AU693782B2 - Recombinant adenoviruses and use thereof in gene therapy for treating eye diseases - Google Patents

Abstract

The use of defective recombinant adenoviruses containing an inserted gene for preparing a pharmaceutical useful for treating eye diseases is disclosed.

Description

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P:\OPERLR\61444-94.149- 29/5/98 -1- RECOMBINANT ADENOVIRUSES AND THEIR USE IN GENE THERAPY FOR THE TREATMENT OF OCULAR PATHOLOGIES The present invention relates to new recombinant viruses, to their preparation and to their use in gene therapy for the transfer of genes to the eye and their expression therein. More especially, the present invention relates to defective recombinant viruses and to their use for the treatment of ocular pathologies.

The treatment of ocular pathologies, and in particular of hereditary diseases, constitutes a problem which has not been solved at the present time. Among these pathologies, there may be mentioned, for example, retinitis pigmentosa, which results from adverse genetic modification and for which no treatment is currently available. Moreover, no suitable treatment is at present available either for non-hereditary pathologies such as postinflammatory complaints (retinal degeneration, and the like). In particular, while an effo't 15 is made to act preventively, in particular using corticoids, no satisfactory means for treating these complaints is currently available.

It is hence important to be able to have tools available permitting a specific, effective and localised treatment of ocular pathologies. The present invention provides an advantageous approach to this Cs, CC Sy

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2 problem, by demonstrating the possibility of treating ocular pathologies by gene therapy.

Gene therapy consists in correcting a deficiency or an abnormality (mutation, aberrant expression, and the like) by introduction of genetic information into the affected cell or organ. This genetic information may be introduced either in vitro into a cell extracted from the organ, the modified cell then being reintroduced into the body, or directly in vivo into the appropriate tissue. In this second case, different techniques exist, including various transfection techniques involving complexes of DNA and DEAE-dextran (Pagano et al., J.Virol. 1 (1967) 891), of DNA and nuclear proteins (Kaneda et al., Science 243 (1989) 375), of DNA and lipids (Felgner et al., PNAS 84 (1987) 7413), the use of liposomes (Fraley et al., J.

Biol. Chem. 255 (1980) 10431), and the like. More recently, the use of viruses as vectors for the transfer of genes has appeared as a promising alternative to these physical transfection techniques.

In this connection, different viruses have been tested for their capacity to infect certain cell populations.

These comprise, in particular, retroviruses (RSV, HMS, MMS, and the like), the HSV virus, adeno-associated i 25 viruses and adenoviruses.

However, hitherto, none of these vectors has been used or described as being usable for the transfer of genes to the eye. The present invention constitutes the f irst dmonstration that it is possible to trea~t ocular pathologies by gene therapy.

A first subject of the invention lies In a method of treating ocular pathologies which comprises the ocular administration of a defective recombinant adenovirus containing an inserted gene.

More especially, defective recombinant viruses derived from viruses capable of infecting and of expressing an inserted gene in the cells of the eye, without giving rise to cytopathological Pha.nomend Or pathogenic effects, are uised according to the present invention.

The present invention is based more especially on the demonstration that adenovirus type viruses are capable of transferring desired genes to the eye and of expressing them therein. The examiples presented later show that et4enoviruses are capable, depending on the mode of administration, of transferring genes to the corneal endotheliuim, to photoreceptor cello, to calls of the optic nerve, to bipolar cells, and the like, effectively,4 for a considerable period and without a cytopathological.

effect. Moreover, in view of the relative ease of access to the different compartments of the eye by microsurgery (nicroinjection), as well. as of the existence of natural barriers in this organ (Deoceniet's membrane, Driuch' u membrane, lens, and the like)e, the present Invention advantageously enables a very targeted transfer of genes to be performed, in accordance with the pathology to be treated. The results presented also show that the expression of a desired gene is stable over a long period (no loss of activity at 50 days).

The to= "detective virus" denotes an adeinovi.rus incapable of replicating autonomously in the target'cell.

Generally, the defective recombinant adenovirus lacks the regions of its genome which are neeed for its replication in the inmfected cell. Theme regions may be either removed (wholly or partially), or rendered non-functional, or replaced by other sequences, and in particular by the inserted gene. Preferably, the defective virus nevertheless retains the sequences of its genome which are needed for encapsidation of the viral particles.

Adenoviruses exist in the form of different serotypes, whose structure and properties vary somewhat.

Nevertheless, these viruses are not pathogenic for man, and in particular non-±mmunosuppressed subjects. In the method of treatment of the invention it In preferred that the defective recombinant adenovirus is a type Ad 2 1* adeziovirus. It is also preferred that defective -Cott, recombinant adenovius is a type Ad 5 adeanovirus. In the case of adenovicusem Ad 5, the sequences needed for replication are the ZIA and Hi regions.

For the purposes of the present ionvention, the tezu "inserted gonam denotes any DNA sequence introduced into the recombinant virus, whose expression in the target call is sought. Typically the inserted gene codes f or a protein or a protein fragment.

It can be, in particular, one (or nore) structural gene(s) coding for a (same) protein(m) or for a portion of a (aome) protein(s). The protein or protein portion thus encoded can be a protein which is homologous with cempect to the target cell (that is to may a protein which in normally expressed in the target cell when the jI r latter does not exhibit any Pathology), Or aL Proteini which is haterologous with respect to the gai.d Cell. zn the former case, expression of the Protein makes it possible, for example, to remedy an '4 9*~

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I i -p 6 insufficient expression in the cell or the expression of a protein which is inactive or poorly active as a result of a modification, or alternatively to overexpress the said protein. In the second case, the protein expressed can, for example, supplement or supply an activity which is deficient in the cell, enabling it to combat a pathology.

Among inserted genes for the purposes of the present invention, there may be mentioned, more especially: genes involved in ocular genetic pathologies, i genes coding for growth factors, cytokines or neurotrophins: the protective or curative role of the expression product of these genes in different ocular pathologies has been demonstrated, and in particular on the deterioration of photoreceptor cells under the effect of light (Lavail et al., PNAS 89 (1992) 11249), genes for regulatory factors (transcription factors, translation factors), genes coding for enzymes, genes coding for proteins having anticancer properties, such as interferons, tumour necrosis factors, and the like, or alternatively, genes coding for antigens permitting a local vaccination (protection) against an eye infection.

i 1 7 As specific, but non-limiting, examples, there may be mentioned: j the ornithine aminotransferase gene involved in gyrate atrophy (Akaki et al., J. Biol.

Chem. 267 (18) (1992) 12950), the rhodopsin gene involved in a form of retinitis pigmentosa (Dryja et al., Nature 343 (1990) 364), the RDS peripherin gene involved in a form of retinitis pigmentosa (Farrar et al., Nature 354 (1991) 478), the tyrosinase gene involved in type B1 oculocutaneous albinism (Giebel et al., Am. J.Hum.

Genet. 48 (1991) 1159), the mitochondrial NDI gene involved in Leber's disease (Howell et al., Am. J. Hum. Genet. 48 (1991) 935), S the gene for the j subunit of cGMP phosphodiesterase, which enables retinal degeneration to be slowed down (Lem et al., PNAS 89 (1992) 4422), the rab geranylgeranyl transferase gene, the deficiency of which appears to be associated with a retinal degeneration in choroidermia (Seabra et al., Science 259 (1993) 377), the basic fibroblast growth factor (bFGF) gene, capable of retarding the degeneration of the photoreceptor cells which is observed in some i,: i I tot

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S S C t S tt .5 'itt CC C hereditary retinal dystrophies (Faktorovich at Z9ature 347 (1L990) 83), the iaterleukin-8 gene, which enables a neovaocularization to be indu~ced in the cornea (b'iter at al., Am. j. pathol. 141. (1992) 1279).

The defecti.ve recombinant adenovirus used in the method of treatment of the invention may be one in which the inserted gene in an antisense sequlence. Thus the term Rinnerted gene"f also denotes antigense ,sequences, whose expression in the target cell enables the expression of genes or the transcription of cellular zLRA8 to be controlled. Such sequences can, for example, be transcribed in the target cell into INAs complseentary to cellular mRN~s and can thus block their translation into Protein.

Generally, the inserted gene also comprises sequences permitting its expression in the infected cell.

The soquences in question can be ones which are naturally responsible for expression of the said gone when these sequences are capable of functioning in the infected cell. They can also e sequences of different origin (responsible for the expression of other proteins, or even synthetic sequences). In particular, they can be sequences originating f rom the genome of the cell which it is desired to infect, or from the genome of the vi.rus used, for examIle the promoters of the ZIA' 2MP gel .es, and the like. in addition, these expveuuion sequences may be modified by the addition og activation, regulatory, and the like, sequences. Moreover, when the inserted gene does not contain expression sequences, it nay be inserted into the genome of the defective virus downstream of such it. I cici c cc tic' cc ccc c V cc cc cc ti t it .c t it it t C C tic a sequence.

In what follows, the constructiou and use of defective recombinant adenoviruoes are described in greater detail.

Defective recombinant adenovixtlses may be prepared by homologous recombination between an adenovirus and a plasmid carrying, inter aim, the gene which it in desired to insert. .Xomologous recombiuatIzLz taken place after cotranhfaction of the maid adenovirux and maid piasnid into a suitable cell line. The call line used should preferably be transformable by the said elements, as~d (Wi contain the sequences capable of complementing the portion of the genome of the defective adenovirus, preferably in integrated form in order to avoid the risks of recombination. As an example of a line, there may be mentioned the human embryonic kidney line 293 (Gralham et al., a7. Gen. Vi.rol. 36 (1977) 59), which contains, in particular, integrated in its genome, the left-hand portion of the genome of an adenovirus Ad (12 Thereafter, the vectors which have multiplied rAre recovered and purified according to standard techniques of molecular biology.

The defective recombinant virus may be in thie form, of an injection, eye lotion, ophthalmic ointment, and the like. The pharmaeutically acceptable vehicles for such formulationi suitable for ocular administration are, in particular, Saline solutions (monosodium or diadium, phosphate, sodium,, potassium calciva or magnesium croride, and the like, or mixtures of such salts), Soft paraffin, liquid paraffin, and the like.

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in the came ot eye lotiona or ophthalmic ointmentbs, it is understood that the therapeutic applicationo may be more limited on account of a weaker diffusion of the detective recombinant virus.

Xn their use for the treatment of ocular pathologies, the defe~tive recombinant, viruses accor~ding to the invention may be administered according to different modes, and in particular by subretinal injection preceded, where appropriate, by a vitrectomy, or by intravitreous injection, the injections beia single or multiple (see Figure Subretinal injectiou may be carried out selectively iA different compartments A of the eye, and, in particular, injection may be carried 1 out in the vitreous, in the anterior chamiber or in the ix t retrabulbaz space. The t t -s tC Uii 'i 11 results presented in the present application show that these different modes of injection enable the different tissues of the eye, and in particular the corneal endothelium, the photoreceptor cells, the bipolar cells, the ganglion cells or alternatively the cells of the oculomotor muscles, to be infected in a targeted manner.

The doses of virus used for the injection may be adapted in accordance with different parameters, and in particular in accordance with the mode of administration used, the pathology in question, the gene to be expressed or alternatively the period of treatment required. Generally speaking, the recomb. nt adenoviruses according to the invention are formulated and administered in the form of doses of between 104 and 1014 pfu/ml, and preferably 106 to 1010 pfu/ml. The term pfu (plaque forming unit) corresponds to the infectious power of a solution of virus, and is determined by infection of a suitable cell culture and measurement, generally after 48 hours, of the number of plaques of infected cells. The techniques of Sdetermination of the pfu titre of P viral solution are well documented in the literature.

In view of the stability of expression of the inserted gene in the target cell, the present invention should make it possible to treat the majority b- ocular pathologies with few injections.

The present invention thus affords a very r I

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-12effective means for the treatment of ocular pathologies, and in particular those whose mechanisms have been elucidated at molecu~lar level. In particular, the involvement of genes has been demonstrated ini gyrate atrophy, in Norrie's disease (Hum. 3Mol. g~enet. 1. (7) (1992) 461), in retinal degeneration (Bowes at al., P2IAS 86 (1989) 9722) in-Leber's disease, in choroidersuja (Cremers et al., Nqature 347 (1990) 674), in degeneration of photoreceptor cells, in retinitis pigmentosa, in albinism, in Keaziis-Sayre syn~drome (Sbhoffner et al., PNAS 86 (1989) 7952), and the like.

The ocular pathologies may be hereditary pathologies ouch as retinitis piglientosa. The present invention is also for the treatment of acquired adverse modification in the cornea resulting from inflamsmatory di&!orders, poot-inflammatozy retinal complaints, and the like.

The present invention also mak~es possible therapy with proteins or peptides, the use of which via the traditional administration routes is very hypothetical on account of their great sensitivity to the mechanism of degradation and elimination from the body, and problms associated with penetration into the cells.

The use of viruses according to the invention permits the direct expression, within the population of~ targeted cells, of the desired protein or polypeptide, which is hence no longer accessible to the mechanisms mentioned above.

The collective results presented in the present application demonstrate that

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13 recombinant adenoviruses, which are defective for replication, constitute especially advantageous vectors for the transfer of genes in vivo to ocular cells. The experiments carried out show the possibility of a stable long-term expression of genes in these cells. In particular, a stable expression is observed 50 days after injection. Furthermore, the broad spectrum of expression in the different ocular cells also constitutes an especially advantageous result, inasmuch as practically all disorders of the retina (in particular retinitis pigmentosa) affect a large area of the retina.

In addition, this treatment can relate both to man and to any animal such as sheep, cattle, domestic animals (dogs, cats, and the like), horses, fish, and the like.

The present invention is described more completely by means of the examples which follow, which are to be considered as illustrative and non-limiting.

Legend to the figures Fiqure 1: Diagrammatic representation of the eye. C cornea; AC anterior chamber; L lens; V vitreous; I iris; ON optic nerve; R retrobulbar space.

Construction of a defective recombinant adenovirus 1 25 (Ad.RSVGal): SThe general procedure enabling recombinant adenoviruses to be prepared has been described in the Sgeneral part of the description.

1 fl 14 The adenovirus Ad.RSV3Gal is a defective recombinant adenovirus (from which the El and E3 regions have been deleted) obtained by homologous recombination in vivo between the mutant adenovirus Ad-d1324 (Thimmappaya et al., Cell 31 (1982) 543) and plasmid pAd.RSV3Gal (Akli et al., 1993).

Plasmid pAd.RSV#Gal contains, in the 5' 3' orientation, the PvuII fragment corresponding to the left-hand end of the adenovirus Ad 5, comprising: the ITR sequence, the origin of replication, the encapsidation signals and the ElA amplifier; the gene coding for -galactosidase under the control of the RSV (Rous sarcoma virus) promoter; a second fragment of the genome of the adenovirus Ad 5, which permits homologous recombination between plasmid pAd.RSVGal and the adenovirus d1324.

After linearization with the enzyme Clal, plasmid pAd.RSV3Gal and the adenovirus d1324 are cotransfected into the line 293 in the presence of calcium phosphate to permit homologous recombination.

The recombinant adenoviruses thus generated are selected by purification on plates. After isolation, 25 the DNA of the recombinant adenovirus is amplified in the cell line 293, thereby leading to a culture supernatant containing the unpurified recombinant defective adenovirus having a titre of approximately 111 0A to I- 6 11__

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S. 1010 pfu/ml.

The viral particles are generally purified by centrifugation on a caesium chloride gradient according to known techniques (see, in particular, Graham et al., Virology 52 (1973) 456). The adenovirus Ad.RSVPGal is stored at -80 0 C in 20 glycerol. Before injection, the adenovirus suspension is diluted to one third in phosphate buffer PBS.

Injection in vivo Protocol 3- to 7-week-old C57B1/6 mice were anaesthetized with Avertin. 10 7 to 108 pfu of recombinant adenovirus Ad.RSVaGal were then injected into each eye, either in the anterior chamber, or in the vitreous, or in the retrobulbar space (see Figure The animals were sacrificed 7 to 50 days after injection by cervical dislocation, and the eyes were recovered and fixed in liquid nitrogen. Sagittal and coronal sections (10-15 gm) are prepared on a cryostat, then stained in the presence of X-gal to disclose S-galactosidase activity, which may be visualized by the appearance of a blue stain in the nucleus of the infected cells, and counterstained with haematoxylin and eosin.

Injection in the anterior chamber After injection of 108 pfu of adenovirus Ad.RSVGal in the space of the anterior chamber, only Sithe cells of the endothelial layer express

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coona setion (1-15fna arepreare on crostt, 4T~I- *LL 1: 16 -galactosidase activity. On the other hand, the epithelial or stromal cells do not exhibit any staining following such an injection. Furthermore, the labelled (infected) cells are distributed evenly in the endothelial layer, irrespective of the time of administration. This result shows that the present invention enables a gene to be transferred to the endothelial cells of the eye and expressed therein.

Intravitreous injections Intravitreous injections were also carried out, with the object of infecting different cell types of the retina. In contrast to the uniform distribution in the endothelial cells after injection in the anterior chamber space, the distribution of positive (infected) cells after intravitreous injection is limited to the half-retina corresponding to the point of injection. The large size of the lens and the viscosity characteristics of the vitreous humour might explain this confined expression. However, when temporal and nasal injections are performed simultaneously, the cells of both half-retinas are infected. Hence these results show that it is possible to transfer a gene to the retina and express it therein. They also show that, depending on the pathology to be treated and, in particular, depending on its distribution on the retina, it is possible to target the transfer on one half-retina only.

Three nuclear layers, corresponding to the 4

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i C 17 ganglion, bipolar and photoreceptor cells, also exhibit an intense staining at three weeks (age at which development of the retina is complete), as well as in adult mice. Despite the presence of the signal permitting the nuclear localization of the LacZ protein, the labelling (and hence infection) of some cells at the injection site is so intense that the staining diffuses into the cytoplasm. For this reason, the layer of nerve fibre corresponding to the axons of the labelled nuclei (which converge to form the optic nerve) is labelled homogeneously.

A careful analysis of the different layers of retinal cells does not reveal any significant decrease in their thickness. Furthermore, the head of the optic nerve is not adversely affected, even at high doses of adenovirus (107 pfu).

Injection in the retrobulbar space To evaluate the possibility of a diffusion of the virus through the sclera, mice were injected in the retrobulbar space. In contrast to the retinal staining, approximately 100 of the fibres of the 4 oculomotor muscles were infected and express P-galactosidase activity.

These collective results clearly demonstrate that recombinant adenoviruses which are defective for replication constitute especially advantageous vectors for the transfer of genes in vivo to ocular cells.

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Claims (9)

1. Method of treating ocular pathologies which comprises the ocular administration of a defective recombinant adenovirus containing an inserted gene.

2. Method according to claim 1, characterised in that the defective recombinant adenovirus lacks the regions of its genome which are needed for its replication in the infected cell.

3. Method according to claim 1 or claim 2, characterised in that the defective recombinant adenovirus is a type Ad 2 adenovirus.

4. Method according to claim 1 or claim 2, characterised in that the defective recombinent adenovirus is a type Ad 5 adenovirus.

5. Method according to any one of claims 1 to 4, characterised in that the inserted gene comprises sequences permitting its expression in the infected cell.

6. Method according to any one of claims 1 to 5, characterised in that the inserted gene codes for a protein or a protein fragment.

7. Method according to any one of claims 1 to 5, characterised in that the inserted gene is an antisense sequence.

8. Method according to claim 1 in which the ocular pathologies are hereditary pathologies such as retinitis pigmentosa. PAOPER\JLR61444-94. 149 29/5/98 -19-

9. A method according to a~y one of claims 1 to 8, substantially as hereinbefore described with reference to the Figures and/or Examples. DATED this 1st day of June, 1998 Rhone-Poulenc Rorer S.A. AND Institut National De La Sante Et De La Recherche Medicale by their Patent Attorneys DAVIES COLLISON CAVE C I