subjected to ~urification.

Exam~le 14 Anti-Sense Aqent for the ~ons-Term Inhibition of VEGF Production Preparation of VEGF-~Ad.RSV for Homologous ~ec. ~ Ation VEGF16s in Bluescript IIKS (Stratagene) was used to ~ro~uce R~nI ~ites. R~n I restriction enzyme sitea were obtA;n~ at both the 5' and 3' ends of VEGF165 by subcloning. VEGFl65 was ~ -~ved from Blue~cri~t II RS
u~ing an Xba I (5' cut)/Rpn I (3' cut) restriction enzyme dige~t, and cloned into ~Gem 7Zf(l) (Promega). A R~n site was then added to the 3' end by cloning VEGFl65 into pGem 3Zf(+) (~ ~ a), using a Hind III (3' cut)//Xba I (5' ~cut) digest.
VEGF was ~ ved from ~Gem 3Zf(l) with a R~n :C
restriction enzyme dige~t and cloned into the unique Kpn I
~ite on pAd.RSV. This plasmid cont~ two ~e~ of the adenovirus genome ~eparated by cloning ~ites for the insertion of foreign DNA. The resulting clones were screened for the pre~ence of sense and antisense clone~, which were used in ho~ lo~ou~ recombination (VEGF-pAd.RSV).
VEGFl65 was ~hown to be present and intact within ~Ad.RSV
by re~triction enzyme cleava~e and sequencing.
VEGF-~Ad.RSV DNA was ~re~ared using the Qia~en Plasmid Midi Kit, as ~er the manufacturer's in~truction~3.
The DNA was linearised by Xmn I restriction enzyme digestion, ~urif iea by ~odium acetate/ethanol precipitation - and resuspended in TE buffer. The DNA was then storea at -20~C until re~uired.

PC~/AU~IW6~1 Gene~ tlon of Ad . RSV-V~GF or ~d . RSV-aV}~GF by ~omolosJou~3 Rec~ h; rl5~ tion The adenovirus ty~e 5 deletion mutant, dl324, wa~
usea to generate the recombinant aaenoviru~ carrying VEGF.
dl324 is unable to replicate due to deletion of the El re~ion and, in addition, carries a partial deletion in the E3 region. In order to generate ~iral particles this mutant was pro~agated in 293 cells, which supply the missing El region in trans. The linearised ~lasmid DNA
pA~RSV-VEGF or ~Ad.RSV-aVEGF was co-tran~fected into 293 cell~ with dl324 viral DNA which has had its extreme left-hand sequences 1~ -ved by a Clal digestion. This re~ es the infecti~ity of al324 ana allows for ea~ier identification of recombinants. After tran~fection using the calcium ~hos~hate ~recipitation method, scre~ni n~ of the resultant plaque~ yielded recombinant AdRSV-VEGF ~irus carrying VEGF in sense or antisense orientation.

Exam~le 15 Construction of a Vehicle for the P~ -n~t Expression of ~arget ~olecules ~ The vehicle aescribed in ~xam~le 14 is suitable for long~term treatment in that it ~roviaes t~ ~ary (maximum one year) ex~ression of the anti-sen~e VEGF DNA
molecule, and consequent protection ~ t neovascularisation. To achie~e in~efinite treatment, we use a vector system which enables the integration of VEGF
in the aDti-sense direction into the 1 -n ~ ~r- present in RP~ cells using an adeno-a~sociated virus (AAV) vector, which mean~ that the ~rotection against neovasculari~ation can be yrovided for the rest of the life of the ~atient, as long a~ the RP~ cells 1~ -i n functional.
Adeno-as~ociated viruse~ are non-pathogenic, are able to infect non-dividing cells, and ha~e a high frequency of integration. We use AAV-2, which is a re~lication defecti~e parvovirus which can readily infect other cell~ such as RP~ cells, and integrate into the genome of the host cell~. Recent characterisation ha~

PCT/AU9C,'~IO~

revealed that AAV-2 ~pecifically targets the long arm of human ch~ -30me 19.
AAV constructs use varyincJ ~romoter secauence~ in r combination with a re~orter gene. The expre~ion of the 5 reporter gene mRNA i~ detected with PCR amplification or in ~itu PCR, and the intecJratiOn of the reporter gene is identified by ch~ -30mal analy~i of RPE cells.
~8ing the a~ro~riate re~triction ~ites, the reporter gene is replaced by anti-~ense VEGF DNA. The new 10 con~truct i8 co-transfected with the com~lementin~ pl~
(pAAV/aa) into kic~ney 293 cells previou~ly infected with adenovirus type 5 to make the rAAVaVEGF construct. The construct produced i~ usea to infect RPE cell~, and the ex~re~sion of anti-sense VEGF i8 detectecl by PCR
15 ampli~ication.

Exam~le 16 Model SYstems for Testing Inhibition In Vi tro Human VEGF is cloned into COS cells to procluce a culture system (V~GF-COS) in which the e~fective inhibi~ion 20 of VEGF expres~ion can be te~ted. The inhibition of VEGF
ex~ression i~ te~tea by Northern and Western blot analy~3es and quantified by immunoas~ay.
The toxicity of increasing co~r~ntration~ of oligonucleotide~ on COS cell~ is asses~ecd with the try~an 25 blue as~ay. The proliferation of COS cell~ is monitorecl with or without increasin~ co~ce~t~ations of olicJonucleotides. The inhibition of the expression of VEGF
in controls and in cultures ~; ~t~; ~eA in the presence of anti-sense oligonucleotiaes is monitored by ~orthern ancl 30 Western blot analyses, ; ocytoch- ;~try and by a quantitative immunoassay.
- RP~ cells are cultured in hy~oxic condition~ ancl the up-regulation of VEGF expression is monitored in the presence o~ increasin~ concentrations of oligonucleotide~
35 for an extended perioa of time. Toxicity, proliferation assay and the monitoring of VEGF expression are performe.d PCT/AU~)61~6~q a~ describea above.
~ EC cells are culture~ in normal an~ hypoxic conditions with or without increasing concentration of oligonucleotide~. In addition to the toxicity, ~rolife~ation a~say and VEGF detection, the effect of anti-sense oligonucleotide- -~;Ated inhibition of VEGF
expression on tube ~ormation i~ analysed. RP2/CEC dual culture~ ~roduced in no ~l and hy~oxic con~ition~ will be ~ub~ected to similar tests. The same model systems are used to asses~ the lon~-term and ~ermanent agent of the invention.

~xam~le 17 In Vi~o Model for Sub-Retinal Neovascular Membrane (SRNVM) In ad~ition to the above examples an animal model for ~tuay of the particular inhibition of the develoc - ~
of SRNV wa~ develo~ed. The mo~el uses la~er treatment of rats to induce sym~toms similar to those ob~erved in as SRNV.
Pigmente~ rats ~Dark Agouti, D~) weighing between 175 and 250 g were anaesthetized with an intramuscular injection of xylazine hydrochloride (2 mg/kg of body wei~ht), ace~romazine maleate (0.5 mg/kg), and ketamine hydrochloride (100 mg/kg of body weight) and given to~ical 0.5% ~ro~arAcA;ne hydrochloride. The ~upil~ were dilated with 2.5% pheny3e~hrine hy~rochloride.
Krypton laser radiation (647 nm) wa~ delivered throu~h a Zeiss sli~ lamp (Coherent Model 920 Photocoas~ulator, Palo Alto, Calif) with a hzlnAhelA
coverslip (22 c 30 mm) serving as a contact lens. Laser ~arameter~ u~ed were a~ follows: a spot size of 100 ~m, a power of lS0 mW, an~ an exposure duration of 0.1 s. An attem~t was made to break Bruch~s membrane, as clinically e~idenced by central bubble formation with or without intraretinal or choroidal hemorrhage. We found that a treatment ~ower of 150 mW most con~istently produced thi~
effect.

PCTfAU9~'!JO~
Wo97/1s330 Approximately 40% of An; ~ 1 ~ treated the above cdescribed way develo~ed growth o~ blood ves~els into t~he retina from the choroid. Thi~ growth i~ accompanied by the upregulation of VEGF expre~ion, proviciing an excellen~
~y~tem to te~t our oligonucleotides and con~tructs.

Exam~le 18 Inhibition of RPE-V~GF Ex~re~ion with Anti-Sen~e Oli~onucleotide~, Ad.RSV.aVFGF
and rAAVaVEGF In Vi~o in Rats Neova culari~ation can be ; n~c~ using pocket implantn in the choroid or the ~ubretinal layer. One of the aisaavantage~ of the~e mociels is that the ~roces~ of neova~culari~ation might not follow the ~ame biochemical ~tep~ which naturally occur in humans sufferincJ from AgMD.
To overcome the~e ciifficultie~ we use an An; ~l model in which choroidal neova~culari~ation is ;~ce~ by VEGF
overex~re~ion in the RPE cell~. ~ing reco_binant acdenoviru~e~ carrying VEGF, for example Acl.RSV.VEGF, fc~r the in vivo trial~ all ~n; ~ ~ ae~cribed above are utili~ea to ~rovide u~ with a wide rancJe of information.
Te~t~ are conducted to ~ - ~trate the expre~3ion of a VEGF
expre~ion over a period of one year. ~sing Northern anci We~tern blot analysi~, VEGF down-regulation i~ monitored ana i~ oh; ~toche_i~try i~ u~ea to ~ - ~trate the down-re~ulation of V~GF exRre~sion in a cell-~pecific ~-nn~.
U~ing the above clescribed animal moaels, choroidal neova~cularisation is monitored by hi~tology and angio~raphy. The~e mociel~ are applicable to all the embo~; ?~t ~ of the invention.

It will be-ap~reciated that the ~resent inven~ion is particularly u~eful in the ~tudy, treatment or ~ ~revention of a~e-related macular degeneration, by virtue of the ~ucces~ful adenoviral ~ene tran~fer to the RPE.
Without wishing to be bound by any proposed mech~n;~m ~or the ob~erved advantage~, the hi~her de~ree of gene expression in the HRPE7 cell~, com~arecl with the F2000 CA 0223~68~ 1998-04-23 WO97/15330 PCT/AU~GI'~066q cell~, may indicate the ability of ~PE cells to ~hagocytose large molecules and hence increase the u~take of aaenovirus. The level of ex~ression of the transgene may also be increasea by increasing the time of exposure or the viral titre.
The com~arison studies between the HRPE7 cell~
and the F2000 fibroblast show that there are marked differences in the pattern of ex~ression between the different cell ty~es under the same conditions. These aifferences could be ex~loited for targeting of aifferent cells, for exam~le RPE. The u~stroke in the time/expression curve for RPE cells (Figure 5) was at 4 hours, while for F2000 cells it was 24 hours. There is, therefore, a window during which RPE cells are t~k;ng up Ad.RSV.~gal. and ex~ressing the transgene at a significantly higher level than F2000 fibroblasts.
Transfection for perioas of less than 24 hours woul~ allow use of this window as a targeting tool (eg. virus ~olutions coula be as~irated from subretinal blebs or the ~itreous ao after 24 hours). The titre/ex~re~sion curves (Figure 5) also show that there was a difference between the cells, with RPE cells beg; ~n; ng to express highly at a lower concentrations. Once again, low conc~nt~ation coula be used to ~referentially tar~et RPE cells. A c- ~;n~tion of lower titres for less than 24 hours woula combine the two effects ana ~rovide targeted delivery.
As shown in some of the 1- ho~; ments, the present invention may also be used in conjunction with adjuvants to kee~ viral toxicity to a minimum by reaucing the titre required to effect gene transfer and expres~ion.
We have shown a consistent and ~ignificant aajuvant effect for adenoviral gene transfer using HA.
This was the case in both phagocytic and non-phagocytic cell lin~. The advanta~e of HA is its presence as a normal com~onent of 1 ~n vitreous and extracellular matrix, ana its long history of thera~eutic acce~tance as a viscoela~tic aid to surgerY~

WO97115330 PCT/AU9G/C A ~1 The important feature of HA in term~ of it~
acting a~ a ~ot~t;~ adjuvant i~ its ability to bind cell membranes and other molecules simultaneously. We propo~e that the HA molecule can bind aaenoviru~ and the cell membrane at the ~ame time, ana therefore increa~e the contact time or concent~ation of virus in the vicinity of the cell membrane u~ing thi3 -.-hAn;l . We have ;~ent;Eied cell ~urface receptor~ ~pecific to HA identified on bot]h F2000 and RPE7, as each cell tested ~o~itive for the ~re~ence of CD44, ~MM and ICAM-l receptor~.
Intere~tingly, the ~MM receptors on RPE showed a nuclear ~istribution, and thi could account for the 31ightly higher a~juvant effect o~ HA in RPE than in F2000. Our preli ;n~y ~tudie~ of in vivo immunofluorescent s~A;n;~g for CD44 show no si~nal in the neuroretina, su~ge~ting 1_hat HA a~sociation of the aaenovirus may al~o be a potentia:L
targeting mechani~m for RP~ i~ vivo.

It will be apparent to the per~on ~killea in the art that while the invention has been described in the Example~, various modifications ana alteration~ to the e~o~; - t~ de~cribea herein may be made without depart~ng from the 3co~e of the inventive concept di~clo~e~ in thi.s specification.

References cited herein are listed on the followi.ng pages, and are incorporate~ herein by thi~ reference.

CA 02235685 l998-04-23 ~ ~ ~

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Claims (61)

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. A. composition comprising (a) a nucleic acid sequence encoding a specific protein and (b) a hyaluronic acid or a derivative thereof, together with a pharmaceutically-acceptable carrier, wherein the nucleic acid is either an anti-sense nucleic acid directed against a target sequence or a sense nucleic acid encoding a specific desired protein.

2. A composition according to Claim 1, in which the nucleic acid is a nucleotide sequence which is in the anti-sense orientation to a target sequence.

3. A composition according to Claim 2, in which the target nucleic acid sequence is a genomic DNA, a cDNA, a messenger RNA or an oligonucleotide.

4. A composition according to Claim 1, in which the nucleic acid is present in a vector comprising a nucleic acid sequence to be transferred into a target cell.

5. A composition according to Claim 4, in which the nucleic acid sequence to be transferred is a genomic DNA, a cDNA, a messenger RNA or an oligonucleotide.

6. A composition according to Claim 5, wherein the vector comprises a sense sequence to be provided to a target cell in order to exert a function.

7. A composition according to Claim 6, in which the vector comprises an anti-sense sequence to be provided to a target cell in order to inhibit the functioning of a nucleic acid present in the target cell.

8. A composition according to any one of Claims 4 to 7, in which the vector is a liposome.

9. A composition according to any one of Claims 4 to 8, in which the vector is a virus.

10. A composition according to Claim 9, in which the virus is an adenovirus, an adeno-associated virus, a herpes virus or a retrovirus.

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11. A composition according to Claim 9, in which the virus is a replication-defective adenovirus.

12. A composition according to Claim 11, where the virus is a replication-defective adenovirus comprising a promoter selected from the group consisting of respiratory syncytial virus promoter, cytomegalovirus promoter, adenovirus major late protein (MLP), VA1 pol III and .beta.-actin promoters.

13. A composition according to Claim 11, wherein the vector is pAd.RSV, pAd.MLP or pAd.VA1.

14. A composition according o Claim 11, wherein the vector is Ad.RSV.aVEGF or Ad.VA1.aVEGF.

15. A composition according to any one of Claims 10 to 14, wherein the vector also comprises a polyandenylation signal sequence.

16. A composition according to Claim 15, wherein the polyadenylation signal sequence is the SV40 signal sequence.

17. A method of treatment of a pathological condition in a subject in need of such treatment, comprising the step of administering an effective dose of a composition according to any one of Claims 1 to 16 to said subject.

18. A method according to Claim 17, in which the composition is administered systemically by injection.

19. A method according to Claim 17, in which the composition is administered topically.

20. A method according to Claim 17, in which the composition is administered directly into the tissue to be treated.

21. A method of preparing a composition according to any one of Claim 1 to 16, comprising the step of binding a nucleic acid or vector to a hyaluronic acid or a derivative thereof, and isolating the thus-formed complex.

22. A composition for treatment of a retinal disease mediated by abnormal vascularization comprising a) an anti-sense nucleic acid sequence directed against vascular endothelial growth factor (VEGF), and b) hyaluronic acid, together with a pharmaceutically-acceptable carrier.

23. A composition according to Claim 22, in which the anti-sense nucleic acid sequence is present in a vector comprising a nucleic acid sequence to be transferred into a target cell

24. A composition according to Claim 23, in which the vector is a virus.

A composition according to Claim 24, in which the virus is an adenovirus, an adeno-associated virus, a herpes virus or a retrovirus.

26 A composition according to Claim 24 or Claim 25, in which the viral vector is a replication-defective recombinant virus.

27. A composition according to Claim 26, where the virus is a replication-defective adenovirus comprising a promoter selected from the group consisting of respiratory syncytial virus promoter, cytomegalovirus promoter, adenovirus major late protein (MLP), VA1 pol III and .beta.-actin promoters.

28 A composition according to Claim 27, wherein the vector is pAd.RSV, pAd.MLP or pAd.VA1.

29. A composition according to Claim 27, wherein the vector is Ad.RSV.XVEGF or Ad.VA1.XVEGF.

30. A composition according to any one of Claims 1 to 29, wherein the vector also comprises a polyadenylation signal sequence.

31. A composition according to Claim 30, wherein the polyadenylation signal sequence is the SV40 signal sequence.

32. A composition for treatment of a retinal disease mediated by abnormal vascularization, comprising an anti-sense nucleic acid sequence corresponding to at least a part of the sequence encoding VEGF, and optionally further comprising one or more adjuvants for increasing cellular uptake, together with a pharmaceutically-acceptable carrier.

33. A composition according to Claim 32, comprising as adjuvant hyaluronic acid or a derivative thereof.

34. A composition according to Claim 32 or Claim 33, wherein the anti-sense sequence has 100% complementarity to a corresponding region of the gene encoding VEGF.

35. A composition for short-term treatment according to Claim 32 or Claim 33, wherein the anti-sense sequence is 16 to 50 nucleotides long.

36. A composition for short-term treatment according to Claim 32 or Claim 33, wherein the anti-sense sequence is 16 to 22 nucleotides long.

37. A composition for short-term treatment according to Claim 32 or Claim 33, wherein the anti-sense sequence is 16 to 19 nucleotides long.

38. A composition according to Claim 32 or Claim 33, wherein a modified oligonucleotide as herein defined is used, and the anti-sense sequence is 7 to 50 nucleotides long.

39. A composition for long-term treatment of a retinal disease mediated by abnormal vascularisation, comprising a recombinant virus comprising an anti-sense nucleic acid sequence corresponding to at least part of the sequence encoding VEGF, together with a pharmaceutically-acceptable carrier, wherein the anti-sense sequence is between 20 nucleotides in length and the full length sequence encoding VEGF.

40. A composition according to Claim 39, further comprising as adjuvant hyaluronic acid or a derivative thereof.

41. A composition according to Claim 39, or Claim 40, wherein the anti-sense sequence is between 50 nucleotides long and the full length sequence of VEGF.

42. A composition according to any one of Claims 22 to 41, wherein the VEGF sequence is that of VEGF from human retinal pigment epithelial cells or choroidal endothelial cells.

43. A composition for treatment of a retinal disease mediated by abnormal vascularisation, wherein said treatment is effective for an indefinite period, comprising a virus comprising an anti-sense DNA corresponding to at least part of the sequence encoding VEGF, together with a pharmaceutically-acceptable carrier, wherein said virus is one capable of integrating the anti-sense sequence into the genome of the target cell.

44. A composition according to Claim 43, further comprising as adjuvant hyaluronic acid or a derivative thereof.

45. A composition according to Claim 43 or Claim 44, wherein the virus is an adeno-associated virus.

46. A composition according to Claim 43, Claim 44 or Claim 45, wherein the anti-sense sequence is between 20 nucleotides long and the full length sequence of VEGF.

47. A composition according to Claim 43, Claim 44 or Claim 45, wherein the anti-sense sequence is between 50 nucleotides long and the full length sequence of VEGF.

48. A method of treatment of a retinal disease mediated by abnormal neovascularisation, comprising the step of administering an effective amount of an anti-sense nucleic acid sequence corresponding to at least part of the sequence encoding VEGF into the eye(s) of a subject in need of such treatment, thereby to inhibit neovascularisation.

49. A composition according to Claim 48, further comprising as adjuvant hyaluronic acid or a derivative thereof.

50. A method according to Claim 48 or Claim 49, wherein the anti-sense sequence is 16 to 50 nucleotides long.

51. A method according to Claim 48 or Claim 49, wherein the anti-sense sequence is 16 to 22 nucleotides long.

52. A method according to Claim 48 or Claim 49, wherein the anti-sense sequence is 16 to 19 nucleotides long.

53. A method according to Claim 48 or Claim 49, wherein a modified oligonucleotide as herein defined is used, and the anti-sense sequence is 7 to 50 nucleotides long.

54. A method of treatment of a retinal disease mediated by abnormal neovascularisation, comprising the step of administering an effective amount of a composition according to any one of claims 22 to 47 to a subject in need of such treatment

55. A method of treatment of a retinal disease mediated by abnormal neovascularisation, comprising the step of administering a composition according to any one of Claims 39 to 42 to the eye(s) of a subject in need of such treatment, thereby to inhibit neovascularisation in the long term.

56. A method of treatment of a retinal disease mediated by abnormal neovascularisation, comprising the step of administering an effective amount of a composition according to Claims 42 to 47 into the eye(s) of a subject in need of such treatment, thereby to inhibit neovascularisation for an indefinite period.

57. A method according to any one of Claims 48 to 56, wherein the retinal disease is selected from the group consisting of age-related macular degeneration, diabetic retinopathy, branch or central retinal vein occlusion, retinopathy of prematurity, rubeosis iridis and corneal neovascularisation.

58. A method of promoting uptake of an exogenous nucleic acid sequence by a target cell, comprising the step of exposing the cell to the nucleic acid, or to a virus or vector comprising the nucleic acid, in the presence of a hyaluronic acid or a derivative thereof.

59. A method according to Claim 58, in which the target cell is a phagocytic cell.

60. A method according to Claim 58 or Claim 59, in which the nucleic acid and hyaluronic acid are administered together in vitro .

61. A method according to Claim 58 or Claim 59, in which the nucleic acid and hyaluronic acid are administered together in vivo.