AU4237599A - Recombinant viruses, preparation and use thereof in gene therapy - Google Patents

Description

AUSTRALIA

PATENTS ACT 1990

ORIGINAL

COMPLETE SPECIFICATION Name of Applicant: Address of Applicant: Rhone-Poulenc Rorer S.A.

Patents Directorate, 20, avenue Raymond-Aron, F-92160 Antony, France Patrick Benoit; Patrice Denefle; and Michel Perricaudet.

DAVIES COLLISON CAVE, Patent Attorneys, 1 Little Collins Street, Melbourne, 3000.

Actual Inventor(s): Address for Service: .0 :00.0 Complete Specification for the invention entitled: "Recombinant viruses, preparation and use thereof in gene therapy" The following statement is a full description of this invention, including the best method of performing it known to us: 1A RECOMBINANT VIRUSES, PREPARATION AND USE IN GENE THERAPY The present invention relates to recombinant vectors of viral origin, to their preparation and to their use, in particular for the treatment and/or prevention of pathologies associated with dyslipoproteinaemias. More especially, it relates to recombinant viruses containing a DNA sequence coding for a lipase involved in lipoprotein metabolism. The 10 invention also relates to the preparation of these vectors, to pharmaceutical compositions containing them and to their therapeutic use, in particular in gene therapy.

Dyslipoproteinaemias are disorders of the metabolism of the lipoproteins responsible for transport of lipids such as cholesterol and triglycerides in the blood and the peripheral fluids.

They lead to major pathologies associated, respectively, with hypercholesterolaemia or hypertriglyceridaemia, such as, in particular, atherosclerosis. Atherosclerosis is a complex disease of polygenic origin, which is defined from a histological standpoint by deposits (lipid or fibrolipid plaques) of lipids and of other blood derivatives in the wall of the large arteries (aorta, coronary arteries, carotid). These plaques, which are more or less calcified according to the degree of 2 progression of the process, may be coupled with lesions, and are associated with the accumulation of fatty deposits in the arteries, consisting essentially of cholesterol esters. These plaques are accompanied by a thickening of the arterial wall, with hypertrophy of the smooth muscle, appearance of foam cells and accumulation of fibrous tissue. The atheromatous plaque protrudes markedly from the wall, endowing it with a stenosing character responsible for vascular occlusions 10 by atheroma, thrombosis or embolism which occur in those patients who are most affected. The dyslipoproteinaemias can hence lead to very serious cardiovascular pathologies such as infarction, sudden death, cardiac decompensation, stroke, and the like.

15 At the present time these pathologies, and especially the hypercholesterolaemias, are treated essentially by means of compounds which act either on cholesterol biosynthesis (hydroxymethylglutarylcoenzyme A reductase inhibitors, statins), or on the uptake and removal of biliary cholesterol (sequestering agents or resins), or alternatively on lipolysis by a mode of action which remains to be elucidated at molecular level (fibrates). Consequently, all the major classes of medicinal products which have been used in this indication (sequestering agents, fibrates or statins) are directed only towards the preventive aspect of atheromatous plaque formation and not, in fact, towards the treatment of atheroma. Current treatments of atheroma following coronary accident are merely palliative, since they do not intervene in cholesterol homeostasis and are surgical procedures (coronary bypass, angioplasty).

The present invention constitutes a novel therapeutic approach to the treatment of pathologies associated with dyslipoproteinaemias. It proposes an advantageous solution to the drawbacks.of the prior art, by demonstrating the possibility of treating pathologies associated with dyslipoproteinaemias by gene therapy, by the transfer and expression in vivo of a gene coding for a lipase involved in lipoprotein S" metabolism. The invention thus affords a simple means permitting specific and effective treatment of these 15 pathologies.

Gene therapy consists in correcting a deficiency or an abnormality (mutation, aberrant expression, and the like) or in providing for the expression of a protein of therapeutic interest by 20 introducing genetic information into the affected cell or organ. This genetic information may be introduced either ex vivo 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 techniques of transfection 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), and 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 gene transfer has been seen to be a promising alternative to these physical transfection techniques. In this connection, different viruses have been tested for their capacity to infect certain cell populations. This applies especially to retroviruses (RSV, HMS, MMS, and the like), the HSV virus, adenoassociated viruses and adenoviruses.

The present invention constitutes a novel ~therapeutic approach to the treatment of pathologies associated with dyslipoproteinaemias, consisting in 15 transferring and expressing in vivo genes coding for lipases involved in lipoprotein metabolism. It is especially advantageous that the Applicant has now shown that it is possible to construct recombinant viruses containing a DNA sequence coding for a lipase 20 involved in lipoprotein metabolism, and to administer these recombinant viruses in vivo, and that this administration permits a stable and effective expression of a biologically active lipase in vivo, and without a cytopathological effect.

The present invention is also the outcome of the demonstration that adenoviruses constitute especially effective vectors for the transfer and expression of such genes. In particular, the present invention shows that the use of recombinant adenoviruses as vectors enables levels of expression of these genes to be obtained which are sufficiently high to produce the desired therapeutic effect.

The present invention thus affords a novel approach to the treatment and prevention of cardiovascular and neurological pathologies associated with dyslipoproteinaemias.

The invention hence provides a defective 10 recombinant virus comprising a nucleic acid sequence coding for all or part of lipoprotein lipase (LPL) or S*of a derivative of the latter.

Also provided is the use of such a virus for the preparation of a pharmaceutical composition intended 15 for the treatment and/or of pathologies associated with dyslipoproteinaemias.

The invention also provides mammalian cells infected with one or more defective recombinant viruses of the invention. Such cells can be implanted in the 20 body, permitting a sustained and effective in vivo release of a biologically active lipase. Thus the invention also provides an implant comprising infected cells of the invention which produce recombinant viruses of the invention, and an extracellular matrix.

Lipoprotein lipase (LPL) is an enzyme which 6 permits the hydrolysis of triglycerides contained in very low density lipoproteins (VLDL) or chylomicrons.

Apolipoprotein CII, which is present at the surface of these particles, is used as cofactor in this hydrolysis. Naturally, LPL is mainly synthesized by adipocytes in the form of a 51-kDa monomeric precursor, which is then glycosylated (58 kDa). In the blood, LPL is active in dimeric form. Up to 80 of freshly synthesized LPL is degraded in the lysosomal compartment before being able to be secreted. After its secretion, LPL is taken up by the luminal face of the cells of the vascular endothelium, to which it binds via glycosaminoglycans. It has a very strong affinity for heparin, which enables LPL to be displaced from its 15 binding site at the surface of the endothelial cell.

Intravenous injection of heparin enables

LPL

concentration and activity to be measured in patients.

LPL is utilized in the vascular cells and also in liver *cells as an uptake agent for lipoprcteins, increasing 20 their retention at the cell surface and thereby promoting their uptake or their modification.

cDNA coding for human LPL has been cloned and sequenced (Wion et al., Science 235 (1987) 1638-1641).

Two forms of messengers coexist, of 3350 and 3750 bases, mainly in adipose and muscle tissue, and originate from the use of 2 polyadenylation sites. They include a long untranslated 3' sequence and code for a preprotein of 475 aa, from which a leader sequence of 27 aa is cleaved to give rise to the mature monomeric protein of 448 residues. The LPL gene has also been cloned (Kirchgessner et al., Proc. Natl. Acad. Sci.

USA, 1987, 262:9647-9651). The synthesis, processing and secretion of LPL are regulated in a complex manner during development and in response to hormonal stimuli.

A sizeable part of this regulation is accomplished at transcriptional level (review in Auwerx et al., Critical Reviews in Clinical Laboratory Sciences, 1992, 29:243-268).

The present invention shows that it is possible to incorporate a DNA sequence coding for LPL in a viral vector, and that these vectors enable a biologically active (dimeric) mature form of LPL to be 15 expressed and secreted effectively. More especially, the invention shows that in vivo expression of active LPL may be obtained by direct administration of an adenovirus or by implantation of a cell which is productive or genetically modified by an adenovirus or 20 by a retrovirus incorporating such a DNA.

The vectors of the invention may be used, in particular, to correct LPL deficiencies due to mutations in the LPL gene. Such deficiencies are relatively common and can reach an incidence of 1:5,000-1:10,000 in some populations Santamarina- Fojo, 1992, Cur. Op. lipid., 3:186-195). These deficiencies can result from a sizeable mutation in the gene, leading to the absence of LPL synthesis or to the synthesis of a truncated or highly modified protein.

The existence has, in effect, been shown in some patients of mutations of the insertion, deletion or nonsense mutation type (review in S. Santamarina-Fojo, 1992, Cur. Op. lipid., 3:186-195). They can also result from a defect at the catabolic site, which may be due to mutations of the missense type in the gene. They can also result from modification both at the heparinbinding site and at the catalytic site. At the 10 heterozygous stage, these deficiencies can represent a considerable proportion of the commonest hyperlipidaemias, including familial hypertriglycerinaemias, combined familial hyperlipidaemias and postprandial hyperlipidaemias.

15 The present invention is especially advantageous, since it enables an expression of LPL which is controlled and without a harmful effect to be induced in organs which are not normally affected by the expression of this protein. In particular, an 20 altogether advantageous release is obtained by implantation of cells which produce vectors of the invention, or are infected ex vivo with vectors of the invention.

The lipase activity produced in the context of the present invention can be a human or animal lipase. The nucleic acid sequence used in the context of the present invention can be a cDNA, a genomic

DNA

(gDNA), an ARN (in the case of retroviruses) or a hybrid construction consisting, for example, of a cDNA into which one or more introns might be inserted. Other possible sequences are synthetic or semi-synthetic sequences. It is especially advantageous to use a cDNA or a gDNA. In particular, the use of a gDNA permits better expression in human cells. To permit their incorporation in a viral vector according to the invention, these sequences are advantageously modified, for example by site-directed mutagenesis, especially for the insertion of suitable restriction sites. The sequences described in the prior art are not, in effect, constructed for a use according to the invention, and prior adaptations may prove necessary in order to obtain substantial expressions. In the context 15 of the present invention, it is preferable to use a nucleic acid sequence coding for a human lipase.

Moreover, it is also possible to use a construction S coding for a derivative of these lipases, especially a derivative of human LPL and HL. HL (hepatic lipase) is localized at the surface of hepatic endothelial cells.

It differs from LPL in its insensitivity to the activating action of apoC-II. HL is involved in the hydrolysis of IDL lipids, and also of HDL2 lipids, bringing about their conversion to HDL3. A derivative of these lipases comprises, for example, any sequence obtained by mutation, c-letion and/or addition relative to the native sequence, and coding for a product retaining lipase activity. These modifications may be carried out by techniques known to a person skilled in the art (see general techniques of molecular biology below). The biological activity of the derivatives thereby obtained may then be readily determined, as described, in particular, in Example 3. The derivatives for the purposes of the invention may also be obtained by hybridization from nucleic acid libraries, using the native sequence or a fragment of the latter as probe.

These derivatives are, in particular, I0 molecules having a greater affinity for their binding sites, molecules displaying greater resistance to 0 proteases, molecules having greater therapeutic efficacy or reduced side effects, or possibly novel oo biological properties. The derivatives also include S" 15 modified DNA sequences permitting improved expression in vivo.

Among preferred derivatives, there may be mentioned, more especially, natural variants, molecules in which some N- or O-glycosylation sites have been 0o 20 modified or eliminated, molecules in which one or more residues have been substituted or molecules in which all the cysteine residues have been substituted (muteins). There may also be mentioned derivatives obtained by deletion of regions having little or no involvement in the interaction with the binding sites of interest or expressing an undesirable activity, and derivatives containing additional residues relative to the native sequence, such as, for example, a secretion 11 signal and/or a junction peptide.

In a first embodiment, the present invention relates to a defective recombinant virus comprising a cDNA sequence coding for a lipase involved in lipoprotein metabolism. In another preferred embodiment of the invention, the DNA sequence is a gDNA sequence.

The vectors of the invention may be prepared from different types of virus. Preferably, vectors derived from adenoviruses, from adeno-associated viruses (AAV), from herpesviruses (HSV) or from retroviruses arc used. It is most especially advantageous to use an adenovirus, for a direct administration or for the ex vivo modification of cells intended to be implanted or a retrovirus, for the 15 implantation of productive cells.

The viruses according to the invention are defective, that is to say they are incapable of replicating autonomously in the target cell. Generally, the genome of the defective viruses used in the context 20 of the present invention hence lacks at least the sequences needed for replication of the said virus in the infected cell. These regions may be either removed (wholly or partially), or rendered non-functional, or substituted by other sequences, and in particular by the nucleic acid sequence coding for the lipase.

Preferably, the defective virus nevertheless retains the sequences of its genome which are needed for encapsidation of the viral particles.

12 As regards adenoviruses more especially, different serotypes, the structure and properties of which vary somewhat, have been characterized. Among these serotypes, it is preferable to use, in the context of the present invention, human adenoviruses type 2 or 5 (Ad 2 or Ad 5) or adenoviruses of animal origin (see Application WO 94/26914). Among adenoviruses of animal origin which are useable in the context of the present invention, adenoviruses of canine, bovine, murine Mavl, Beard et al., Virology 75 (1990) 81), ovine, porcine, avian or alternatively simian SAV) may be mentioned.

Preferably, the adenovirus of animal origin is a canine adenovirus, and more preferably a CAV2 adenovirus 15 [Manhattan or A26/61 (ATCC VR-800) strain, for example]. It is preferable to use adenoviruses of human or canine or mixed origin in the context of the invention. Preferably, the defective adenoviruses of the invention comprise the ITRs, a sequence permitting 20 encapsidation and the sequence coding for the lipase.

Advantageously, in the genome of the adenoviruses of the invention, the El region at least is rendered nonfunctional. Still more preferably, in the genome of the adenoviruses of the invention, the El gene and at least one of the genes E2, E4, L1-L5 are non-functional. The viral gene of interest may be rendered non-functional by any technique known to a person skilled in the art, and in particular by total elimination, substitution, partial deletion or addition of one or more bases in the gene or genes of interest. Such modifications may be obtained in vitro (on the isolated DNA) or in situ, for example by means of genetic engineering techniques, or alternatively by treatment by means of mutagenic agents. Other regions may also be modified, and in particular the E3 (WO 95/02697), E2 (WO 94/28938), E4 (WO 94/28152, WO 94/12649, WO 95/02697) and (WO 95/02697) regions. According to a preferred embodiment, the adenovirus according to the invention comprises a deletion in the El and E4 regions, and the sequence coding for LPL is inserted in the inactivated El region. According to another preferred embodiment, it comprises a deletion in the El region, into which 15 are inserted the E4 region and the sequence coding for LPL (see FR 94/13355).

The defective recombinant adenoviruses according to the invention may. be prepared by any technique known to a person skilled in the art (Levrero 20 et al., Gene 101 (1991) 195, EP 185,573; Graham,

EMBO

J. 3 (1984) 2917). In particular, they may be prepared by homologous recombination between an adenovirus and a plasmid carrying, inter alia, the DNA sequence coding for the lipase. Homologous recombination takes place after cotransfection of the said adenovirus and said plasmid into a suitable cell line. The cell line used should preferably be transformable by the said elements, and (ii) contain the sequences capable of 14 complementing the portion of the genome of the defective adenovirus, preferably in integrated form in order to avoid risks of recombination. As an example of a line, there may be mentioned the human embryonic kidney line 293 (Graham et al., J. Gen. Virol. 36 (1977) 59) which contains, in particular, integrated in its genome, the left-hand portion of the genome of an adenovirus (12 or lines capable of complementing the El and E4 functions as are described, S. 10 in particular, in Applications Nos. WO 94/26914 and WO 95/02697.

Thereafter, the adenoviruses which have Smultiplied are recovered and purified according to standard techniques of molecular biology, as 15 illustrated in the examples.

SAdeno-associated viruses (AAV) are, for their part, relatively small-sized DNA viruses which integrate stably and in a site-specific manner in the genome of the cells they infect. They are capable of 20 infecting a broad range of cells without inducing an effect on growth, morphology or cell differentiation.

Moreover, they do not appear to be implicated in pathologies in man. The AAV genome has been cloned,.

sequenced and characterized. It comprises approximately 4,700 bases, and contains at each end an inverted repeat region (ITR) of approximately 145 bases, serving as origin of replication for the virus. The remainder of the genome is divided into 2 essential regions carrying the encapsidation functions: The left-hand portion of the genome, which contains the rep gene involved in the viral replication and expression of the viral genes; the right-hand portion of the genome, which contains the cap gene coding for the capsid proteins of the virus.

The use of vectors derived from AAV for the transfer of genes in vitro and in vivo has been described in the literature (see, in particular, 10 WO 91/18088; WO 93/09239; US 4,797,368, US 5,139,941, EP-488,528). These applications describe different constructions derived from AAV, in which the rep and/or :cap genes are deleted and replaced by a gene of interest, and their use for transferring the said gene 15 of interest in vitro (into cells in culture) or in vivo (directly into a body). However, none of these documents describes or suggests the use of a recombinant AAV for the transfer and expression of a lipase in vivo or ex vivo, or the advantages of such a transfer. The defective recombinant AAVs according to the invention may be prepared by cotransfection, into a cell line infected with a human helper virus (for example an adenovirus), of a plasmid containing the sequence coding for the lipase flanked by two inverted repeat regions (ITR) of AAV, and a plasmid carrying the encapsidation genes (rep and cap genes) of AAV. The recombinant AAVs produced are then purified by standard techniques.

Regarding herpes viruses and retroviruses, the construction of recombinant vectors has been amply described in the literature: see, in particular, Breakfield et al., New Biologist 3 (1991) 203; EP 453242, EP 178220, Bernstein et al., Genet. Eng.; 7 (1985) 235; McCormick, BioTechnology 3 (1985) 689, and the like.

In particular, retroviruses are integrative viruses which infect dividing cells. The retrovirus 10 genome essentially comprises two LTRs, an encapsidation sequence and three coding regions (gag, pol anc env) In the recombinant vectors derived from retroviruses, the gag, pol and env genes are generally deleted wholly or partially, and replaced by a heterologous nucleic 15 acid sequence of interest. These vectors may be produced from different types of retrovirus such as, in particular, MoMuLV (Moloney murine leukaemia virus; also designated MoMLV), MSV (Moloney murine sarcoma virus), HaSV (Harvey sarcoma virus), SNV (spleen necrosis virus), RSV (Rous sarcoma virus) or alternatively Friend virus.

To construct recombinant retroviruses containing a sequence coding for LPL according to the invention, a plasmid containing, in particular, the LTRs, the encapsidation sequence and the said coding sequence is generally constructed, and then used to transfect a so-called encapsidation cell line capable of providing in trans the retroviral functions which are deficient in the plasmid. Generally, the encapsidation lines are hence capable of expressing the gag, pol and env genes. Such encapsidation lines have been described in the prior art, and in particular the line PA317 (US 4,861,719), the line PsiCRIP (WO 90/02806) and the line GP+envAm-1 2 (WO 89/07150).

Moreover, the recombinant retroviruses can contain modifications in the LTRs to eliminate transcriptional activity, as well as extended encapsidation sequences 0 containing a portion of the gag gene (Bender et al., J.-Virol. 61 (1987) 1639). The recombinant retroviruses Iproduced are then purified by standard techniques.

To implement the present invention, it is most especially advantageous to use a defective recombinant adenovirus. The results given below demonstrate, in effect, the especially advantageous properties of adenoviruses for expressing in vivo a protein having lipase activity. The adenoviral vectors according to the invention are especially advantageous for a direct administration of a purified suspension in vivo, or for the ex vivo transformation of cells, in particular autologous cells, for the purpose of their implantation. Furthermore, the adenoviral vectors according to the invention possess, in addition, considerable advantages such as, in particular, their very high efficiency of infection, enabling infection to be carried out using small volumes of viral suspension. In an especially preferred embodiment, an adenovirus containing, in addition to the gene coding for the lipase, a gene coding for an apolipoprotein is used according to the invention. The lipase is preferably hepatic lipase and the apolipoprotein is preferably selected from apoA-I and apoAIV. The two genes are advantageously used in the form of a bicistronic construction which is introduced into an adenoviral vector according to the protocol described above for the construction of an adenovirus containing a single gene. Advantageously, the invention relates to a recombinant adenovirus containing a gene coding for HL and a gene coding for ApoA-I, inserted into the El .:region. The adenovirus construction containing a gene coding for an apo has been described in Application 15 PCT/FR94/00 422 which is incorporated herein by reference.

According to another especially advantageous embodiment of the invention, a line is used which produces retroviral vectors containing the sequence coding for the lipase, for implantation in vivo. The lines which are usable for this purpose are, in particular, PA317 (US 4,861,719), PsiCrip (WO 90/02806) and GP+envAm-1 2 (US 5,278,056) cells, modified to permit the production of a retrovirus containing a nucleic acid sequence coding for a lipase according to the invention.

Advantageously, in the vectors of the invention, the sequence coding for the lipase is placed under the control of signals permitting its expression in infected cells. These signals can be ones for homologous or heterologous expression, that is to say signals different from the ones naturally responsible for the expression of the lipase. They can, in particular, be sequences responsible for the expression of other proteins, or synthetic sequences. In particular, they can be sequences of eukaryotic or viral genes or derived sequences, stimulating or 10 repressing the transcription of a gene, specifically or non-specifically and inducibly or non-inducibly. As an example, they can be promoter sequences originating from the genome of the cell which it is desired to infect, or from the genome of a virus, and in 15 particular the promoters of the adenovirus E1A and MLP genes, the CMV, RSV LTR promoter, and the like. Among eukaryotic promoters, there may also be mentioned the ubiquitous promoters (HPRT, vimentin, a-actin, tubulin, and the like), the promoters of intermediate filaments (desmin, neurofilaments, keratin, GFAP, and the like), the promoters of therapeutic genes (MDR, CFTR, factor VIII type, and the like), tissue-specific promoters (pyruvate kinase, villin, promoter of the fatty acidbinding intestinal protein, a-actin promoter of smooth muscle cells, promoters specific to the liver; Apo AI, Apo AII, human albumin, and the like) or alternatively promoters responding to a stimulus (steroid hormone receptor, retinoic acid receptor, and the like). In addition, these expression sequences may be modified by adding activation, regulatory, and the like, sequences.

Moreover, when the inserted gene does not contain expression sequences, it may be inserted into the genome of the defective virus downstream of such a sequence.

In a particular embodiment, the invention relates to a defective recombinant virus comprising a nucleic acid sequence coding for a lipase involved in 10 lipoprotein metabolism, under the control of a promoter chosen from RSV LTR or the CMV early promoter.

More preferably, the nucleic acid sequence used also comprises signals permitting secretion of the lipase by infected cells. To this end, the nucleic acid 15 sequence generally contains, upstream of the coding sequence, a signal sequence directing the lipase synthesized into the pathways of secretion of the infected cell. This signal sequence can be the natural signal sequence of the lipase synthesized, but it can also be any other signal sequence which is functional in the infected cell, or an artificial signal sequence.

As stated above, the present invention also relates to any use of a virus as described above for the preparation of a pharmaceutical composition intended for the treatment and/or prevention of pathologies associated with dyslipoproteinaemias.

The present invention also relates to a pharmaceutical composition comprising one or more defective recombinant viruses as are described above.

These pharmaceutical compositions may be formulated with a view to topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular, transdermal, and the like, administration- Preferably, the pharmaceutical compositions of the invention contain a pharmaceutically acceptable vehicle for an injectable formulation, in particular for an intravenous injection, such as, for example, into the 10 patient's portal vein. The formulations can be, in particular, isotonic steryl solutions, or dry, in :particular lyophilized, compositions which, on adding sterilized water or physiological saline, as the case may be, enable injectable solutions to be produced.

Direct injection into the patient's portal vein is advantageous, since it enables the infection to be targeted to the liver, and thus the therapeutic effect to be concentrated in this organ.

The doses of defective recombinant virus used for the injection may be adapted in accordance with different parameters, and in particular in accordance with the viral vector, the mode of administration used, the pathology in question or alternatively the desired period of treatment. Generally speaking, the recombinant 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 infecting a suitable cell culture and measuring, generally after 48 hours, the number of plaques of infected cells. The techniques of determination of the pfu titre of a viral solution are well documented in the literature.

As regards retroviruses, the compositions according to the invention can contain the productive cells directly, with a view to their implantation.

10 In this connection, another subject of the invention relates to any mammalian cells infected with one or more defective recombinant viruses as are described above. More especially, the invention relates to any human cell population infected with these 15 viruses. The cells in question can be, in particular, fibroblasts, myoblasts, hepatocytes, keratinocytes, endothelial cells, glial cells, and the like.

The cells according to the invention can originate from primary cultures. ThLy may be removed by any technique known to a person skilled in the art and then set up in culture under conditions permitting their proliferation. As regards-fibroblasts, more especially, the latter may be readily obtained from biopsies, for example according to the technique described by Ham [Methods Cell. Biol. 21a (1980) 255].

These cells may be used directly for infection with the viruses, or stored, for example by freezing, to establish autologous banks with a view to subsequent use. The cells according to the invention can also be secondary cultures obtained, for example, from preestablished banks (see, for example, EP 228458, EP 289034, EP 400047, EP 456640) The cells in culture are then infected with the recombinant viruses to endow them with the capacity to produce a biologically active lipase. Infection is carried out in vitro according to techniques known to a person skilled in the art. In particular, depending on the cell type used and the desired number of copies of virus per cell, a person skilled in the art can adapt the multiplicity of infection and, where appropriate, o the number of infection cycles carried out. It should be obvious that these steps must be performed under 15 suitable conditions of sterility when the cells are intended for administration in vivo. The doses of recombinant virus used for infecting the cells may be adapted by a person skilled in the art in accordance with the desired objective. The conditions described above for in vivo administration may be applied to infection in vitro. For infection with retroviruses, it is also possible to coculture the cells which it is desired to infect with cells producing the recombinant retroviruses according to the invention. This makes it possible to eliminate the need to purify the retroviruses.

Another subject of the invention relates to an implant comprising mammalian cells infected with one

I

I>

24 or more defective recombinant viruses as are described above, or cells which produce recombinant viruses, and an extracellular matrix. Preferably, the implants according to the invention comprise 105 to 1010 cells.

More preferably, they comprise 106 to 108 cells.

More especially, in the implants of the invention, the extracellular matrix comprises a gelling compound and, where appropriate, a support permitting anchorage of the cells'.

10 For the preparation of the implants according to the invention, different types of gelling agents may be employed. The gelling agents are used for inclusion of the cells in a matrix having the constitution of a gel, and to promote anchorage of the cells to the 15 support, where appropriate. Different cellular adhesion agents may hence be used as gelling agents, such as, in particular, collagen, gelatin, glycosaminoglycans, fibronectin, lectins, and the like. Preferably, collagen is used in the context of the present invention. The collagen may be of human, bovine or murine origin. More preferably, type I collagen is used.

As stated above, the compositions according to the invention advantageously comprise a support permitting anchorage of the cells. The term anchorage denotes any form of biological and/or chemical and/or physical interaction bringing about adhesion and/or binding of the cells to the support. Moreover, the

-N

cells can either coat the support used or enter the interior of this support, or both. It is preferable, in the context of the invention, to use a non-toxic and/or biocompatible solid support. In particular, polytetrafluoroethylene (PTFE) fibres or a support of biological origin may be used.

The implants according to the invention may be implanted in different sites of the body. In particular, the implantation may be performed in the 10 peritoneal cavity, in the subcutaneous tissue (subpubic region, iliac or inguinal fossae, and the like), in an 0*.j organ, a muscle, a tumour or the central nervous system, or alternatively under a mucosa. The implants according to the invention are especially advantageous 15 in that they enable the release of the lipase in the body to be controlled: this is, in the first place, determined by the multiplicity of infection and by the number of cells implanted. The release can then be controlled either by withdrawing the implant, which stops the treatment permanently, or by the use of regulable expression systems enabling the expression of the therapeutic genes to be induced or repressed.

The present invention thus affords a very effective means for the treatment or prevention of pathologies associated with dyslipoproteinaemias, especially obesity, hypertriglyceridaemia or, in the field of cardiovascular complaints, myocardial infarction, angina, sudden death, cardiac decompensation and stroke.

In addition, this treatment can be applied equally well 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 will be described more completely by means of the examples which follow, which are to be considered to be illustrative and nonlimiting.

10 Lecrend to the figures Figure 1: Structure of the vector pXL241 8 i Figure 2: Structure of the vector pXL2419 Figure 3: Structure of the vector pXL CMV-LPL Figure 4: Structure of the vector pXL RSV-LPL :I 15 Figure 5: Structure of the vector pXL RSV-LPLc General techniques of molecular biology The methods traditionally used in molecular 6 biology, such as preparative extractions of plasmid DNA, centrifugation of plasmid DNA in a caesium chloride gradient, agarose or acrylamide gel electrophoresis, purification of DNA fragments by electroelution, phenol or phenol/chloroform extraction of proteins, ethanol or isopropanol precipitation of DNA in a saline medium, transformation in Escherichia coli, and the like, are well known to a person skilled in the art and are amply described in the literature [Maniatis T. et al., "Molecular Cloning, a Laboratory Manual", Cold Spring Harbor Laboratory, Cold Spring

M

Harbor, 1982; Ausubel F.M. et al. (eds), "Current Protocols in Molecular Biology", John Wiley Sons, New York, 1987].

Plasmids of the pBR322 and pUC type and phages of the M13 series are of commercial origin (Bethesda Research Laboratories).

To carry out ligation, the DNA fragments may be separated according to their size by agarose or acrylamide gel electrophoresis, extracted with phenol 10 or with a phenol/chloroform mixture, precipitated with ethanol and then incubated in the presence of phage T4 DNA ligase (Biolabs) according to the supplier's recommendations.

The filling in of 5' protruding ends may be 15 performed with the Klenow fragment of E.coli

DNA

polymerase I (Biolabs) according to the supplier's specifications. The destruction of 3' protruding ends is performed in the presence of phage T4 DNA polymerase (Biolabs) used according to the manufacturer's recommendations. The destruction of 5' protruding ends is performed by a controlled treatment with S1 nuclease.

Mutagenesis directed in vitro by synthetic oligodeoxynucleotides may be performed according to the method developed by Taylor et al. [Nucleic Acids Res.

13 (1985) 8749-8764] using the kit distributed by Amersham.

The enzymatic amplification of DNA fragments by the so-called PCR [Polymerase-catalysed Chain Reaction, Saiki R.K. et al., Science 230 (1985) 1350- 1354; Mullis K.B. and Faloona Meth. Enzym. 155 (1987) 335-350] technique may be performed using a DNA thermal cycler (Perkin Elmer Cetus) according to the manufacturer's specifications.

The verification of the nucleotide sequences may be performed by the method developed by Sanger et al. [Proc. Natl. Acad. Sci. USA, 24 (1977) 5463-5467] 10 using the kit distributed by Amersham.

Examples Example 1. Construction of the vector pXL2418 carrying the gene coding for LPL under the control of the cytomegalovirus (CMV) early promoter (Figure 1) This example describes the construction of a vector comprising a cDNA sequence coding for LPL, under the control of a promoter consisting of the cytomegalovirus (CMV) early promoter, as well as a region of the Ad5 adenovirus genome permitting homologous recombination. This vector was constructed as described below.

1.1. Construction of the vector pXL2375: The vector pXL2375 contains, in particular, a region of the adenovirus genome and a DNA sequence coding for apolipoprotein AI under the control of the CMV promoter. More especially, the CMV promoter used extends as far as the donor 5' splicing site linked to the 107 bp nearest the 3' end of the synthetic intron 29 described by O'Gorman et al. (Science 251 (1991) 1351).

The construction of this vector has been described in detail in copending Application FR 93/05125. It is understood that similar constructions may be carried out by a person skilled in the art.

1.2. Construction of the cDNA sequence coding for LPL Plasmid pHLPL 26-1 described by Wion et al.

(Science 235 (1987) 1638-1641) contains an incomplete 10 sequence of LPL cDNA. Thus, this plasmid contains bases 272 to 1623 of LPL cDNA flanked by two EcoRI sites, cloned to the EcoRI site of a plasmid pGEM1 (Promega).

The EcoRI fragment of pHLPL 26-1 containing the partial LPL cDNA was recloned into the EcoRI site 15 of a plasmid pMTL22 (Chambers et al., Gene, 1988, 68:138-149), in the orientation placing the 5' bases of the cDNA on the same side as the BglII site of pMTL22.

The resulting plasmid was called pXL2402.

The RNA of human adipose tissues was then extracted according to the technique of Chromczynski and Sacchin (Anal. Biochem. 162 (1987) 156-159). From this RNA preparation, an amplification was carried out by RT-PCR so as to isolate the missing portion of the LPL cDNA. To this end, the following primers were used: Sq4541: TTA GAT CTA TCG ATA GAT GGA GAG CAA AGC CCC TG (SEQ ID No. 1) This primer makes it possible to introduce a BglII site as well as a Clal site upstream of the ATG.

4 Sq3810: TAC ATT CCT GTT ACC GTC CAG CCA TGG ATC (SEQ

ID

No. 2) The 260-bp PCR fragment obtained after amplification cycles was then cloned into plasmid pCR-II (Invitrogen) and sequenced for verification. It was then introduced via the BglII and NcoI sites into the vector pXL2402, which reconstitutes a complete cDNA preceded by a Clal site and followed by a SalI site.

The resulting plasmid was called pXL2417.

0 1.3. Construction of the vector pXL2418.

Lastly, the LPL cDNA was inserted into plasmid pXL2375 between the SalI and Clal sites, following excision of the Apo Al cDNA with these same two enzymes. The plasmid obtained was designated 15 pXL2418 (Figure 1).

Example 2. Construction of the vector pXL2419 carrying the gene coding for LPL under the control of the promoter of the Rous sarcoma virus LTR (RSV

LTR)

(Figure 2) This example describes the construction of a vector comprising a cDNA sequence coding for LPL, under the control of a promoter consisting of the Rous sarcoma virus LTR (RSV LTR), as well as a region of the adenovirus genome permitting homologous recombination. This vector was constructed as described below.

2.1. Construction of the vector pXL224 4 The vector pXL224 4 contains, in particular, a region of the adenovirus genome and a DNA sequence coding for apolipoprotein AI under the control of the RSV LTR promoter.

2.2. Construction of a cDNA sequence coding for LPL The cDNA sequence coding for LPL used in this example is that described in Example 1.2.

2.3. Construction of the vector pXL2419 The LPL cDNA was inserted into plasmid 10 pXL2244 between the SalI and Clal sites, following excision of the Apo Al cDNA with these same two enzymes. The plasmid obtained was designated pXL2419 (Figure 2).

Example 3: Construction of the vectors pXL RSV-LPL and 15 pXL CMV-LPL The vector pRC-CMV LPL contains a fragment of 0: LPL cDNA extending from bases 1 to 2388 of the sequence published by Wion et al., cloned at the HindIII and XbaI sites of the expression vector pRC-CMV (Invitrogen). The HindIII site was modified to a Clal site by inserting the oligonucleotide AGC TAC ATC GAT GT (SEQ ID No. The LPL cDNA and the polyadenylation site of bovine growth hormone (initially contained in pRC-CMV) are finally extracted from the pRCMV-LPL by SphI cleavage, treatment with T4 polymerase and Clal cleavage. The fragment thereby obtained is cloned inuo the vectors pXL241 8 (Example 1) and pXL241 9 (Example 2) cut with Clal and EcoRV, generating the vectors pXL CMV-LPL (Figure 3) and pXL RSV-LPL (Figure 4), respectively.

Example 4: Construction of the vector pXL RSV-LPLc This example describes the construction of a vector which is usable to generate recombinant viruses containing a short cDNA coding for LPL.

A shorter cDNA (bases 146 to 1635 of the sequence of Wion et al.) was cloned from the RNA of human adipose tissue. The primers ATC GGA TCC ATC GAT GCA GCT CCT CCA GAG GGA CGC (SEQ ID No. 4) and ATC TCT AGA GTC GAC ATG CCG TTC TTT GTT CTG TAG (SEQ ID No. which create, respectively, a BamHI site and a Call site at the 5' end of the cDNA, as well as an XbaI site and a Sail site at the 3' end of the LPL cDNA, were 15 used.

This PCR fragment is cloned into PCR II, and o Sits sequence verified in its entirety. The LPL cDNA is then released via the BamHI and XbaI sites and cloned into an expression vector pcDNA3 (Invitrogen) for 20 verification of the expression, generating plasmid pcDNA3-LPLc.

The ClaI-SalI fragment containing the LPL cDNA is finally cloned at the same sites into plasmid pXL RSV-LPL (Example 3) to generate the shuttle plasmid pXL RSV-LPLc (Figure Example 5: Functionality of the vectors of the invention: demonstration of an LPL activity The capacity of the vectors of the invention A 33 to express a biologically active form of LPL in a cell culture was demonstrated by transient transfection of 293 CosI cells. To this end, cells (2 x 10 6 cells per dish 10 cm in diameter) were transfected (8 pg of vector) in the presence of Transfectam. The expression of the sequence coding for LPL and production of a biologically active protein are demonstrated either in terms of mass using an immunoenzymatic test or in terms of lipase activity 5.1. Measurement of LPL in terms of mass An Immulon I ELISA plate (Dynatech) is coated with anti-bovine LPL monoclonal antibodies crossreacting with human LPL (20 Ag/ml in PBS, The potential sites remaining in the wells are then 15 blocked (saturated) by incubation in the presence of 1 gelatin for 1 hour at room temperature. The samples to be measured are then incubated for 1 hour at 37 0

C.

Visualization is carried out with an anti-LPL serum diluted to 10 1g/ml, 100 Al/well, followed by a peroxidase-labelled antiserum. Peroxidase activity is detected using a TMB substrate (Kirkegaard and Perry Laboratories Inc. kit) and reading of the absorbance at 490 nm.

5.2. Measurement of LPL activity Total lipase activity is measured on a substrate composed of an emulsion of 0.3 mg of triolein (Sigma), 75nCi of tri(l-14C)oleoylglycerol Amersham), 18 mg of BSA (Fraction V, Sigma) and 25 1 of normal human plasma as a source of ApoCII, all these constituents in a final volume of 500 1 of 0.223M Tris pH 8.5. Generally speaking, activity is measured on 100 Al of supernatant of transfected cells or 50 gl of post-heparin plasma.

After 1 hour of incubation at 370C, the reaction is stopped by adding 3.25 ml of extraction buffer (chloroform/methanol/heptane, 10:9:7 v/v/v) and 0.75 ml of carbonate/borate buffer pH 10.5, and the organic phase counted to determine the amount of fatty acids liberated.

To determine the activity specifically associated with LPL, the measurement of hepatic lipase *e activity is carried out in the presence of a 1M concentration of NaCl (which inhibits LPL), and then subtracted from the total activity. It is also possible to inhibit lipoprotein lipase activity with a specific monoclonal antibody (Babirak et al., Atheriosclerosis, 1989, 9:326-334) 20 Plasmids pXL RSV-LPL and pXL CMV-LPL were tested by transfection into CosI cells by comparison with plasmid pRC CMV-LPL. The results are presented in Table 1.

Table 1 Expression vector Activity in the supernatant Day 1 pRC-CMV-LPL pXL RSV-LPL pXL CMV-LPL 24.5 15.1 22.9 Plasmid pcDNA-LPLC was tested by transfection into 293 cells by comparison with an expression vector pcDNA3 containing the same cDNA as the vector pRC-CMV-LPL. The results are presented in Table 2.

LO Table 2 Expression Activity in the Activity in the vector supernatant supernatant Day 1 Day 2 pcDNA3-LPL 106.4 mU/ml 106.7 mU/ml pcDNA3-LPLc 114 mU/ml 109.6 mU/ml Example 6. Construction of a recombinant adenovirus Ad-CMV.LPL containing a sequence coding for LPL lipase The plasmids prepared in Examples 1 to 4 are linearized and cotransfected for recombination with the deficient adenoviral vector, in helper cells (line 293) providing in trans the functions encoded by the El (E1A and E11B) regions of adenovirus.

More especially, the adenovirus Ad.CMV.LPL is obtained by homologous recombination in vivo between the adenovirus Ad.RSVOgal (Stratford-Perricaudet et al., J. Clin. Invest 90 (1992) 626) and plasmid pXL2418 or pXL CMV-LPL according to the following protocol: The linearized plasmid pXL2418 or pXL CMV-LPL and the adenovirus labelled Ad.RSVgal linearized with Clal are cotransfected into line 293 in the presence of calcium phosphate to permit homologous recombination. The recombinant adenoviruses thus generated are selected by plaque purification. After isolation, the recombinant 10 adenovirus is amplified in the cell line 293, yielding a culture supernatant containing the unpurified recombinant defective adenovirus having a titre of approximately 1010 pfu/ml.

The viral particles are then purified by 15 centrifugation on a caesium chloride gradient according to known techniques (see, in particular, Graham et al., Virology 52 (1973) 456). The adenovirus Ad-CMV.LPL may be stored at -800C in 20 glycerol.

The same protocol is reproduced with plasmid 20 pXL2419 or pXL RSV-LPL or pXL RSV-LPLc, yielding the recombinant adenovirus Ad.RSV.LPL or Ad.RSV.LPLc.

Example 7: In vivo transfer of the LPL gene by a recombinant adenovirus This example describes the transfer of the LPL gene in vivo by means of an adenoviral vector according to the invention.

The adenoviruses injected are the adenoviruses Ad-CMV.LPL and Ad.LTR.LPL prepared in 37 Example 5, used in purified form (3.5 x 106 pfu/Al), in saline phosphate solution (PBS). These viruses are injected into C57B1/6 mice intravenously using the tail vein, the retro-orbital sinus or the portal vein. The expression of an active form of LPL is demonstrated under the conditions described in Example Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of S, integers or steps.

**r SEQUENCE

LISTING

GENERAL

INFORMATION:

APPLICANT:

NAME: RHONE-POULENC RORER

S.A.

STREET: 20, avenue Raymond

ARON

CITY:

ANTONY

COUNTRY:

FRANCE

POSTAL CODE: 92165 (ii) TITLE OF INVENTION: RECOMBINANT

VIRUSES,

10 PREPARATION AND USE IN GENE THERAPY (iii) NUMBER OF SEQUENCES: (iv) COMPUTER READABLE

FORM:

i" MEDIUM TYPE: Tape COMPUTER: IBM PC compatible OPERATING SYSTEM:

PC-DOS/MS-DOS

SOFTWARE: patentIn Release Version #1.25 (EPO) a 2) INFORMATION FOR SE ID NO: 1: SEQUENCE CHARACTERISTIC.: LENGTH: 35 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix)

FEATURE:

OTHER INFORMATION: /product= Sq45 4 1 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: TTAGATCTAT CGATAGATGG AGAGCAAAGC CCCTG INFORMATION FOR SEQ ID NO: 2: SEQUENCE

CHARACTERISTICS:

LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: OTHER INFORMATION: /product= Sq381 0 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: TACATTCCTG TTACCGTCCA

GCCATGGATC

INFORMATION FOR SEO ID NO: 3: SEQUENCE CHARACTERISTICS: LENGTH: 14 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: AGCTACATCG

ATGT

INFORMATION FOR SEQ ID NO: 4: SEQUENCE

CHARACTERISTICS:

LENGTH: 36 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: ATCGGATCCA TCGATGCAGC TCCTCCAGAG GGACGC 36 NFOMATON OR SEQ ID NO: (i SEQUENCE

CHARACTERISTICS:

LENGTH: 36 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY:. linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: ATCTCTAGAG TCGACATGCC GTTCTTTGTT CTGTAG 36

Claims (28)

1. Defective recombinant virus comprising a nucleic acid sequence coding for for all or part of lipoprotein lipase (LPL) or of a derivative of the latter.

2. Virus according to claim 1, characterized in that the DNA sequence is a cDNA sequence.

3. Virus according to claim 1, characterized in that the DNA sequence is a gDNA Ssequence. S:

4. virus according to any one of claims 1 .to 3, characterized in that the DNA sequence codes for human LPL. 15

5. Virus according to any one of claims 1 to 4, characterized in that the DNA sequence is placed under the control of signals permitting its expression in infected cells. S.

6 Virus according to claim characterized in that the expression signals are chosen from viral promoters.

7. Virus according to claim 6, characterized in that the expression signals are chosen from E1A, MLP, CMV and RSV LTR promoters.

8. Defective recombinant virus comprising a cDNA sequence coding for lipoprotein lipase, under the control of a promoter chosen from the RSV LTR promoter and the CMV early promoter.

9. Virus according to any one of the preceding claims, characterized in that it comprises a sequence enabling lipoprotein lipase to be directed into the pathways of secretion of the infected cell.

Virus according to claim 9, characterized in that the secretion sequence is the native sequence of lipoprotein lipase.

11. Virus according to any one of claims 1 to 10, characterized in that it lacks the regions of its genome which are needed for its replication in the .t target cell.

12. Virus according to any one of claims 1 to 11, characterized in that it is a human adenovirus 15 type Ad 2 or Ad 5 or a canine adenovirus type CAV-2.

13. Virus according to any one of claims 1 to 11, characterized in that it is an adeno-associated virus.

14. Virus according to any -ne of claimS 1 to 1, characterized in that it is a retrovirus.

Virus according to any one of claims 1 to 11, characterized in that it is a herpesvirus (f4SV).

16. Use of a virus according to any one of claims 1 to 15 for the preparation of a pharmaceutical composition intended for the treatment and/or prevention of pathologies associated with dyslipoproteinaemias.

17. Pharmaceutical composition comprising one or more defective recombinant viruses according to any one of claims 1 to 16 and a pharmaceutically acceptable vehicle.

18. Pharmaceutical composition according to claim 17,characterized in that it is in injectable form.

19. Pharmaceutical composition according to claim 17 or 18, characterized in that it comprises 0 between 10' and 10" pfu/ml, and preferably 106 to 0* o 1 0 pfu/ml, of defective recombinant adenoviruses.

;20. Mammalian cell infected with one or more defective recombinant viruses according to any one of claims 1 to 15

21. Cell according to claim characterized in that it is a human cell. 000a S0

22. Cell according to claim 20 or 21, 000. characterized in that it is a human cell of fibroblast, f myoblast, hepatocyte, endothelial cell, glial cell or *O 0 keratynocyte type.

23. Implant comprising infected cells according to claims 20 to 22 or cells which produce recombinant viruses according to any one of claims 1 to 17, and an extracellular matrix.

24. Implant according to claim 23, characterized in that the extracellular matrix comprises a gelling compound preferably chosen from 44 collagen, gelatin, glycosaminoglycals, fibronectin and lectins.

Implant according to claims 23 Or 24, characterized in that the extraceilular matrix also comprises a support permitting anchorage of the infected cells.

26. Implant according to claim characterized in that the support preferably consists of polytetrafluoroethylele fibres.

27. Defective recombinant adenovirus according to claim 12, containing, in addition, a gene :coding for an apolipoprotein.

28. Method of treating and/or pr-eventing a pathology associated with dyslipoproteinaemias is comprising administering a virus according to any one of claims I to Dated this .30th day of July 1999. *****Rhone-Poulenlc Rorer S.A. By its Patent Attorneys Davies Collison Cave