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WO1997015330A1 - Acide hyaluronique utilise comme porteur d'adn pour une therapie genique et adn anti-sens du facteur de croissance de l'endothelium vasculaire pour le traitement d'une vascularisation anormale de la retine - Google Patents

Acide hyaluronique utilise comme porteur d'adn pour une therapie genique et adn anti-sens du facteur de croissance de l'endothelium vasculaire pour le traitement d'une vascularisation anormale de la retine Download PDF

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WO1997015330A1
WO1997015330A1 PCT/AU1996/000664 AU9600664W WO9715330A1 WO 1997015330 A1 WO1997015330 A1 WO 1997015330A1 AU 9600664 W AU9600664 W AU 9600664W WO 9715330 A1 WO9715330 A1 WO 9715330A1
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vegf
sequence
composition according
nucleic acid
βequence
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PCT/AU1996/000664
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English (en)
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Piroska Elizabeth Rakoczy
Ian Jeffrey Constable
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Hyal Pharmaceutical Australia Limited
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Priority claimed from AUPN6161A external-priority patent/AUPN616195A0/en
Priority claimed from AUPN9047A external-priority patent/AUPN904796A0/en
Application filed by Hyal Pharmaceutical Australia Limited filed Critical Hyal Pharmaceutical Australia Limited
Priority to JP9516137A priority Critical patent/JP2000507915A/ja
Priority to EP96934189A priority patent/EP0859636A4/fr
Priority to NZ320006A priority patent/NZ320006A/en
Priority to AU72664/96A priority patent/AU721060B2/en
Priority to KR1019980702900A priority patent/KR19990066967A/ko
Publication of WO1997015330A1 publication Critical patent/WO1997015330A1/fr

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    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1136—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against growth factors, growth regulators, cytokines, lymphokines or hormones
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
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    • C12Y304/23005—Cathepsin D (3.4.23.5)
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2310/31—Chemical structure of the backbone
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    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
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    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/025—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a parvovirus

Definitions

  • This invention relates to use of hyaluronic acid to target active agents which ablate the function of targeted genes in the control or treatment of disease.
  • this invention relates to a method and composition for treating ocular diseases, in particular retinal disease involving neovascularisation of the choroid and/or retina. It makes use of the phagocytic characteristic of specific cells in the eye to provide an 0 effective manner of delivering an active agent to the target, for either short term or long term treatment of neovascularisation.
  • the methods and compositions of the invention are useful for delivering DNA, RNA, anti-sense nucleotides, peptides or other therapeutic agents to 5 phagocytic cells or surrounding cells.
  • Hyaluronic acid is a large, complex oligosaccharide consisting of up to 50 000 pairs of the 0 basic disaccharide glucuronic acid- ⁇ (l-3) N-acetylglucos- amine ⁇ (l-4) . It is found in vivo as a major component of the extracellular matrix. Its tertiary structure is a random coil of about 50 nm in diameter.
  • HA has the ability to bind a large amount of 5 water, which in vivo makes it a viscous hydrated gel with viscoelastic properties. It is found in this form in the mammalian eye, both in the vitreous and in the extracellular matrix.
  • HA has been used in the treatment of certain 0 diseases and conditions of the human body both systemically and topically, because of its ability to target an active agent to sites where the disease or condition is localised (International Patent Publications No. WO 91/04058 and No. WO 93/16733) . It has been shown that HA forms depots, for 5 example at the injured carotid artery (relative to uninjured contralateral arteries) and in colorectal tumours growing in experimental animals, and is retained in the skin of such animals.
  • the sites of the deposits are areas of high HA receptor expression, indicating that HA targets specifically to tissues that are expressing high levels of these receptors, particularly to tissues undergoing unusual proliferation and migration, including tissues responding to injury, inflammation, development, and tumorigenesis.
  • the characteristic of HA which is important to its action as a potential adjuvant i ⁇ its ability simultaneously to bind to other molecules and to bind to cell membranes.
  • Cell surface receptors specific for HA have been identified, including the histocompatibility antigen CD-I4, the receptor for hyaluronic acid-mediated motility (RHAMM), intercellular adhesion factor (ICAM), and some homologous proteins in the CD44 family.
  • CD-I4 histocompatibility antigen
  • RHAMM receptor for hyaluronic acid-mediated motility
  • IAM intercellular adhesion factor
  • the binding of virus to the cell membrane facilitated by HA would allow the usual endocytotic mechanisms of viral uptake to be more effective.
  • a variety of ocular diseases such as macular degeneration and diabetic retinopathy are characterised by neovascularisation of the choroid and/or retina. This process is the major cause of blindness in patients suffering from these conditions.
  • SRNVM subretinal neovascular membrane
  • Various treatments are available, but all are unreliable.
  • Laser photocoagulation is the most acceptable type of treatment, but it still suffers from the disadvantages that damage by the laser rays causes dense, permanent scotoma (Schachet, 1994; Ibanez et al, 1995 and Hudson et al, 1995) resulting in temporary loss of vision, and inability to prevent progression of the condition in the long term because of recurrence of the neovascular membrane.
  • this treatment provides an advantage only in terms of preventing profound visual loss.
  • the other main method of treating diabetic retinopathy is the control of blood glucose and blood pressure.
  • the efficacy of such forms of treatment is limited by the motivation and compliance of the patient involved.
  • VEGF Vascular endothelial cell growth factor
  • VEGF mRNA levels were elevated in the hypoxic region of the retina following induction of retinal vein occlusion.
  • Stimulation of VEGF expression by hypoxia has also been observed in other animal models (Pierce et al, 1995; Miller et al, 1994), and in vitro in all types of cell cultures (Simorre-Pinatel et al, 1994; Hata et al, 1995 and Thiema et al, 1995).
  • anti-sense sequences are DNA sequences which, when transcribed, result in synthesis of RNA whose sequence is antiparallel to the sequence encoding the protein.
  • anti-sense sequences have been tested in a number of viral diseases.
  • anti-sense oligodeoxynucleotide ⁇ can be introduced into target cells; such short sequences are not themselves transcribed, but inhibit transcription and/or subsequent translation of the corresponding sense DNA sequence in the target cell.
  • flanking sequences of the target RNA are important in determining specificity (Wagner et al, 1996) .
  • the targeting may also be achieved by virus- mediated DNA transfer, using viruses such as the Sendai virus.
  • Sendai virus is an RNA virus which has been shown to deliver DNA and proteins into cells with more than 95% efficiency (Kaneda et al, 1987).
  • DNA nuclear protein complex in liposomes i ⁇ directly introduced into the cytoplasm of the cell by the fusion activity of Sendai virus.
  • the DNA is delivered rapidly into the nucleus with nuclear protein.
  • Sendai virus-mediated gene transfer occurs by fusion of the virus with the cell membrane, and bypasses the endocytic pathway. Recently, highly efficient delivery of anti-sense or plasmid DNA into target cells by Sendai virus has been observed.
  • both the anti-sense and plasmid DNAs retained their activity not only in culture but also in vivo (Kaneda et al, 1987) .
  • the use of this virus is limited by the fact that there are no suitable constructs available at present to use as vectors.
  • the transferred DNA can only be expressed for a limited period of time since the gene transfer is mediated by fusion.
  • Retroviruses have been widely used for somatic tis ⁇ ue gene therapy (Boris-Lawrie and Temin, 1993) . They can target and infect a wide variety of host cells with high efficiency, and the transgene DNA integrates into the host genome. Theoretically, the integration of the DNA will provide permanent production of the transgene which could result in permanent rescue of the cells.
  • retroviruses cannot infect non-dividing cells (Salmons and G ⁇ inzburg, 1993) .
  • the retrovirus particles are unstable in vivo, which makes it difficult to achieve high virus titre with inoculation.
  • the inability of retroviruses to infect non-dividing cells means that they cannot be selected a ⁇ candidate ⁇ for gene transfer in the eye, as the most important target cells such as photoreceptors and RPE cells are non-dividing cells.
  • herpes simplex virus vectors have been limited by their poor efficiency of infection (Culver et al, 1992) .
  • Two types of vectors have been developed, namely the replication defective recombinants and the plasmid-derived amplicons. The latter requires a helper virus.
  • the toxic genes can be removed from the herpes simplex virus with difficulty, the constructs remain cytotoxic (Johnson et al, 1992) .
  • the long term expres ⁇ ion of the sequences inserted has been unsuccessful to date, and there are problems with the regulation and stability of the constructs.
  • the application of modified herpes simplex viruses to the eye in gene therapy poses major concerns because of their pathogenicity. Herpes zoster virus infection causes serious infections in the eye, frequently resulting in blindness requiring corneal transplantation.
  • Adenoviruses have been widely u ⁇ ed for gene transfer in both non-dividing and proliferating cells. They can accommodate DNA up to 7.5 kb, and provide efficient transfection and high viral titre.
  • the main advantage of using these rather than retroviruses is the ability to infect a wide range of non-dividing target cells (Kozarsky and Wilson, 1993).
  • Replication-defective adenoviruses are considered to be relatively safe, in that these viruses are common pathogens in humane, usually causing relatively benign conditions such as colds.
  • the vectors carry tumour genes with a deletion mutation, lowering the possibility of becoming oncogenic (Siegfried, 1993) .
  • adenoviru ⁇ e ⁇ were u ⁇ ed to treat individuals suffering from cystic fibrosis.
  • the main disadvantage of adenoviru ⁇ es is their transient gene expression. This is a result of the lack of integration of the transgene into the cellular genome.
  • few attempts at gene delivery to non- dividing cells have been successful. The first successful gene transfer into the brain, which consists of non- dividing cells, was reported in 1993 using adenoviruses (Le Gal La Salle et al, 1993).
  • a method of stimulating proliferation of non-malignant cells i ⁇ comprises the in vitro treatment of cells with an anti-sense nucleotide corresponding to the retinoblastoma (Rb) gene to inhibit expression of the Rb gene product, resulting in suppres ⁇ ion of the expression of proteins which inhibit cell growth.
  • Rb retinoblastoma
  • the proliferated cells can then be re-implanted if desired, and the cells may be genetically engineered to replace a ⁇ pecific gene prior to re-implantation.
  • there i ⁇ no reference to use of this anti-sense sequence to treat conditions of the eye.
  • the invention of US-5324654 i ⁇ directed to establishing cell lines capable of long-term proliferation and to treatment of conditions such as muscular dystrophy and diabetes, caused by failure to express a gene.
  • the advances in gene therapy referred to above have led to further ⁇ tudie ⁇ of the delivery and expre ⁇ ion of transgenes into target cells, such as the ⁇ -galactosidase transgene into the retina (Bennett et al, 1994, Li et al, 1994 and Mashmour et al, 1994) using recombinant adenovirus as a delivery system.
  • the retinal pigment epithelium (RPE) is a non-renewable single cell layer in the eye, situated between the neural retina and the choroid.
  • the cells of the RPE are phagocytic neuroepithelial cells which form the outer most layer of the retina.
  • the phagocytic properties of these cells have long been known, and have been reviewed (Bok and Young, 1979) . High levels of transgene expression within 3 days in the RPE layer and within two weeks in the photoreceptor cells of the neural retina in young animale were observed. The expression of the reporter gene was followed up to 9 weeks. In older animals, neither subretinal nor intravitreal injections induced the expre ⁇ ion of the ⁇ - galactosidase transgene in the photoreceptor cells (Li et al, 1994).
  • Cytotoxicity has been observed in association with use of adenoviruses as a transport vector for retinal gene therapy. This cytotoxicity has been shown to be dose- dependent (Mashmour, 1994) and poses another difficulty in using such a vector. In order to decrease the dose of a given vector but retain its transfer efficiency, an adjuvant may be used. Adjuvants such as lipofectin have been shown to increase the uptake of "naked" DNA by cells. Even though HA has been widely u ⁇ ed in eye surgery a ⁇ a replacement for vitreous humour lost during the surgical procedure, we are not aware of any suggestion in the art that HA promotes uptake of any pharmaceutical agent into any cells or ti ⁇ sues in the eye.
  • HA has been suggested to promote penetration of pharmaceutical agents such as antibiotics or anti-cancer agents, as set out in Australian Patent Application No. 52274/93 by Norpharmco, this specification does not sugge ⁇ t that HA promotes uptake of any agent, let alone DNA or viruses, by individual cells of any type. In particular, this specification does not teach the use of HA via intra ⁇ ocular injection.
  • RPE cells will increase the uptake of molecules such as oiigonucleotides and viruses, following injection into the vitreous space in vivo. These RPE cells show increased uptake of virus compared to other cell types.
  • Our findings enable the induction of both long-term and short-term inhibition of VEGF expression in retinal or choroid epithelial cells, and hence inhibition of neovascularisation of the retina or the development of SRNVM.
  • the invention provides a compo ⁇ ition comprising a nucleic acid and a hyaluronic acid or a derivative thereof, together with a pharmaceutically- acceptable carrier.
  • the nucleic acid may be a DNA or RNA, and/or may be a nucleotide sequence which is in the anti-sense orientation to a target sequence.
  • the target sequence is a nucleic acid sequence which is implicated in the causation or exacerbation of a pathological condition.
  • This target nucleic acid sequence may be a genomic DNA, a cDNA, a messenger RNA or an oligonucleotide. Where the target nucleic acid sequence i ⁇ a genomic DNA, it may be present in a coding region, or in a regulatory region, such as a promoter sequence.
  • the nucleic acid may be present in a vector comprising a nucleic acid sequence to be transferred into a target cell.
  • the nucleic acid sequence may be genomic DNA, cDNA, messenger RNA, or an oligonucleotide.
  • the nucleic acid may either be a sense ⁇ equence to be provided to a target cell in order to exert a function, or may be an anti-sense sequence to be provided to inhibit the functioning of a nucleic acid present in the target cell.
  • the vector comprising the DNA to be transferred may be a virus, ⁇ uch a ⁇ an adenovirus, an adeno-as ⁇ ociated virus, a herpes viru ⁇ or a retrovirus.
  • the use of all of the ⁇ e cla ⁇ e ⁇ of viru ⁇ a ⁇ vector ⁇ for gene therapy has been extensively canvassed in the art.
  • the vector may be a liposome.
  • the invention also provide ⁇ 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 ⁇ aid subject.
  • 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 mammal ⁇ ⁇ uch as cattle, horse, sheep, goats, cats and doge.
  • the nucleic acid or vector may simply be mixed with the hyaluronic acid, or may optionally be physically or chemically coupled to hyaluronic acid.
  • Methods for attaching DNA to hyaluronic acid have been disclosed in "Synthesis of Sulfonated Hyaluronan Derivatives containing Nucleic Acid Bases, Chemistry Letters, 1994 2027-2030 and "Transport Performance of Nucleosides Through Nucleic Acid Base ⁇ Conjugated to Hyaluronan"; Chirachanchai, S., Wada, T., Inaki, Y. and Takemoto, K, Chemistry Letters. 1995 2_ 121- 122.
  • this aspect of the invention provides compositions and methods for treatment of a retinal disease mediated by abnormal vascularization, in which the nucleic acid i ⁇ 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.
  • VEGF vascular endothelial growth factor
  • Many forme 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 u ⁇ ed. The only requirement i ⁇ that the HA be of a degree of purity and sterility to be suitable for pharmaceutical use; preferably the HA is also pyrogen-free.
  • HA high molecular weight preparation of HA may require dilution prior to use.
  • commercially-available HA products suitable for use in the invention are those supplied by Hyal Pharmaceutical Corporation, Mis ⁇ i ⁇ auga, which is a 2% solution of HA having a mean average molecular weight of about 225,000; sodium haluronate produced by Life CoreTM Biomedical, Inc.; Pro Vise (Alcon Laboratories); and "HEALON” (Pharmacia AB, Uppsala) .
  • the term derivative ⁇ of HA encompasses homologues, analogues, complexes, esters and fragments and sub-unite of HA.
  • Derivatives of HA which may be u ⁇ ed in the invention include pharmaceutically-acceptable salts thereof, or fragments or subunits of HA.
  • the person skilled in the art will readily be able to determine whether a given preparation of HA, or a particular derivative, complex etc. of HA, i ⁇ suitable for use in the invention.
  • 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.
  • VEGF vascular endothelial growth factor
  • 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.
  • the VEGF i ⁇ most preferably human retinal pigment epithelial (RPE) or choroidal endothelial VEGF.
  • thi ⁇ aspect of the invention is directed to treatment for such retinal disease in the short term (up to about two months), the long-term (up to about one year), and indefinitely (for the life of the patient) .
  • the invention provides one or more anti-sense oiigonucleotides having 100% complementarity to a corresponding region of the VEGF gene.
  • the oligonucleotide should have 16 to 50 nucleotides, preferably 16 to 22, and more preferably 16 to 19 nucleotides. Modified oiigonucleotides of the kind described by Wagner et al (1996) may be used, and enable the lower limit of sequence length to be reduced to 7 nucleotide ⁇ .
  • the invention provides a recombinant virus comprising VEGF DNA in the anti- ⁇ en ⁇ e direction.
  • Thi ⁇ VEGF DNA i ⁇ a long sequence, which for the purpose ⁇ of thi ⁇ ⁇ pecification i ⁇ to be understood to represent a VEGF sequence of greater than 20 nucleotides in length, preferably greater than 50 nucleotide ⁇ , ranging up to the full length ⁇ equence of VEGF.
  • the recombinant viru ⁇ i ⁇ accumulated in RPE cells, and produces anti-sense VEGF in situ, thereby inhibiting VEGF expression in the RPE cell.
  • the invention provides a viru ⁇ compri ⁇ ing VEGF DNA in the anti- ⁇ en ⁇ e direction in which the viru ⁇ i ⁇ one capable of integrating the anti- ⁇ en ⁇ e ⁇ equence into the genome of the target cell.
  • the virus is an adeno-associated or similar virus.
  • this VEGF DNA i ⁇ of at least 20 nucleotide ⁇ , preferably greater than 50 nucleotides.
  • the adeno- as ⁇ ociated or similar virus facilitates integration of anti- ⁇ en ⁇ e VEGF DNA into the RPE cell genome, thu ⁇ enabling expression of anti-sense VEGF for as long as the cell remains functional.
  • Eye di ⁇ ea ⁇ e ⁇ which may be treated using the compositions and methods of the invention include, but are not limited to, age-related macular degeneration (ARMD) and diabetic retinopathy.
  • AMD age-related macular degeneration
  • Other ocular conditions and tissues in which neovascularisation occurs for example branch or central retinal vein occlusion, retinopathy of prematurity (also known as retrolental fibroplasia) , rubeosis iridie or corneal neovascularisation, may also be treated by the invention.
  • the invention provides a method of prevention or amelioration of a retinal disea ⁇ e mediated by abnormal neovascularisation, comprising the step of administering an effective amount of an anti- ⁇ en ⁇ e nucleic acid sequence directed against VEGF into the eye, thereby to inhibit neovascularisation.
  • the anti-sense ⁇ equence may be carried in a replication-defective recombinant viru ⁇ , a ⁇ a vector or vehicle.
  • the vector preferably comprises replication- defective adenovirus carrying promoters such as the respiratory syncytial virus (RSV), cytomegalovirus (CMV), adenovirus major late protein (MLP), VAl pol III or ⁇ -actin promoters.
  • the vector may aleo comprise a polyadenylation signal sequence such a ⁇ the SV40 signal sequence.
  • the vector is pAd.RSV, pAd.MLP, or pAd.VAl.
  • the vector is Ad.RSV.aVEGF or Ad.VAl.aVEGF.
  • human VEGF is subcloned into the vector, in order to create the restriction ⁇ ite ⁇ necessary for insertion, to form an adenovirus plasmid carrying VEGF or partial sequence ⁇ thereof in an anti- ⁇ en ⁇ e direction, which can then be linearized by restriction enzyme digestion.
  • the linearized plasmid can then be co-transfected with a linearized replication defective adenovirus, in a suitable permissive host cell such a ⁇ the kidney 293 cell line.
  • compositions of the invention may be delivered into the eye by intra-vitreal or sub-retinal injection, preferably in an appropriate vehicle or carrier. Such methods of administration and vehicle ⁇ or carriers for such injection are known in the art.
  • ex vivo delivery of the compositions of the invention may be achieved by removal of RPE cells from the patient to be treated, culturing the cells and subjecting them to infection in vitro with a replication-defective adenovirus or an adeno-associated virus as defined above. RPE cells carrying the virus are then injected into the ⁇ ub-retinal layer of the eye of the patient.
  • Figure la shows the results of GeneScan analysis of persistence of anti-sense oiigonucleotides in vivo in the retina following a single intra-vitreal injection.
  • Figure lb shows a confocal microscopic image of the retina of a RCS-rdy + rat at different times following injection of CATSCF.
  • Figure 2 is a graphical representation of the number of phagosomes in the RPE layers of Long-Evans rats. Doses were as follows: Low 6.6 ⁇ g, medium 66 ⁇ g and high 132 ⁇ g of CATSC anti-sense oligonucleotide. Each column shows the mean and standard deviation of the number of phago ⁇ omes in five randomly selected areas in the rat retinae.
  • Figure 3 is a graphical representation of the number of phagosome ⁇ in the RPE layers of RCS-rdy-f- rats. Experimental animals were injected with 66 ⁇ g of sense oiigonucleotides (Sl) and 66 ⁇ g of antisense oligonucleotide (CATSC) .
  • Sl sense oiigonucleotides
  • CATSC antisense oligonucleotide
  • Figure 4 shows the effect of increasing the titre of adenoviral vector on the number of cells expre ⁇ sing the adenoviral transgene. In all cases, the incubation period was 16 hours.
  • RPE7 denotes Human retinal pigment epithelial cells from a 7 year old donor;
  • F2000C denotes F2000 fibroblastic cells.
  • the C suffix on the F2000 key indicates that the counts for the F2000 cell expression have been corrected for direct comparison with the RPE7 cells.
  • Figure 5 shows the effect of increasing the time of incubation with the adenoviral vector on the number of cell ⁇ expressing the adenoviral transgene. In all cases, the concentration of the adenoviral vector was 2 x 10 £ p.f.u./ml.
  • the C suffix on the F2000 key indicates that the counts for the F2000 cell expression have been corrected for direct comparison with the RPE7 cells.
  • Figure 6 i ⁇ a graphical representation of the effect of Hyaluronic Acid (HA) on the number of RPE7 cells expressing an adenoviral transgene 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 indicates one standard deviation.
  • Figure 7 i ⁇ a graphical representation of the effect of Hyaluronic Acid (HA) on the number of F2000 cells expressing an adenoviral transgene 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 indicates one standard deviation.
  • Figure 8 shows the immunofluorescent staining of HA receptors in RPE7 and F2000 fibroblasts 8a.
  • RHAMM staining on F2000 fibroblasts a. CD44 staining on RPE7; 8b. ICAM staining on RPE7; 8c. RHAMM staining on RPE7; 8d. CD44 ⁇ taining on F2000 fibroblasts; 8e. ICAM staining on F2000 fibroblasts; 8f. RHAMM staining on F2000 fibroblasts.
  • Figure 9 show ⁇ micrograph ⁇ of choriocapillary endothelial cell ⁇ isolated from porcine eye, illustrating their characteristic appearance (top panel), presence of Factor Vlll-related antigen (middle panel), and ability to take up acetylated low-density lipoprotein into the cytoplasm (bottom panel) .
  • Figure 10 show ⁇ the effects of a variety of hyaluronic acid preparations on tube formation by choriocapillary endothelial cell ⁇ .
  • Figure 11 show ⁇ the alkaline phosphatase staining of CD44 antigen in retinal pigment epithelium cells. In each case the epithelium is at the bottom of the picture with choroid above.
  • Pigment epithelium layer bleached to remove melanin granules.
  • Figure 12 show ⁇ the results of DNA PCR and RT-PCR analysis of transfection of a retinal pigment epithelial cell line with VEGF 165 .
  • Figure 13 show ⁇ the effect of VEGF 165 produced by transfected RPE cells on tube formation by choriocapillary endothelial cells.
  • Example 1 Accumulation of Antisense Oiigonucleotides in the RPE Cell Layer Human retinal pigment epithelial cells were cultured and on the third passage were used for in vitro experiments. Confluent cultures were incubated with bovine rod outer segments (ROS) to mimic the in vivo situation.
  • ROS bovine rod outer segments
  • a fluorescein-labelled anti-sense oligonucleotide complementary to human cathepsin S was added to the medium of these cells and after 7 days of incubation, the cell ⁇ were harvested. The presence of fluorescein- labelled oiigonucleotides within the RPE cells was detected by fluorocytometry (FACS) . A GeneScan DNA analyser was used to assess the presence and stability of the oiigonucleotides in the cells. The fluorescence of cultured RPE cells was increased by about 100-fold, demonstrating the presence of the anti-sense oiigonucleotides within the RPE cell ⁇ . The ⁇ e results are summarised in Table 1.
  • Example 2 Cellular Distribution of Oiigonucleotides in Retinal Cell ⁇ and Stability of Oiigonucleotides Following Injection Into Eyes
  • One nmole of CATSCF was injected into the vitreous humour of 6-week old non-pigmented RCS-rdy + rats, and the movement of the oiigonucleotides were followed by confocal fluoromicroscopy. Fluorescein (1 nmole) was also injected as a control. Animals were euthanised 2 hours, 3 days and 7, 14 and 28 and 56 days after injection.
  • the injected eyes were enucleated, frozen, sectioned and immediately used for confocal microscopy without fixation.
  • Figure lb in which g represents the ganglion cell layer, i the inner nuclear layer, o the outer nuclear layer, and r the retinal pigment epithelial layer.
  • the panels show retinas 2 hours (B), 3 days (A), 7 days (C), 28 days (D) and 56 days (E) after injection of 10 nmol CATSCF, and 3 daye (F) after injection of FITC a ⁇ a control.
  • the ⁇ e re ⁇ ults demonstrate that following intravitreal injection, oiigonucleotides accumulate in the RPE cells.
  • the oiigonucleotides are present in the RPE layer up to 56 days, and remain in a biologically active form during this period of time.
  • Oiigonucleotides Female sixty day-old pigmented rats of the Long- Evans strain were obtained from Charles River Breeding Laboratories, Wilmington, MA. Sixty day old non-pigmented RCS-rdy + rats were obtained from our colony. The animals were acclimatised to a 12 hr light/ 12 hr dark lighting cycle, with an average illuminance of 5 lux for at least 10 daye prior to experimentation. Animals were anae ⁇ theti ⁇ ed by intraperitoneal injection of sodium pentobarbital (50 mg/kg body weight) . Intravitreal injections through the pare plana were made using a 32 gauge needle.
  • Histological data were obtained by light microscopy. Semi-thin 1 ⁇ m sections were cut using a LKB 2088 Ultratome (LKB-Sher, Sweden) with a diamond knife and stained with toluidine blue. The number of phagosomee that accumulated in the RPE cell ⁇ of each ⁇ pecimen injected with ⁇ aline, low ( 6 .6 ⁇ g) , medium (66 ⁇ g) or high 132 ⁇ g) dose of CATSC and 66 ⁇ g of Sl sense oligonucleotide was determined. From each eye, five sets of counts were made at 40 fold magnification and the standard deviation was calculated.
  • Each set consisted of the total number of phagosomee in 250 ⁇ m length of RPE from 6 different randomly selected areas. The number of phagosomes that accumulated in the RPE of the control eyes, low medium and high do ⁇ es of CATSC were analysed and graphically represented. Comparisons were made using the analysis of variance following the general linear models procedure of the SAS R (version 6) statistical package (SAS Institute Inc., USA) .
  • the re ⁇ ults show that we successfully tested an anti- ⁇ en ⁇ e oligonucleotide (CATSC) in two strains of rats.
  • the number of phagosome-like inclusion bodies present in control Long-Evans and RCS rdy + rats was not significantly different, 35.84-11.6 and 47.29+14.8 (mean ⁇ SD), respectively.
  • the intravitreal injection was non- traumatic.
  • Light microscopic examination of the retinas of the saline injected eyes revealed no damage to the outer layers of the retina, and there was no increase in the number of phagosome-like inclusion bodies in the RPE layer when compared to the control non-injected animals.
  • Long- Evans rats were used to identify the minimum amount of CATSC required to induce biological changes in the RPE layer.
  • RCS-rdy4- rats injected with 66 ⁇ g of CATSC also demonstrated a statistically significant increase in the number of phagosome-like inclusion bodies, ie 204.204-39.3 when compared to the 47.204*14.8 in controls.
  • the injection of 66 ⁇ g of sense oligonucleotide (Sl) did not increase the number of phagosomee ( Figure 3) pre ⁇ ent in the RPE Layer, (34.44*12.54).
  • Electron microscopic examination of the RPE layer of a CAT SC-injected eye revealed no significant changes in the morphology of RPE cells. Melanin granules appeared smaller and less concentrated due to regional differences. Individual mitochondrial profiles were smaller in the treated group than in the controls, although the number was greater in the treated than in the untreated animals. Electron microscopic examination confirmed that the structures of the undigested material was similar to that of phagosomee.
  • the numerous phagosomee seen in the RPE layer of rate treated with CATSC were paranuclear, and contained mainly compacted phospholipid membranes, resembling undigested photoreceptor outer segment (POS) and confirming their photoreceptor origin. There were no other morphological changes observed in the POS layer, except for the disorganised appearance of the apices in treated animals.
  • Example 4 Gene Transfer to the RPE Cell Layer The nature and dynamics of gene transfer using an adenoviral vector were examined. The effects of adjuvants on the uptake of the adenovirus wa ⁇ also studied.
  • Human RPE cultures were obtained from a 7-year old Caucasian donor and prepared as described in Rakoczy et al (1992) .
  • Human F2000 fibroblast cells were cultured, harvested and pooled in Minimal Eagles Medium (MEM, Multicel TM Trace Bio ⁇ ciences, Australia), with 10% FBS (MultiserTM, Trace Bioscience ⁇ ) and containing 125 ⁇ l gentamicin (Delta West, Bentley, Australia) per 100 ml medium.
  • MEM Minimal Eagles Medium
  • FBS MultiserTM, Trace Bioscience ⁇
  • gentamicin Delta West, Bentley, Australia
  • Ad.RSV. ⁇ gal Replication-deficient Adenovirus 5 carrying a RSV promoter and ⁇ -Galactosidase gene (Ad.RSV. ⁇ gal) (Stratford-Perricaudet, 1992) was cultured and purified as described by Graham and Prevac, 1991. Ad.RSV. ⁇ gal was added to each well as a 1 ml aliquot, in MEM, at a concentration of 4xl0 6 p.f.u./ml. for the time-based trials, giving a final concentration of 2xl0 6 p.f.u./ml.
  • concentrations of 8xl0 3 , 4xl0 4 , 8xl0 4 , 2.4xl0 5 , 4x10 s p.f.u./ml were added to the wells in a 1 ml aliquot, making the total volume 2 ml in each well (the final viral concentration is half of that added) .
  • All of the trials examining the effect of increasing viral titres involved incubation of the culture with the viral suspension for a fixed period of 16 hours. Experiments were terminated by removing the medium from each well and fixing the cells with 0.5 ml of 0.5% glutaraldehyde.
  • Viral Vector HRPE7 and F2000 cells were aliquoted into 24 well plates. The cells were incubated as described in Example 4, and allowed to reach 95% of confluence. Solutions of 0.001% to 0.005% buffered eodium hyaluronate (HA) (1% Hyaluronic acid from rooster comb; HEALON, Pharmacia AB, Upp ⁇ ala, Sweden) were prepared with MEM. A dose of 10 ⁇ l of viral solution at a concentration of 4 x 10 6 p.f.u. was added to 10 ml of each of the diluted HA solutions and 10 ml of MEM for the control, and incubated for 30 minute ⁇ at 25°C with intermittent gentle shaking. To separate wells of the 24 well plate, 1 ml of each of the test and control solutions was added. There were four parallel samples for each test concentration and for the control, which were counted and averaged.
  • HA eodium hyaluronate
  • the probability of the significance of the increase in number of HRPE7 cells expressing the gene, when 0.005% HA is used, compared with the control, is 0.0097, which shows a level of significance of p ⁇ 0.01.
  • the significance reflects the large difference between the means (20592 (test) v 14705 (control)) and the separation of the means by more than two standard deviations.
  • the t test probability of the significance of the increase in number of RPE7 cells expre ⁇ ing the gene, when 0.001% HA i ⁇ u ⁇ ed compared with the control, is 0.02931, which show ⁇ a level of significance of p ⁇ 0.05.
  • the reduced significance reflects the smaller difference between the means (19168 (test) v 14705 (control)).
  • the protocols for examining the effect of HA on the expres ⁇ ion of a tranegene in F2000 fibrobla ⁇ t ⁇ were the same a ⁇ that for HRPE7.
  • the numbers of cells expressing transgenes were significantly less than for HRPE7, which is consistent with the results demonstrated in Example 4.
  • the mean number of cells expressing in each well for adenovirus alone was 3780 (SD ⁇ IOO) .
  • the mean number of expressing cells was 4391 per well
  • the high significance here reflects the large difference between the means (4391(test) v 3790(control) ) and the small variation within the two samples.
  • the standard deviation is 214(test) and 111(control) .
  • i ⁇ a greater variation in the raw figures, and the standard deviation is higher than for the 0.005% sample (355 v 214), which accounts for the higher p value.
  • Preliminary trials of chondroitin sulphate and lipofectamine as adjuvants were also carried out in order to assess the likely efficacy. These agents had no significant effect on gene expres ⁇ ion in HRPE7 cells.
  • the figures represent the effect of HA concentration on the uptake and expression of ⁇ -gal transgene. Increasing virus concentration resulted in an increase in the number of ⁇ -gal expressing cells.
  • the numbers represent the number of RPE cells staining positive for ⁇ -gal following 16 hours incubation of virus in the presence of HA in a 24 well plate (cc 2x10" pfu/ml) .
  • Example 6 Effect of HA Molecular Weight on the Uptake and Expression of the ⁇ gal Gene ⁇ eing a Viral Vector Adenovirus with a ⁇ -galactoeidase marker gene and a RSV promoter (Adv.RSV. ⁇ gal) was cultured in cells of the K293 embryonic human kidney cell line. Supernatant was collected, and the concentration of virus was determined by serial dilution with 4 replicates of each dilution. The concentration of the virus wa ⁇ calculated to be 5 x 10 s pfu/ml. The viru ⁇ was suspended in MEM medium with 10% fetal bovine serum (FBS) and 125 ⁇ l/100ml gentamicin.
  • FBS fetal bovine serum
  • HRPE Human Retinal Pigment Epithelial Cells
  • Healon GV (MW approx. 5 000 000) Each of the preparations was diluted to a solution of 0.002% in MEM without FBS.
  • the virus solution as above was mixed in a 1:1 ratio with the adjuvant solution giving a final viral concentration of 2.5 x 10 8 pfu and an HA concentration of 0.001%.
  • the two solutions were incubated in this mixture for 30 minutes at room temperature with gentle shaking.
  • the control solution consisted of a mixture of the virus with MEM without FBS with no HA present.
  • Example 7 Demonstration of HA Receptors on the cell membrane of HRPE7 and F2000.
  • Polyclonal RHAMM (Receptor for Hyaluronan Mediated Motility) antibodies were kindly provided by Dr E Turley, Manitoba Institute of Cell Biology, Canada. The antibody was used at a dilution of 1:75.
  • Monoclonal Intercellular Adhesion Molecule 1 (ICAM-1) antibodies (Boehringer-Mannheim) were u ⁇ ed at a concentration of 4 ⁇ g/ml and monoclonal homing receptor CD44 antibody (CD44) wa ⁇ u ⁇ ed at a concentration of 4 ⁇ g/ml (Boehringer Mannheim Biochemica, Germany) .
  • Monoclonal anti-human IgG antibody and rat non-immune serum were kindly provided by Dr M Baine ⁇ , Lions Eye Institute, Perth, Australia. They were u ⁇ ed at a concentration of 4 ⁇ g/ml and a dilution of 1:75 respectively.
  • Anti-Mouse IgG (Fab specific)-FITC conjugate secondary antibody was used at a 1:64 dilution and anti-Rabbit IgG (whole molecule)-FITC conjugate secondary antibody wa ⁇ u ⁇ ed at a 1:100 dilution (Sigma Immunochemicals, St Louie, Missouri) .
  • HRPE7 and F2000 fibroblast cells were cultured in
  • the secondary antibody to the monoclonal antibodies was antimou ⁇ e IgG and the polyclonal wa ⁇ anti-rabbit IgG.
  • the ⁇ econdary antibodie ⁇ were applied to ti ⁇ ue without primary antibody a ⁇ a further control.
  • Immunohistochemical staining for CD44 using a monoclonal antibody demonstrated positive staining for both HRPE7 cells and F2000 fibroblasts, as shown in Figures 8a and 8b respectively.
  • the staining had a distribution consistent with the cell surface, as the staining pattern was the same a ⁇ the cellular outline of cultured ti ⁇ ue.
  • Immunohistochemical ⁇ taining using a monoclonal antibody for ICAM-1 demonstrated positive staining for both HRPE7, and F2000 fibroblast ⁇ , a ⁇ shown in Figures 8c and 8d respectively.
  • the ⁇ taining had a similar distribution to that of CD44, but the signal was slightly weaker.
  • the same controls as for CD44 were u ⁇ ed for ICAM-1 ⁇ taining, and were al ⁇ o negative.
  • Hank's balanced salt ⁇ olution (Hank'e BSS) without calcium or magnesium, medium Hams F12, minimum essential medium with Earlee salts (EMEM) , foetal calf serum (FCS), penicillin-streptomycin, amphetericin B, and trypsin-EDTA were obtained from Australian Biosearch (Perth, Western Australia) .
  • Collagenase A, endothelial cell growth supplement (ECGF) , mouse anti-human monoclonal antibody against factor VIII-related antigen, and anti- mouse Ig-fluorescein were acquired from Boehringer Mannheim Australia Pty. Ltd. (Perth, Western Australia) .
  • Gelatin, heparin, ascorbic acid were purchased from Sigma Chemical Company (Sydney, Australia), acetylated low-density lipoprotein (Dil-ac-LDL, l,l'-dioctadecyl-3,3,3' ,3'- tetramethyl-indocarbo-cyanine perchlorate) from Biomedical Technologies, Inc. (Stoughton, Massachusetts), Matrigel from Collaborative Research (Bedford, Massachusetts), recombinant human vascular endothelial cell growth factor (VEGF) from Pepro Tech EC Ltd. (Rocky Hill, New Jersey), ProVisc (MW 1.9 x IO 6 ) from Alcon Laboratories, Healon
  • Porcine eyes were obtained from a local abattoir 2-4 hours after death of the animals.
  • the choriocapillary endothelial cells (CECs) were isolated as previously described (Morse et al, 1990, Sakomoto et al, 1995) . Briefly, Hank's balanced ealt ⁇ olution (Hank'e BSS) without calcium or magnesium, but with 0.1 % collagenase A was used to release endothelial cells at 37°C for 1 hour. After washing twice in Hank's BSS, the cells were plated in 1 % gelatin-coated 75-cm 2 cell culture flasks in 5% C0 2 , 95% air at 37°C.
  • the growth medium consisted of Hams F12 plus 10% fetal calf serum (FCS), 100 U penicillin-100 ⁇ g streptomycin/ml, 2.5 ⁇ g/ml amphotericin B, 37.5 ⁇ g/ml endothelial cell growth supplement (ECGS), heparin 100 ⁇ g/ml, and ascorbic acid 25 ⁇ g/ml.
  • FCS fetal calf serum
  • ECGS endothelial cell growth supplement
  • heparin 100 ⁇ g/ml 37.5 ⁇ g/ml
  • ascorbic acid 25 ⁇ g/ml.
  • a glass pipette which had been drawn through a flame to produce a bead tip was u ⁇ ed to remove and crush any non- endothelial colonies within the circles (Folkman et al., 1979).
  • This technique was carried out under a phase contrast microscope (xlO phase objective) in a laminar flow hood. The medium was changed twice to remove floating cell ⁇ . Thi ⁇ procedure wa ⁇ repeated three to five times to enrich the primary cells for endothelial cell ⁇ before they became confluent.
  • the cell ⁇ were identified a ⁇ va ⁇ cular endothelial cell ⁇ by typical cobblestone morphology, presence of factor Vlll-related antigen (Sakomoto et al, 1995), and positive staining (uptake) with Dil-ac-LDL (Folkman et al, 1979).
  • the tube formation assay was performed a ⁇ previously described (Haralabopoulos, et al, 1994. Briefly, Matrigel (16.1 mg protein/ml) wa ⁇ prepared from the Engelbreth-Holm Swarm tumour wa ⁇ u ⁇ ed to coat 24 well cluster plates (250 ⁇ l/well) as recommended by the product sheet.
  • the CECs (passage 3-7) were lifted from flasks by 0.25% trypsin-0.02% EDTA, suspended in 5% MEM, and added to the coated wells (50,000 cells/well in 0.5 ml medium) .
  • photographe were taken with a phase-contra ⁇ t microecope after six hours.
  • capillary endothelial cells have a characteristic appearance that distinguishes them from other cell types. In addition they were characterized by staining for factor Vlll-related antigen, and assaying for the ability to phagocytize Dil-ac-LDL. More than 95% of the CECs showed a positive reaction to factor VIII- related antigen. Almost every cell showed uptake of Dil-ac-LDL into the cytoplasm, as shown in Figure 9. This indicates that at least 95% of the cells were choriocapillary endothelial cells (CEC cell ⁇ ) .
  • CEC cell ⁇ choriocapillary endothelial cells
  • Tube formation was measured u ⁇ ing a Computer Imaging Analyzer System (Professional Image Proceeding for Windows, Matrox Inspector) .
  • the slide photographs were ⁇ canned into a computer and the background adjueted to obtain the beet contra ⁇ t between the tubee and Matrigel.
  • Tube formation wa ⁇ then quantified by mea ⁇ uring the total tube area of each photograph. The results were expressed as the mean and the standard error of the percentage of tube area in the presence of 7.5% FCS alone (the final concentration) and were analyzed by Student's t-test for at least two experiments.
  • a human Eye Bank donor eye was di ⁇ ected following the removal of the cornea. After di ⁇ carding the anterior segment the vitreous wa ⁇ carefully removed, leaving behind some parts of the neural retina and the complete layer of pigment epithelium attached to the choroid. The eye cap was filled with 2.5% glutaraldehyde for fixation. Sections of the fixed tissue were subjected to paraffin embedding. Paraffin blocks were cut and sections were transferred on to glass histochemical slides, dewaxed in xylane and ethanol, and washed in distilled water and Tris-buffered ⁇ aline pH 7.2 (TBS).
  • TBS Tris-buffered ⁇ aline pH 7.2
  • the ⁇ e results demonstrate that the retinal pigment epithelial cell ⁇ preferentially express HA receptors, thus facilitating an enhanced uptake of HA complexes.
  • NIH 3T3 cells were seeded on to 6-well tissues culture plates at a concentration of 2 X 10 s in 2 ml DMEM supplemented with 10% fetal bovine serum (FBS) . The cells were incubated overnight at 37°C until they became 70% confluent. Having reached confluency, the cells were waehed twice with serum and antibiotic-free medium.
  • Lipofection reagent (10 ⁇ l) (GIBCO-BRL) diluted in 100 ⁇ l of OPTI-MEM (GIBCO-BRL) were gently mixed and incubated at room temperature for 15 minutes. Following incubation, an additional 800 ⁇ l of OPTI-MEM wa ⁇ added to the mixture.
  • Thi ⁇ diluted mixture was gently overlaid onto the washed NIH 3T3 cells.
  • the cells were incubated for 16-20 hrs before the transfection media was removed and replaced with DMEM supplemented with 10% FBS. After a further 48 hrs incubation the cells were trypsinised and subcultured at 1:5 in media containing 10% FBS and Geneticin 418 (GIBCO ⁇ BRL) at 1 ng/ml concentration. Succes ⁇ fully transfected cell ⁇ eelected with Geneticin 418 were maintained in media supplemented with FBS and Geneticin 418 as described above. Confluent transformed cultures were frozen for storage and subcultured for further analysis.
  • cathepsin D The presence of cathepsin D in the tran ⁇ formed NIH 3T3 cells was detected with polyclonal antibody against cathepsin D, using a conventional cytochemical technique and an alkaline phosphataee-labelled ⁇ econd antibody.
  • the immunocytochemistry results sugge ⁇ t that NIH 3T3 cell ⁇ carrying cathepsin D in the sense direction up-regulated cathepsin D production, while those carrying cathepsin D in the anti- ⁇ en ⁇ e direction down regulated cathepsin D production.
  • the human RPE cell line 407A (Davis et al, 1995), was maintained at 37°C in a humidified environment containing 5% C0 2 .
  • the culture medium consisted of Minimal Essential Medium (MEM, Trace Bio ⁇ ciences, Sydney, NSW, Australia) supplemented with 10% FCS (Trace Biosciences, Sydney, NSW, Australia) and 100 lU/ l Penicillin/100 ⁇ g/ml Streptomycin (P/S) (ICN Pharmaceuticals Inc, Costa Mesa, CA, USA).
  • VEGF 16S in Bluescript KS was obtained from Dr Georg Breier, Max Planck Institut, Germany (Breier et al, 1992) .
  • VEGF 1 ⁇ S was inserted into the Bam HI site of pH ⁇ APr-1-neo ( Figure 1) (Gunning et al, 1987) . This cloning was performed via pGem 7Zf(4-) (Promega, Madison,
  • VEGF-pH ⁇ APr-1-neo clone was identified by Eco RI digeetion (Promega, Madison, WI, USA) .
  • VEGF-pH ⁇ APr-1-neo DNA was prepared using the Qiagen Plasmid Midi Kit (Qiagen GmbH, Hilden, Germany) . The extraction was carried out as described in the manufacturer's protocol, and the reeulting pellet wa ⁇ re ⁇ u ⁇ pended in 500 ⁇ l TE buffer (10 mM Trie HCL, pH 8.0, 1 mM EDTA).
  • Lipofectin Gibco BRL, Gaithersburg, MD, USA
  • the cells were incubated at 37°C overnight in a humidified environment and 5% C0 2 , then 4 ml MEM with 10% FCS and P/S was added. The cells were re-incubated for 24 hours before 1 mg/ml Geneticin (Gibco BRL, Gaithersburg, MD, USA) wa ⁇ included in the cell culture medium. After one week a ⁇ erie ⁇ of discrete colonies was selected, and grown in 1 mg/ml Geneticin until established. The concentration of Geneticin wa ⁇ then decrea ⁇ ed to 300 ⁇ g/ml cell culture medium.
  • Geneticin Gabco BRL, Gaithersburg, MD, USA
  • a control cell line consisting of 407A cells transfected with pH ⁇ APr-1-neo only (407A-pH ⁇ APr-l-neo) was also produced using Lipofectin. Both cell lines were maintained in MEM containing 10% FCS, P/S and 300 ⁇ g/ml Geneticin.
  • Primers were selected to allow ⁇ pecific amplification of transfected mouse VEGF l ⁇ 5 , without background amplification of human VEGF 1C5 from the human 407A cell line.
  • the sequences of mouse VEGF 165 and human VEGF 1(5 as listed on the GenBank database were compared using the IBI Pustell Analy ⁇ is Software (IBI Ltd, Cambridge, England) . 19mer region ⁇ which were less than 70% homologous with human VEGF 1C5 were selected from mouse VEGF 165 .
  • Primer sequence ⁇ were: "VEGFM01”, 115-134 bp on mou ⁇ e VEGF l ⁇ s , 5' -AGG AGA GCA GAA GTC CCA T; "VEGFM02", 300- 318 bp on mou ⁇ e VEGF l ⁇ 5 5'-CGT CAG AGA GCA ACA TCA C. Analysis of primer sequences by the Basic Local Alignment Search Tool (BLAST, National Centre for Biotechnology
  • Cells were harvested using 0.25% trypsin/0.05% EDTA. Samples of 2 x IO 6 cells were collected and washed with PBS, then incubated for 3 hours, 37°C, in the presence of 100 ng/ml Proteinase K (Boehringer Mannheim, Mannheim, Germany) and 0.5% w/v Sodium Dodecyl Sulphate (SDS) (BDH Chemicals Australia Pty Ltd, Kilsyth, VIC, Australia) . D ⁇ A wa ⁇ isolated by phenol/chloroform extraction and sodium acetate/ethanol precipitation. DNA pellets were resuspended in 100 ⁇ l TE buffer.
  • SDS Sodium Dodecyl Sulphate
  • PCR reagents including Ultra Pure Water, were obtained from Biotech International Ltd. (Bentley, WA, Australia) .
  • the PCR reaction mixture consisted of 5 ⁇ l 5X Polymerisation Buffer, 25 mM MgCl 2 , 1U Tt Plus DNA Polymerase, 50 ng VEGFMOl, 50 ng VEGFM02 and Ultra Pure Water to 25 ⁇ l. 1 ⁇ l of each DNA sample was used for PCR.
  • a positive control containing 20 ng VEGF-pH ⁇ APr-1-neo DNA, and a negative control containing Ultra Pure Water in the place of DNA were included.
  • PCR reactions were carried out using a Perkin Elmer GeneAmp PCR System 2400 Thermocycler (Perkin-Elmer Corporation, Norwalk, CT, USA) . Cycles used were 1 cycle of 92°C for 5 minutes, 55°C for 1 minute, 74°C for 1 minute; 35 cycles 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 product ⁇ were eiectrophoresed on a 2% agarose gel, and visuali ⁇ ed by ethidium bromide staining.
  • RT PCR Reverse Transcription PCR
  • RNA wa ⁇ extracted using RNAzolB Biotecx Laboratories Inc., Houston, Texa ⁇ , USA.
  • the procedure wa ⁇ carried out a ⁇ de ⁇ cribed in the manufacturer's protocol, with RNA being extracted directly from confluent 25 cm 3 flasks of cells (4 x IO 6 cells per flask) .
  • the resulting pellets were resuspended in 50 ⁇ l Diethyl Pyrocarbonate (DEPC) (BDH Ltd, Poole, Dorset, England) treated water.
  • RT PCR was performed using the GeneAmp Thermostable rTt Reverse Transcriptase RNA PCR Kit
  • the RT PCR positive control contained 20 ng of VEGF-pH ⁇ APr-1-neo DNA. The negative control received Ultra Pure Water in the place of RNA. Control ⁇ for DNA contamination were produced by the addition of rTt DNA Polymerase after completion of the Reverse Transcription step.
  • RT PCR products were precipitated using sodium acetate/ethanol. Samples were washed in 70% ethanol and resuspended in TE buffer to 1/5 the PCR reaction volume. PCR products were eiectrophoresed on a 2% agarose gel and visualised by ethidium bromide staining.
  • VEGF l ⁇ 5 was successfully cloned into the Bam HI site of pH ⁇ APr-1-neo. The identity of the clone was confirmed using a Bam HI digest which yielded two fragments of 10.0 kb, corresponding to pH ⁇ APr-1-neo, and 656 bp, corresponding to mouse VEGF 165 . Eco RI digestion of the VEGF-pH ⁇ Apr-1-neo clone produced two fragments of 5.7 kb and 5.0 kb, confirming that VEGF was in the sense orientation. VEGF-pH ⁇ Apr-1-neo was transfected into the 407A cell line using Lipofectin.
  • mice VEGF 1SS DNA in the transfected 407A cell line was confirmed using DNA PCR.
  • DNA was extracted from VEGF-pH ⁇ Apr-1-neo transfected 407A colonies, along with DNA from the control 407A-pH ⁇ Apr-l-neo cell line.
  • PCR of the VEGF- ⁇ H ⁇ Apr-1-neo transfected 407A DNA re ⁇ ulted in the production of a 200 bp DNA fragment in every colony te ⁇ ted. This fragment was the size predicted from the position of the primers on the mouse VEGF 165 gene, and agreed with the fragment size produced from the VEGF-positive control.
  • RNA samples were shown to be free of contaminating DNA by omission of the cDNA synthesis step during RT PCR. The re ⁇ ults are shown in Figure IIB. RT PCR using untransfected 407A RNA did not produce any ⁇ ignal.
  • the assay was performed as described in Example 8.
  • CEC adhered to the Matrigel support within 1 hour of seeding. After 2 to 3 hours of culture, the CEC had migrated rapidly to form a reticular network of aligned cells, and subsequently began to form capillary-like structures on the surface of Matrigel. By 24 hours the CEC had the appearance of an anastomosing network, which is typical of vascular tubules.
  • the quantitative analysis of tube formation obtained from computer images, i ⁇ summarised in Figure 13.
  • RPE- VEGF Vascular Endothelial Growth Factor
  • VEGF i ⁇ a highly conserved molecule which is highly homologous between different ⁇ pecies.
  • a murine VEGF cDNA clone, available in our laboratory, is u ⁇ ed to ⁇ creen the human RPE cDNA library in order to identify the full length human RPE-VEGF clone. Positive clones are analysed by restriction enzyme analysis and finally by DNA sequencing. Full length RPE-VEGF clones are analysed to elucidate their genomic structure (initiation sequences, start and stop codons, putative exons etc.).
  • Clones carrying the full length RPE-VEGF are analysed for the expres ⁇ ion of VEGF protein with in vitro translation. The identified clones are used to derive the anti-sense molecule for insertion into the vehicle system, and for the selection of the anti-sense oiigonucleotides.
  • Anti- ⁇ en ⁇ e DNA technology enables the sequence- specific inhibition of target molecules without affecting the non-targeted functions of the cell.
  • a panel of 16 to 19-mer oiigonucleotides, 100% complementary to parts of the VEGF gene, is selected from the 5' and 3' ends of the DNA ⁇ equence.
  • Sense and scrambled sequence ⁇ are al ⁇ o u ⁇ ed as control.
  • Phosphorothioate-protected oiigonucleotides are syntheeized on a DNA ⁇ ynthe ⁇ izer and subjected to purification.
  • VEGF-pAd.RSV for Homologous Recombination VEGF 165 in Bluescript IIKS (Stratagene) wa ⁇ u ⁇ ed to produce Kpnl sites.
  • Kpn I restriction enzyme ⁇ ite ⁇ were obtained at both the 5' and 3' ends of VEGF 165 by subcloning.
  • VEGF 165 wa ⁇ removed from Bluescript II KS using an Xba I (5' cut)/Kpn I (3 1 cut) restriction enzyme digest, and cloned into pGem 7Zf(4*) (Promega).
  • a Kpn site was then added to the 3' end by cloning VEGF 165 into pGe 3Zf(+) (Promega), using a Hind III (3' cut)//Xba I (5' cut) digest.
  • VEGF was removed from pGem 3Zf(4-) with a Kpn I restriction enzyme digest and cloned into the unique Kpn I site on pAd.RSV.
  • Thi ⁇ pla ⁇ mid contains two segments of the adenovirus genome separated by cloning sites for the insertion of foreign DNA.
  • the resulting clones were screened for the presence of sense and antisense clones, which were used in homologous recombination (VEGF-pAd.RSV) .
  • VEGF 165 was shown to be pre ⁇ ent and intact within pAd.RSV by restriction enzyme cleavage and sequencing.
  • VEGF-pAd.RSV DNA was prepared using the Qiagen Plasmid Midi Kit, as per the manufacturer's instructions. The DNA was linearised by Xmn I restriction enzyme digestion, purified by sodium acetate/ethanol precipitation and resuspended in TE buffer. The DNA was then stored at -20°C until required. Generation of Ad.RSV-VBGF or Ad.RSV-aVBGF by Homologous Recombin tion
  • the adenovirus type 5 deletion mutant, dl324, was used to generate the recombinant adenovirus carrying VEGF.
  • dl324 is unable to replicate due to deletion of the El region and, in addition, carries a partial deletion in the E3 region.
  • this mutant was propagated in 293 cells, which supply the missing El region in trans.
  • the linearised plasmid DNA pAdRSV-VEGF or pAd.RSV-aVEGF was co-transfected into 293 cells with dl324 viral DNA which has had its extreme left- hand sequence ⁇ removed by a Clal digeetion. Thi ⁇ reduces the infectivity of dl324 and allows for easier identification of recombinants.
  • screening of the resultant plaques yielded recombinant AdRSV-VEGF virus carrying VEGF in sense or antisense orientation.
  • Example 14 Expres ⁇ ion of Target Molecules
  • the vehicle described in Example 14 is suitable for long-term treatment in that it provides temporary (maximum one year) expres ⁇ ion of the anti- ⁇ en ⁇ e VEGF DNA molecule, and consequent protection against neovascularisation.
  • AAV adeno-associated virus
  • Adeno-associated viruses are non-pathogenic, are able to infect non-dividing cells, and have a high frequency of integration.
  • AAV-2 which is a replication defective parvovirus which can readily infect other cell ⁇ ⁇ uch a ⁇ RPE cell ⁇ , and integrate into the genome of the ho ⁇ t cells. Recent characterisation has - 51 - revealed that AAV-2 specifically targets the long arm of human chromosome 19.
  • AAV construct ⁇ use varying promoter sequences in combination with a reporter gene.
  • the expres ⁇ ion of the reporter gene mRNA is detected with PCR amplification or in situ PCR, and the integration of the reporter gene is identified by chromosomal analysis of RPE cells.
  • the reporter gene is replaced by anti-sense VEGF DNA.
  • the new construct i ⁇ co-transfected with the complementing plasmid (pAAV/ad) into kidney 293 cells previously infected with adenovirus type 5 to make the rAAVaVEGF construct.
  • the conetruct produced i ⁇ u ⁇ ed to infect RPE cell ⁇ , and the expression of anti-sense VEGF is detected by PCR amplification.
  • VEGF-COS culture system
  • the toxicity of increasing concentrations of oiigonucleotides on COS cells is assessed with the trypan blue assay.
  • the proliferation of COS cells is monitored with or without increasing concentrations of oiigonucleotides.
  • the inhibition of the expres ⁇ ion of VEGF in control ⁇ and in cultures maintained in the preeence of anti- ⁇ en ⁇ e oiigonucleotides is monitored by Northern and Western blot analyses, immunocytochemistry and by a quantitative immunoassay.
  • RPE cells are cultured in hypoxic conditions and the up-regulation of VEGF expres ⁇ ion i ⁇ monitored in the preeence of increa ⁇ ing concentrations of oiigonucleotides for an extended period of time. Toxicity, proliferation assay and the monitoring of VEGF expression are performed a ⁇ described above.
  • CEC cells are cultured in normal and hypoxic conditions with or without increasing concentration of oiigonucleotides.
  • the effect of anti ⁇ sense oligonucleotide-mediated inhibition of VEGF expres ⁇ ion on tube formation i ⁇ analy ⁇ ed RPE/CEC dual cultures produced in normal and hypoxic conditions will be subjected to similar tests. The same model systems are used to assess the long-term and permanent agents of the invention.
  • SRNV Membrane
  • Pigmented rate (Dark Agouti, DA) weighing between 175 and 250 g were anae ⁇ thetized with an intramuscular injection of xylazine hydrochloride (2 mg/kg of body weight), acepromazine maleate (0.5 mg/kg), and ketamine hydrochloride (100 mg/kg of body weight) and given topical 0.5% proparacaine hydrochloride. The pupils were dilated with 2.5% phenylephrine hydrochloride.
  • Laser parameters used were a ⁇ follows: a spot size of 100 ⁇ m, a power of 150 mW, and an exposure duration of 0.1 s.
  • An attempt was made to break Bruch's membrane, as clinically evidenced by central bubble formation with or without intraretinal or choroidal hemorrhage.
  • a treatment power of 150 mW most consi ⁇ tently produced this effect.
  • Neovascularisation can be induced using pocket implants in the choroid or the subretinal layer.
  • One of the disadvantages of these models is that the process of neovascularisation might not follow the same biochemical steps which naturally occur in humans suffering from ARMD.
  • To overcome these difficulties we use an animal model in which choroidal neovascularisation is induced by VEGF overexpression in the RPE cells.
  • VEGF vascular endothelial growth factor
  • Tests are conducted to demonstrate the expres ⁇ ion of a VEGF expression over a period of one year.
  • VEGF down-regulation is monitored and immunohistochernistry is used to demonstrate the down- regulation of VEGF expres ⁇ ion in a cell-specific manner.
  • immunohistochernistry is used to demonstrate the down- regulation of VEGF expres ⁇ ion in a cell-specific manner.
  • choroidal neovascularisation is monitored by histology and angiography.
  • the present invention is particularly useful in the study, treatment or prevention of age-related macular degeneration, by virtue of the successful adenoviral gene transfer to the RPE.
  • the higher degree of gene expre ⁇ ion in the HRPE7 cells may indicate the ability of RPE cell ⁇ to phagocytoee large molecules and hence increase the uptake of adenovirus.
  • the level of expression of the transgene may also be increased by increasing the time of expo ⁇ ure or the viral titre.
  • viru ⁇ solutions could be aspirated from subretinal blebs or the vitreou ⁇ after 24 hours) .
  • the titre/expres ⁇ ion curves ( Figure 5) also show that there was a difference between the cell ⁇ , with RPE cell ⁇ beginning to express highly at a lower concentrations. Once again, low concentration could be used to preferentially target RPE cells. A combination of lower titres for less than 24 hours would combine the two effects and provide targeted delivery.
  • the present invention may al ⁇ o be u ⁇ ed in conjunction with adjuvants to keep viral toxicity to a minimum by reducing the titre required to effect gene transfer and expre ⁇ ion.
  • HA adenoviral gene transfer using HA. This wa ⁇ the case in both phagocytic and non-phagocytic cell lines.
  • the advantage of HA is its presence as a normal component of human vitreous and extracellular matrix, and its long history of therapeutic acceptance as a viscoelastic aid to surgery.
  • the important feature of HA in terms of its acting a ⁇ a potential adjuvant is its ability to bind cell membranes and other molecules simultaneously.
  • the HA molecule can bind adenovirus and the cell membrane at the same time, and therefore increase the contact time or concentration of virus in the vicinity of the cell membrane using this mechanism.

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Abstract

Cette invention porte sur des méthodes de thérapie génique ainsi que sur les compositions correspondantes, y compris une thérapie anti-sens. Dans un mode de réalisation, les compositions contiennent de l'acide hyaluronique afin de stimuler l'absorption d'acide nucléique par les cellules cibles. A titre d'exemple, cette invention trouve une application dans le traitement de maladies rétiniennes causées par une néoformation de vaisseaux sanguins.
PCT/AU1996/000664 1995-10-23 1996-10-22 Acide hyaluronique utilise comme porteur d'adn pour une therapie genique et adn anti-sens du facteur de croissance de l'endothelium vasculaire pour le traitement d'une vascularisation anormale de la retine WO1997015330A1 (fr)

Priority Applications (5)

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JP9516137A JP2000507915A (ja) 1995-10-23 1996-10-22 遺伝子療法のためのｄｎａ担体としてのヒアルロン酸および異常な網膜血管新生を治療するためのｖｅｇｆアンチセンスｄｎａ
EP96934189A EP0859636A4 (fr) 1995-10-23 1996-10-22 Acide hyaluronique utilise comme porteur d'adn pour une therapie genique et adn anti-sens du facteur de croissance de l'endothelium vasculaire pour le traitement d'une vascularisation anormale de la retine
NZ320006A NZ320006A (en) 1995-10-23 1996-10-22 An adjuvant composition comprising hyaluronic acid for gene therapy either in the sense or antisense direction
AU72664/96A AU721060B2 (en) 1995-10-23 1996-10-22 Hyaluronic acid as DNA carrier for gene therapy and VEGF antisense DNA to treat abnormal retinal vascularization
KR1019980702900A KR19990066967A (ko) 1995-10-23 1996-10-22 비정상적인 망막 혈관형성을 치료하기 위한 유전자 치료법과vegf 안티센스용 dna 운반체로써 사용되는 히알루론산

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AUPN6161A AUPN616195A0 (en) 1995-10-23 1995-10-23 Method and composition for treatment of ocular diseases
AUPN6161 1995-10-23
AUPN9047A AUPN904796A0 (en) 1996-04-01 1996-04-01 Method and composition for treatment of ocular diseases
AUPN9047 1996-04-01

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Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998017320A1 (fr) * 1991-07-03 1998-04-30 Hyal Pharmaceutical Corporation Utilisation de hyaluronan dans la therapie genique
WO1998013024A3 (fr) * 1996-09-27 1998-06-18 Christopher Marriott Systeme hyaluronique d'administration de medicaments
WO1999024056A1 (fr) * 1997-11-12 1999-05-20 The Victoria University Of Manchester Regulation de l'angiogenese oculaire
WO2001097857A1 (fr) * 2000-06-20 2001-12-27 Sumitomo Pharmaceuticals Company, Limited Preparations destinees au transfert d'oligonucleotides
WO2001048231A3 (fr) * 1999-12-28 2002-04-04 Novartis Ag Procede de mise en oeuvre d'une expression persistante de transgenes
US6448093B1 (en) 1999-11-15 2002-09-10 Jederstroem Gustaf Hydrophobe biomolecular structure
EP1480656A1 (fr) * 2002-02-15 2004-12-01 Research Development Foundation Tranduction adenovirale induite par l'acide hyaluronique
WO2004105784A1 (fr) * 2003-05-29 2004-12-09 The University Of Manchester Agonistes des slrp de classe iii servant a reduire la formation de vaisseaux sanguins
US6875753B1 (en) 1996-03-14 2005-04-05 The Governors Of The University Of Alberta Methods for cell mobilization using in vivo treatment with hyaluronan (HA)
US7867490B2 (en) 2005-06-30 2011-01-11 Vib Vzw Treatment of liver cirrhosis and its complications
US8742091B2 (en) 2001-06-20 2014-06-03 Dainippon Sumitomo Pharma Co., Ltd. Method of promoting nucleic acid transfer
US8758748B2 (en) 2005-03-24 2014-06-24 Thrombogenics N.V. Anti-angiogenic therapy
US9089552B2 (en) 2011-12-01 2015-07-28 Thrombogenics Nv Improving trabeculectomy outcome by administering an anti-placental growth factor antibody
US10106601B2 (en) 2008-10-02 2018-10-23 Vib Vzw Inhibition of PLGF to treat philadelphia chromosome positive leukemia

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP5255277B2 (ja) * 2004-10-05 2013-08-07 ユニバーシティ オブ ピッツバーグ オブ ザ コモンウェルス システム オブ ハイヤー エデュケーション 網膜疾患の検診装置
CN1742623A (zh) * 2005-07-13 2006-03-08 凌沛学 透明质酸磷脂复合物及其制备方法
KR101420751B1 (ko) * 2011-12-12 2014-07-17 서강대학교산학협력단 망막 질환 치료제 후보물질의 스크리닝 방법

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU6090386A (en) * 1985-11-29 1987-06-04 Genzyme Biosurgery Corporation Drug delivery systems based on hyaluronan, derivatives thereof and their salts and methods of producing same
WO1990002774A1 (fr) * 1988-09-09 1990-03-22 Pier Auge Gel aqueux a base d'acide hyaluronique et d'acide desoxyribonucleique utilisable en cosmetique, et procede de preparation
WO1992013063A1 (fr) * 1991-01-18 1992-08-06 Oncogene Science, Inc. Procedes permettant de moduler par transcription l'expression de genes de facteurs de croissance et de genes de recepteurs de facteurs de croissance
AU5227493A (en) * 1989-09-21 1994-03-03 Jagotec Ag Treatment of conditions and disease
WO1995004142A2 (fr) * 1993-07-27 1995-02-09 Hybridon, Inc. Inhibition par des oligonucleotides antisens de l'expression du facteur de croissance endotheliale vasculaire
WO1996000286A1 (fr) * 1994-06-27 1996-01-04 Toagosei Co., Ltd. Compose d'acide nucleique antisens
WO1996023065A2 (fr) * 1995-01-26 1996-08-01 Hybridon, Inc. Inhibition de la neoformation de vaisseaux sanguins au moyen d'oligonucleotides specifiques de vegf
WO1996027006A2 (fr) * 1995-03-02 1996-09-06 Hybridon, Inc. Oligonucleotides antisens specifiques du facteur de croissance de l'endothelium vasculaire humain

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU6090386A (en) * 1985-11-29 1987-06-04 Genzyme Biosurgery Corporation Drug delivery systems based on hyaluronan, derivatives thereof and their salts and methods of producing same
WO1990002774A1 (fr) * 1988-09-09 1990-03-22 Pier Auge Gel aqueux a base d'acide hyaluronique et d'acide desoxyribonucleique utilisable en cosmetique, et procede de preparation
AU5227493A (en) * 1989-09-21 1994-03-03 Jagotec Ag Treatment of conditions and disease
WO1992013063A1 (fr) * 1991-01-18 1992-08-06 Oncogene Science, Inc. Procedes permettant de moduler par transcription l'expression de genes de facteurs de croissance et de genes de recepteurs de facteurs de croissance
WO1995004142A2 (fr) * 1993-07-27 1995-02-09 Hybridon, Inc. Inhibition par des oligonucleotides antisens de l'expression du facteur de croissance endotheliale vasculaire
WO1996000286A1 (fr) * 1994-06-27 1996-01-04 Toagosei Co., Ltd. Compose d'acide nucleique antisens
WO1996023065A2 (fr) * 1995-01-26 1996-08-01 Hybridon, Inc. Inhibition de la neoformation de vaisseaux sanguins au moyen d'oligonucleotides specifiques de vegf
WO1996027006A2 (fr) * 1995-03-02 1996-09-06 Hybridon, Inc. Oligonucleotides antisens specifiques du facteur de croissance de l'endothelium vasculaire humain

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CHEMICAL REVIEWS, 1990, Vol. 90, No. 4, UHLMAN and PEYMAN, "Antisense Oligonucleotides", pages 544-579. *
See also references of EP0859636A4 *

Cited By (26)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6475795B1 (en) 1991-07-03 2002-11-05 Meditech Research, Ltd. Use of hyaluronan in gene therapy
WO1998017320A1 (fr) * 1991-07-03 1998-04-30 Hyal Pharmaceutical Corporation Utilisation de hyaluronan dans la therapie genique
US7446100B2 (en) 1996-03-14 2008-11-04 The Governors Of The University Of Alberta Methods for cell mobilization using in vivo treatment with hyaluronan (HA)
US6875753B1 (en) 1996-03-14 2005-04-05 The Governors Of The University Of Alberta Methods for cell mobilization using in vivo treatment with hyaluronan (HA)
US6890901B2 (en) 1996-09-27 2005-05-10 Jagotec Ag Hyaluronic drug delivery system
WO1998013024A3 (fr) * 1996-09-27 1998-06-18 Christopher Marriott Systeme hyaluronique d'administration de medicaments
WO1999024056A1 (fr) * 1997-11-12 1999-05-20 The Victoria University Of Manchester Regulation de l'angiogenese oculaire
US7338931B2 (en) 1999-11-15 2008-03-04 Gustaf Jederstrom Hydrophobic biomolecular structure
US6448093B1 (en) 1999-11-15 2002-09-10 Jederstroem Gustaf Hydrophobe biomolecular structure
US6926910B2 (en) * 1999-11-15 2005-08-09 Jederstroem Gustaf Hydrophobe biomolecular structure
WO2001048231A3 (fr) * 1999-12-28 2002-04-04 Novartis Ag Procede de mise en oeuvre d'une expression persistante de transgenes
JP2003522133A (ja) * 1999-12-28 2003-07-22 ノバルティス アクチエンゲゼルシャフト 持続性導入遺伝子発現を実現する方法
CN1310679C (zh) * 1999-12-28 2007-04-18 诺瓦提斯公司 获得持续的转基因表达的方法
WO2001097857A1 (fr) * 2000-06-20 2001-12-27 Sumitomo Pharmaceuticals Company, Limited Preparations destinees au transfert d'oligonucleotides
US8742091B2 (en) 2001-06-20 2014-06-03 Dainippon Sumitomo Pharma Co., Ltd. Method of promoting nucleic acid transfer
US7144870B2 (en) 2002-02-15 2006-12-05 Research Development Foundation Hyaluronic acid mediated adenoviral transduction
EP1480656A4 (fr) * 2002-02-15 2007-07-18 Res Dev Foundation Tranduction adenovirale induite par l'acide hyaluronique
EP1480656A1 (fr) * 2002-02-15 2004-12-01 Research Development Foundation Tranduction adenovirale induite par l'acide hyaluronique
US7358224B2 (en) 2003-05-29 2008-04-15 The University Of Manchester Class III SLRP agonists for the reduction of blood vessel formation
US7910567B2 (en) 2003-05-29 2011-03-22 The University Of Manchester Opticin nucleic acid administration reduces blood vessel formation
WO2004105784A1 (fr) * 2003-05-29 2004-12-09 The University Of Manchester Agonistes des slrp de classe iii servant a reduire la formation de vaisseaux sanguins
US8758748B2 (en) 2005-03-24 2014-06-24 Thrombogenics N.V. Anti-angiogenic therapy
US9085617B2 (en) 2005-03-24 2015-07-21 Thrombogenics N.V. Anti-angiogenic therapy
US7867490B2 (en) 2005-06-30 2011-01-11 Vib Vzw Treatment of liver cirrhosis and its complications
US10106601B2 (en) 2008-10-02 2018-10-23 Vib Vzw Inhibition of PLGF to treat philadelphia chromosome positive leukemia
US9089552B2 (en) 2011-12-01 2015-07-28 Thrombogenics Nv Improving trabeculectomy outcome by administering an anti-placental growth factor antibody

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