IL112993A - Recombinant viruses, their preparation and their use in gene therapy - Google Patents
Recombinant viruses, their preparation and their use in gene therapyInfo
- Publication number
- IL112993A IL112993A IL112993A IL11299395A IL112993A IL 112993 A IL112993 A IL 112993A IL 112993 A IL112993 A IL 112993A IL 11299395 A IL11299395 A IL 11299395A IL 112993 A IL112993 A IL 112993A
- Authority
- IL
- Israel
- Prior art keywords
- afgf
- implant according
- sequence
- adenovirus
- cell
- Prior art date
Links
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/18—Growth factors; Growth regulators
- A61K38/1825—Fibroblast growth factor [FGF]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
- C07K14/50—Fibroblast growth factor [FGF]
- C07K14/501—Fibroblast growth factor [FGF] acidic FGF [aFGF]
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Description
6205/J/99 112993/2 CELL IMPLANTS FOR GENE THERAPY, CONTAINING RECOMBINANT aFGF 1 12993/2 1 The present invention relates to gene therapy, and particularly to gene therapy mediated by implants containing cells bearing a gene of interest. Specifically, the present invention refers to an implant comprising mammalian cells infected with a defective recombinant adenovirus, which lacks a region of its genome necessary for its replication in a host cell, containing a DNA sequence encoding acidic fibroblast growth factor (aFGF) and a secretion sequence 5' to and in the same reading frame as the DNA sequence encoding the aFGF and an extracellular matrix.
Neurodegenerative diseases represent a substantial part of health expenditure, in Western countries, a part which is increasingly rising as a result of the ageing of the population. As examples of these conditions, there may be mentioned especially Alzheimer's disease, Parkinson's disease, Huntington's chorea, amyotrophic lateral sclerosis and the like. The pathological signs and the aetiology of these diseases are quite varied, but they all result from a gradual loss of neuron cells in the central nervous system, sometimes in highly localized structures such as the black substance in Parkinson's disease. Although _§ome palliative pharmacological treatments are already available, their effects are relatively limited". The present invention describes a new, particularly advantageous, therapeutic approach for the treatment of these diseases. More particularly, the present invention describes vectors which make it possible to promote directly the survival of the neuron cells implicated in these pathologies, by the effective and localized expression of certain trophic factors.
Trophic factors are a class of molecules having properties for stimulating neuritic growth or the survival of nerve cells. The first factor possessing neurotrophic properties, NGF (Nerve Growth Factor) , was characterized about forty years ago (for a review, see Levi- ontalcini and Angelleti, Physiol. Rev. 48 (1968) 534) . It was only recently that other neurotrophic factors were identified, and especially the brain-derived neurotrophic factor (BDNF) (Thoenen, Trends in NeuroSci. 14 (1991) 165), CNTF and the like. The Applicant was interested more particularly in the acidic fibroblast growth factor (aFGF) . aFGF is a protein of about 134 to 155 amino acids and a molecular weight of between 15 and 17 kD depending on the forms. aFGF was initially described for its fibroblast growth properties, which made it valuable in the treatment of certain pathologies such as especially cicatrisation. Recently, it has been shown that aFGF was subjected to a retrograde transport in several neuronal populations, whose nigro-striatal (Fergusson et Johnson, J. Comp. Neurol. 313 (1991) 693), and that it could allow, in vitro, the survival of the dopaminergic neurons of the mesencephalon (Knusel et al. J. Neurosci. 10 (1990) 558). WO 93/08828 described intravenous administration of neurotrophic factors such as bFGF, aFGF, NGF, CNTF, BDNF, NT3, NT4, IGF-I and IGF-II, in individuals who suffered neuronal damage due to ischemia, hypoxia, or neurodegeneration. In addition, EP 388226 described the treatment of senile dementia through the use of aFGF, or substances like glucose, which cause aFGF secretion in the brain and peripheral tissues.
However, although its properties are advantageous, the therapeutic application of aFGF is confronted with various obstacles. In particular, the absence of bioavailability of aFGF limits any- therapeutic use. Moreover, there is no effective means allowing aFGF to be delivered in a durable and localized manner to certain desired regions of the body. Finally, it is essential that the aFGF delivered is active and can exert a therapeutic activity in vivo.
The present invention provides a particularly advantageous solution to these problems . The present invention indeed consists in the development of particularly effective vectors to deliver in vivo and locally, therapeutically active quantities of aFGF.
Le Gal La Salle and colleagues (Science 259:988-90, 1993 and Genetics 118, page 229, Abstract 118: 141155w ) described a replication-deficient adenoviral vector that contained a reporter gene encoding beta-galactosidase, which was expressed in almost all sympathetic neurons and astrocytes in culture, with no cytopathic effects. 3a In copending Application No. PCT/EP93/02519, it has been shovm that adenoviruses could be used for transferring genes in vivo into the nervous system. The present invention relates to new constructs which are particularly adapted and effective for transferring a specific gene into the nervous system. More particularly, the present invention relates to a recombinant adenovirus comprising a DNA sequence encoding acidic fibroblast growth factor (aFGF) , to its preparation, and to its use for the treatment and^or prevention of neurodegenerative diseases.
The Applicant has now shown that it is possible to construct recombinant adenoviruses containing a sequence encoding aFGF, to administer these recombinant adenoviruses in vivo, and that this administration allows a stable and localized expression of therapeutically active quantities of aFGF in vivo, and in particular in the nervous system, and without cytopathological effect. The particularly advantageous properties of the vectors of the invention stem especially from the construction used (defective adenovirus, deleted of certain viral regions) , the promoter used for the expression of the sequence encoding aFGF (preferably viral or tissue-specific promoter) , and the methods for administering the said vector, allowing the efficient expression, and in the appropriate tissues, of aFGF. The present invention thus provides viral vectors which can be used directly in gene therapy, which are particularly adapted and efficient for directing the expression of aFGF in vivo. The present invention thus offers a particularly advantageous new approach for the treatment and/or prevention of neurodegenerative diseases.
- A. first subject of the invention therefore consists in a defective recombinant adenovirus comprising a DNA sequence encoding acidic fibroblast growth factor . (aFGF) or a derivative thereof.
The subject of the invention is also the, use of such a defective recombinant adenovirus for the preparation of. a pharmaceutical composition intended for the treatment or prevention of neurodegenerative diseases .
The acidic fibroblast growth factor (aFGF) produced within the framework of the present invention may be human aFGF or animal aFGF. In particular, the DNA sequence encoding human aFGF has, been cloned and sequenced (Jaye et al . , Science 273 (1986) 541). Prior to their incorporation into an adenovirus vector according to the invention, these sequences are advantageously modified, for example by site-directed mutageneses, in particular for the insertion of appropriate restriction sites. The sequences described in the prior art are indeed not constructed for use according to the invention, and prior adaptations may prove necessary, in order to obtain substantial expressions (see Example 1.2.). Within the framework of the present invention, it is preferable to use a DNA sequence encoding human acidic fibroblast growth factor (haFGF) . Moreover, as indicated above, it is also possible to use a construct encoding a derivative of aFGF, in particular a derivative of human aFGF. Such a derivative comprises for example any sequence obtained by mutation, deletion and/or addition compared to the native sequence, and encoding a product conserving at least one of the biological properties of aFGF (trophic and/or differentiator effect) . These modifications can be carried out by techniques known to persons skilled in the art (see general molecular biology techniques below and Example 2) . A derivative may, for example. 6 have a sequence at least 80%, at least 90%, at least 95% or at least 99% identical to the sequence of the 154 amino acid form of human aFGF. The biological activity of the derivatives thus obtained can then be easily determined, as indicated especially in Example 3. The derivatives according to the invention can also be obtained by hybridization from nucleic acid libraries, using as probe the native sequence or a fragment thereof .
These derivatives are especially molecules having a higher affinity for their binding sites, sequences permitting an enhanced expression in vivo, molecules having greater resistance to proteases, molecules having greater therapeutic efficacy or fewer side effects, or possibly new biological properties.
Among the preferred derivatives, there may be mentioned more particularly natural variants of aFGF. Thus, as described for example in Patent US 4,868,113, various forms of aFGF exist, and especially a form comprising 154 amino acids, a form comprising 140 amino acids, and a form comprising 134 amino acids. It is understood. that the term aFGF comprises these various forms. Other preferred derivatives are especially molecules in which one or more residues have been substituted, derivatives obtained by deletion of regions having no, or little, involvement in the interaction with the binding sites considered or expressing an undesirable activity, and derivatives 7 containing, compared with the native sequence, additional residues, such as for example a secretion signal and/or a joining peptide.
In a particularly advantageous manner, the sequence used within the framework of the present invention also contains a secretion signal which makes it possible to direct the synthesized aFGF in the secretory pathways of the infected cells, so that the synthesized aFGF is released more efficiently in the extracellular compartments and can activate its receptors. The secretion signal used may be a heterologous or even artificial secretion signal.
Advantageously, a secretion signal which is functional in nerve cells such as the secretion signal for a cytokine, is used. By way of example, there may be mentioned the secretion signal for human fibroblast interferon which allows high secretion of aFGF.
The DNA sequence encoding the acidic fibroblast growth factor used within the framework of the present invention may be a cDNA, a genomic DNA (gDNA) or a hybrid construct consisting for example of a cDNA in which one or more introns could be inserted. This may also be synthetic or semisynthetic sequences. In a particularly advantageous manner, a cDNA or a gDNA is used. In particular, the use of a gDNA can permit an enhanced expression in human cells.
In a first embodiment of the invention, the adenovirus therefore comprises a cDNA sequence encoding 8 acidic fibroblast growth factor (aFGF) . In another, preferred embodiment of the invention, the adenovirus comprises a gDNA sequence encoding acidic fibroblast growth factor (aFGF) . Advantageously, the DNA sequence encodes aFGF preceded by a heterologous secretion signal which is functional in nerve cells.
Advantageously, the sequence encoding aFGF is placed under the control of signals permitting its expression in nerve cells. Preferably, they are heterologous expression signals, that is to say signals different from those which are naturally responsible for the expression of aFGF. They may be in particular sequences responsible for the expression of other proteins, or of synthetic sequences. In particular, they may be promoter sequences of eukaryotic or viral genes. For example, they may be promoter sequences derived from the genome of the cell which it is desired to infect. Likewise, they may be promoter sequences derived from the genome of a virus, including the adenovirus used. In this respect, there may be mentioned for example the E1A, MLP, CMV, RSV-LTR promoters and the like. In addition, these expression sequences can be modified by the addition of activation or regulatory sequences or sequences permitting a tissue-specific expression. It may indeed be , particularly advantageous to use expression signals which are active specifically or predominantly in the nerve cells, so that the DNA sequence is expressed and produces its effect only when the virus has actually infected a nerve cell. In this respect, there may be mentioned for example neuron-specific enolase promoters, GFAP promoters and the like.
In a first specific embodiment, the invention relates to a defective recombinant adenovirus comprising a cDNA sequence encoding human acidic fibroblast growth factor (haFGF) under the control of the RSV-LTR promoter.
In another specific embodiment, the invention relates to a defective recombinant adenovirus comprising a gDNA sequence encoding human acidic fibroblast growth factor (haFGF) under the control of the RSV-LTR promoter.
The Applicant has indeed shown that the Rous sarcoma virus (RSV) LTR promoter allowed durable and substantial expression of aFGF in the cells of the nervous, especially central nervous, system.
Still in a preferred embodiment, the invention relates to a defective recombinant adenovirus, having a DNA sequence encoding human acidic fibroblast growth factor (haFGF) under the control of a promoter allowing predominant expression in the nervous system.
A particularly preferred embodiment of the present invention consists in a defective recombinant adenovirus comprising the ITR sequences, a sequence allowing encapsulation, a DNA sequence encoding acidic fibroblast growth factor (haFGF) or a derivative thereof under the control of a promoter allowing predominant expression in the nervous system and in which the El gene and at least one of the E2, E4. and L1-L5 genes is non- functional .
The defective adenoviruses according to the invention are adenoviruses which are incapable of replicating autonomously in the target cell. Generally, the genome of the defective adenoviruses used within the framework of the present invention therefore lacks at least the sequences necessary for the replication of the said virus in the infected cell. These regions can be either removed (completely or partly), or rendered non-functional, or substituted by other sequences and especially by the DNA sequence encoding aFGF.
Preferably, the defective virus of the invention conserves the sequences in its genome which are necessary for the encapsulation of the viral particles. Still more preferably, as indicated above, the genome of the defective recombinant virus according to the invention comprises the ITR sequences, a sequence allowing encapsulation, the non-functional El gene and at least one non-functional E2, E4 or L1-L5 gene .
There are various adenovirus serotypes, ^.whose structure and properties vary somewhat. Among these serotypes, the use of the type 2 or 5 human adenoviruses (Ad 2 or Ad 5) or of adenoviruses of 11 animal origin (see Application FR 93 05954) is preferred within the framework of the present invention. Among the adenoviruses of animal origin which can be used within the framework of the present invention, there may be mentioned adenoviruses of canine, bovine, murine (Example: Mavl, Beard et al., Virology 75 (1990) 81) , ovine, procine, avian or alternatively simian (Example: SAV) origin. Preferably, the adenovirus of animal origin is a canine adenovirus, or, more preferably, a CAV2 adenovirus [Manhattan strain or A26/61 (ATCC VR-800) for example] .
Preferably, adenoviruses of human or canine or mixed origin are used within the framework of the invention.
The defective recombinant adenoviruses according to the invention can be prepared by any technique known to a person skilled in the art (Levrero et al., Gene 101 (1991) 195, EP 185 573; Graham, EMBO J. 3 (1984) 2917) . In particular, they can be prepared by homologous recombination between an adenovirus and a plasmid carrying, inter alia, the DNA sequence encoding aFGF. The homologous recombination occurs after co-transfection of the said adenoviruses and plasmid into an appropriate cell line. The cell line used should preferably (i) be transformable by the said elements, and (ii) contain the sequences capable of complementing the defective adenovirus genome part, preferably in integrated form in order to avoid risks of recombination. As an example of a cell line, there may 12 be mentioned the human embryonic kidney line 293 (Graham et al . , J. Gen. Virol. 36 (1977) 59) which contains especially, integrated in its genome, the left hand part of the genome of an Ad5 adenovirus (12%) . Strategies for constructing vectors derived from adenoviruses have also been described in Applications Nos. FR 93 05954 and FR 93 08596 which are incorporated herein by way of reference.
Next, the adenoviruses which have multiplied are recovered and purified according to conventional molecular biology techniques as illustrated in the examples .
As indicated above, the present invention also relates to any use of an adenovirus as described above for the preparation of a pharmaceutical composition intended for the treatment and/or prevention of neurodegenerative diseases. More particularly, it relates to any use of these adenoviruses for the preparation of a pharmaceutical composition intended for the treatment and/or prevention of Parkinson's disease, Alzheimer's disease. Amyotrophic Lateral Sclerosis (ALS) , Huntington's disease, epilepsy and vascular dementia.
The present invention also relates to a pharmaceutical composition containing one or more , defective recombinant adenoviruses as described above. These pharmaceutical compositions can be formulated for topical, oral, parenteral, intranasal, intravenous, 13 intramuscular, subcutaneous, intraocular or transdermal administration and the like. Preferably, the pharmaceutical compositions of the invention contain a pharmaceutically acceptable vehicle for an injectable formulation, especially for a direct injection into the patient's nervous system. This may be in particular isotonic sterile solutions, or dry, especially freeze-dried, compositions which, upon addition, depending on the case, of sterilized water or physiological saline, permit the preparation of injectable solutions. Direct injection into the patient's nervous system is advantageous since it makes it possible to concentrate the therapeutic effect at the level of the affected tissues. Direct injection into the patient's central nervous system is advantageously carried out by means of a stereotaxic injection apparatus. The use of such an apparatus makes it possible, indeed, to target the injection site with great precision.
In this respect, the invention also relates to a method for treating neurodegenerative diseases comprising the administration to a patient of a recombinant adenovirus as defined above. More particularly, the invention relates to the method for treating neurodegenerative diseases comprising the stereotaxic administration of a recombinant adenovirus as defined above.
The doses of defective recombinant adenovirus used for the injection can be adjusted according to 14 various parameters, especially according to the mode of administration used, the pathology concerned, or alternatively the desired duration of treatment.
Generally, 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 10s to 1010 pfu/ml. The term pfu (plague forming unit) corresponds to the infectivity of a virus solution, and is determined by infecting an appropriate cell culture, and then measuring, generally after 48 hours, the number of plagues of infected cells. The techniques for determining the pfu titre of a viral solution are well documented in the literature.
Another subject of the invention relates to any mammalian cell infected by one or more defective recombinant adenoviruses as described above. More particularly, the invention relates to any human cell population infected by these adenoviruses. They may be in particular fibroblasts, myoblasts, hepatocytes, keratinocytes, endothelial cells, glial cells and the like.
The cells according to the invention can be ' obtained from primary cultures. They can be collected by any technique known to a person skilled in the art and then cultured under conditions permitting their proliferation. As regards more particularly fibroblasts, these can be easily obtained from biopsies, for example according to the technique described by Ham [Methods Cell. Biol. 21a (1980) 255]. These cells can be used directly for infection by the adenoviruses, or preserved, for example by freezing, for establishing autologous libraries, for subsequent use. The cells according to the invention may also be secondary cultures obtained for example from pre-established libraries.
The cultured cells are then infected with recombinant adenoviruses, so as to confer on them the capacity to produce aFGF. The infection is carried out in vitro according to techniques known to persons skilled in the art. In particular, according to the type of cells used and desired number of virus copies per cell, persons skilled in the art can adjust the multiplicity of infection and optionally the number of cycles of infection performed. It is clearly understood that these steps should be carried out under appropriate sterile conditions when the cells are intended for administration in vivo. The recombinant adenovirus doses used for the infection of the cells can be adjusted by persons skilled in the art according to the desired aim. The conditions described above for the administration in vivo can be applied to infection in vitro.
Another subject of the invention relates to an implant comprising mammalian cells infected with one or more defective recombinant adenoviruses as described above, and an extracellular matrix. Preferably, the 16 implants according to the invention comprise 10s to 1010 cells. More preferably, they comprise 10s to 108.
More particularly, in the implants of the invention, the extracellular matrix comprises a gelling compound and optionally a support permitting anchorage of the cells.
For the preparation of the implant according to the invention, various types of gelling agents can be used. The gelling agents are used for the inclusion of the cells in a matrix having the constitution of a gel, and to enhance the anchorage of the cells on the support, where appropriate. Various cell adhesion agents can therefore be used as gelling agents, such as especially collagen, gelatin, glycosaminoglycans, fibronectin, lectins, and the like. Preferably, collagen is used in the framework of the present invention. This may be collagen of human, bovine or murine origin. More preferably, type I collagen is used.
As indicated above, the compositions according to the invention advantageously comprise a support by permitting anchorage of the cells. The term anchorage designates any form of biological and/or chemical and/or physical interaction resulting in the adhesion and/or binding of the cells on to the support. Moreover, the cells can either cover the support used, or penetrate inside this support, or both. The use of a solid, non-toxic and/or biocompatible support is 17 preferred within the framework of the invention. In particular, it is possible to use polytetra-fluoroethylene (PTFE) fibres or a support of biological origin.
The implants according to the invention can be implanted at different sites in the body. In particular, the implantation can be carried out in the peritoneal cavity, in the subcutaneous tissue (suprapubian region, iliac and inguinal fossae, and the like) , in an organ, a muscle, a tumour, the central nervous system or alternatively under a mucous membrane. The implants according to the invention are particularly advantageous in the sense that they make it possible to control the release of the therapeutic product in the body: this release is first determined by the multiplicity of infection and by the number of implanted cells. Next, the release can be controlled either by the removal of the implant, which permanently stops the treatment, or by the use of regulable expression systems, which make it possible to induce or to repress the expression of the therapeutic genes.
The present invention thus offers a very effective means for the treatment or prevention of neurodegenerative diseases. It is most particularly adapted to the treatment of Alzheimer's, Parkinson's, Huntington's or ALS diseases. The adenoviral vectors-according to the invention have, in addition, substantial advantages linked especially to their very 18 high efficiency of infection of the nerve cells, which makes it possible to carry out infections using small volumes of viral suspension. Furthermore, the infection by the adenoviruses of the invention is highly localized at the site of injection which avoids the risks of diffusion to the neighbouring cerebral structures .
In addition, this treatment may apply both to man and to any animal such as ovines, bovines, domestic animals (dogs, cats and the like), horses, fish and the like.
The present invention will be more completely described with the aid of the following examples which should be considered as illustrative and non-limiting. Legend to the figures Figure 1: Representation of the vector pXL2244 Figure 2 : Representation of the vector pSh-Ad-aFGF.
General molecular biology technigues The methods conventionally used in molecular biology, such as preparative extractions of plasmid DNA, centrifugation of plasmid DNA in caesium chloride gradient, agarose or acrylamide gel electrophoresis, purification of DNA fragments by electroelution, jahenol or phenol-chloroform extraction of proteins, ethanol or isopropanol precipitation of DNA in saline medium, transformation in Escherichia coli and the like, are well known to persons skilled in the art and are widely described in the literature [Maniatis T. et al., "Molecular Cloning, a Laboratory Manual", Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1982; Ausubel F.M. et al. (eds) , "Current Protocols in Molecular Biology", John Wiley & Sons, New York, 1987].
The pBR322- and pUC- type plasmids and the phages of the M13 series are of commercial origin (Bethesda Research Laboratories) .
For the ligations, the DNA fragments can be separated according to their size by agarose or acrylamide gel electrophoresis, extracted with phenol or with a phenol/chloroform mixture, precipitated with ethanol and then incubated in the presence of phage T4 DNA ligase (Biolabs) according to the recommendations of the supplier.
The filling of the protruding 5' ends can be performed with the Klenow fragment of E. coli DNA polymerase I (Biolabs) according to the specifications of the supplier. The destruction of the protruding 3' ends is performed in the presence of phage T4 DNA polymerase (Biolabs) used according to the recommendations of the manufacturer. The destruction of the protruding 5' ends is performed by a controlled treatment with SI nuclease.
Site-directed mutagenesis in vitro by synthetic oligodeoxynucleotides can 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 the DNA fragments by the so-called FCR technique [Polymerase-catalyzed Chain Reaction, Saiki R.K. et al., Science 230 (1985) 1350-1354; Mullis K.B. and Faloona F.A., Meth. Enzym. 155 (1987) 335-350] can be performed using a DNA thermal cycler (Perkin Elmer Cetus) according to the specifications of the manufacturer.
The verification of the nucleotide sequences can be performed by the method developed by Sanger et al. [Proc. Natl. Acad. Sci. USA, 74 (1977) 5463-5467] using the kit distributed by Amersham.
Examples Example 1: Construction of the vector pSh-Ad-aFGF This example describes the construction of a vector comprising a DNA sequence encoding aFGF preceded by a heterologous secretion sequence under the control of a promoter consisting of the Rous sarcoma virus LTR (RSV-LTR) . 1.1. Starting vector (pXL2244) : The plasmid pXL2244 contains the ApoAI cDNA under the control of the RSV virus LTR promoter, as well as the Ad5 adenovirus sequences (Figure 1) . It was constructed by inserting a Clal-EcoRV fragment containing the cDNA encoding preproApoAI into the vector pLTR RSV-/Sgal (Stratford-Perricaudet et al., J. Clin. Invest. 90 21 (1992) 626), digested with the same enzymes. 1.2. Construction of a cDNA sequence encoding aFGF.
In order to allow the construction of vectors according to the invention, a cDNA sequence encoding human aFGF was constructed as follows: - a cDNA sequence encoding human aFGF comprising 134 amino acids (aFGF134) was isolated from the plasmid p J26 (Jaye et al., J. Biol. Chem. 262 (1987) 16612) . The plasmid pMJ26 contains a cDNA sequence encoding human aFGF preceded by the sequence of the EcoRI restriction site. The sequence of the plasmid in this region is the following (EcoRI site underlined and start codon in bold) : ... GAATTCATG...
After digestion with the EcoRI enzyme, the ends of the restriction site were made blunt by treating with mung bean nuclease. The fragments obtained possess the following ends (start codon in bold) : ...GAATTCATG... (pMJ26) ...G CATG ... (EcoRI + mung bean nuclease) This sequence was then modified by inserting a secretion sequence. The introduction of this sequence is particularly advantageous since it allows more efficient extracellular release of the aFGF synthesized by the cells infected with the vectors of the invention. More particularly, the sequence obtained above was modified by insertion, at the 5' end and in phase with the aFGF, of a double-stranded synthetic oligonucleotide corresponding to the secretion sequence for human fibroblast interferon (Taniguchi et al . , Gene 10 (1980) 11) . Each strand of this oligonucleotide was chemically synthesized by means of a nucleic acid synthesizer and then hybridized in order to reconstitute the double-stranded DNA. The sequence used contains the following 70 bp chain: GGCTCGAG ATG ACC AAC AAG TGT CTC CTC CAA ATT GCT CTC CTG TTG TGC Met Thr Asn Lys Cys Leu Leu Gin lie Ala Leu Leu Leu Cys TTC TCC ACT ACA GCT CTT TC Phe Ser Thr Thr Ala Leu Se This oligonucleotide comprises, at the 5' end of the secretion sequence, a Xhol site (underlined in the sequence above) . This double-stranded oligonucleotide was ligated to the aFGF-encoding sequence digested with EcoRI and mung bean nuclease as described above. Ά clone having the insert in the correct orientation was then isolated and checked by sequencing. The complete fragment present in this clone, containing the aFGF-encoding sequence preceded by the secretion sequence, was then isolated by digestion with the Xhol and Bglll enzymes (Bglll -site situated after the stop codon) , are then ligated to the vector p267 (Jaye et al., EMBO J. 7 (1988) 963) previously opened with the same enzymes. The resulting 23 vector was designated pMJ35.
The sequence prepared above was then modified by the PCR technique, using as primer the following oligonucleotides : ' oligonucleotide: 5 ' -GCGATCGATCTCGAGATGACCAACAAG-3 ' 3' oligonucleotide: 5 ' -CTCGGTACCTCTTTAATCAGAAGAGAC-3 ' The amplification steps by PCR on the plasmid pMJ35 make it possible to introduce appropriate restriction sites on either side of the chimeric sequence "secretion/aFGF sequence". More specifically, these steps allow the incorporation of a Clal site at the 5' end (sequence underlined with a single line in the 5' oligonucleotide, which indicates its position relative to the start codon underlined with a double line) and of a Kpnl site at the 3' end (underlined with a single in the 3' oligonucleotide) . The sites created permit the insertion of the sequence under expression conditions into the vectors of the invention. 1.3. Construction of the vector pSh-Ad-aFGF This example describes the construction of the vector pSh-Ad-aFGF containing the sequence encoding aFGF preceded by a secretion sequence, under the control of the RSV virus LTR, as well as Ad5 adenovirus sequences permitting the recombination in vivo.
The PCR fragment prepared in Example 1.2. was digested with the enzymes Clal and Kpnl, and the resulting 0.5 kb fragment, containing the sequence encoding aFGF preceded by the secretion signal for human fibroblast interferon, was then isolated and purified by LMP (Low Melting Point) agarose gel electrophoresis. In parallel, the vector pXL2244 was digested with the same Clal and Kpnl restriction enzymes, and then precipitated after inactivation of the latter. The resulting linear vector, previously isolated and purified by agarose gel electrophoresis, and the 0.5 kb fragment were then ligated in order to generate the vector pSh-Ad-aFGF (Figure 2) . The entire nucleotide sequence of the aFGF insert was checked by dideoxynucleotide sequencing.
Example 2. Functionality of the vector pSh-Ad-aFGF The capacity of the vector pSh-Ad-aFGF to express in cell culture a biologically active form of aFGF was demonstrated by transient transfection of COSl cells. For that, the cells (2 x 10s cells per dish 10 cm in diameter) were transfected (8 μg of vector) in the presence of Transfectam. After 48 hours, the cell culture supernatant was harvested. Serial dilutions (1/200 and 1/50) of this supernatant were then added to NIH 3T3 cell cultures. The trophic effect on these cultures was observed by incorporation of radio labelled thymidine. In parallel, the production of aFGF by the transfected cells was also demonstrated by immunodetections.
Example 3. Construction of a recombinant adenovirus Ad-aFGF containing a sequence encoding aFGF The vector pSh-Ad-aFGF was linearized and cotransfected with a deficient adenoviral vector, into the helper cells (line 293) providing in trans the functions encoded by the adenovirus El regions (E1A and E1B) .
More specifically, the adenovirus Ad-aFGF was obtained by homologous recombination in vivo between the mutant adenovirus Ad-dll324 (Thimmappaya et al., Cell 31 (1982) 543) and the vector pSh-Ad-aFGF, according to the following procedure: the plasmid pSh-Ad-aFGF and the adenovirus Ad-dll324, linearized with the enzyme Clal, were cotransfected into line 293 in the presence of calcium phosphate, so as to allow the homologous recombination. The recombinant adenoviruses thus generated were selected by plague purification. After isolation, the recombinant adenovirus DNA was amplified in the cell line 293, which gives a culture supernatant containing the unpurified recombinant defective adenovirus having a titre of about 1010 pfu/ml.
The viral particles were then purified by caesium chloride gradient centrifugation according to known techniques (see especially Graham et al., Virology 52 (1973) 456) . The adenovirus Ad-aFGF can be preserved at -80°C in 20% glycerol. 26 Example 4. Functionality of the adenovirus Ad-aFGF The capacity of the adenovirus Ad-aFGF to infect cultured cells and to express in the culture medium a biologically active form of aFGF is demonstrated by infecting human 293 and rat PC12 lines. The presence of active aFGF in the culture supernatant was then determined under the same conditions as in Example 2.
These studies make it possible to demonstrate that the adenovirus does indeed express a biologically active form of aFGF in cell culture.
Example 5: Transfer in vivo of the aFGF gene by a recombinant adenovirus to rats with lesion of the fimbria-fornix This example describes the transfer of the aFGF gene in vivo by means of an adenoviral vector according to the invention. It shows, on an animal model of lesion of the fimbria-fornix, that the vectors of the invention make it possible to induce the expression in vivo of therapeutic quantities of aFGF.
In previously anaesthetized rats, the septo-hippocampal route (fimbria- fornix) was sectioned at the level of the left hemisphere. This mechanical lesion was made with the aid of a retractable surgical knife. The stereotaxic coordinates used to this effect are, relative to the bregma: AP.-1.7; ML: +1.5; V:-5.5 to -0.5. 27 The aFGF recombinant adenovirus was injected immediately after the lesion, into the median nucleus of the septum and into the dorsal part of the deafferentated hippocampus (hippocampus on the lesion side) . More particularly, the injected adenovirus is the adenovirus Ad-aFGF prepared in Example 3.1., used in purified form (3.5 x 10s pfu/μΐ) , in a phosphate buffered saline solution (PBS) .
The injections are carried out with the aid of a cannula (external diameter 280 μπι) connected to a pump. The rate of injection is fixed at 0.5 μΐ/min, after which, the cannula remains in place for 4 additional minutes before being withdrawn. The volumes of injection into the hippocampus and the septum are respectively 3 μΐ and 2 μΐ. The adenovirus concentration injected is 3.5 x 10s pfu/μΐ.
For injection into the hippocampus, the stereotaxic coordinates are the following: AP = -4; ML = 3.5; V = -3.1 (the AP and ML coordinates are determined relative to the bregma, the V coordinate relative to the surface of the cranial bone at the level of the bregma.
For the injection into the septum, the stereotaxic coordinates are the following: AP = 1; ML = 1; V = -6 (the AP and ML coordinates are determined relative to the bregma, the V coordinte relative to the surface of the cranial bone at the level of the bregma. Under this condition, the cannula 28 is at an angle of 9 degrees relative to the vertical (in the mediolateral direction) in order to avoid the median venous sinus .
The therapeutic effects of the administration of the adenovirus according to the invention have been demonstrated by three types of analysis: a histological and immunohistochemical analysis, a quantitative analysis and a behavioural analysis.
Histological immunohistochemical analysis The mechanical lesion of the fimbria-fornix induces a loss of cholinergic neurons (visualized in immunohistology by an anti-choline acetyl transferase, ChAT, antibody) in the median septum, as well as cholinergic denervation in the hippocampus (detected in histochemistry by the acetyl choline esterase, AChE, activity) .
Histological analysis of the injected brains is carried out 3 weeks after the intracerebral injection of the adenovirus Ad-aFGF. For that, the animals are sacrificed, under anaesthesia, by intracardiac infusion of 4% paraformaldehyde. After removal, postfixing and cryoprotection, the brain is sectioned using a cryomat along the coronal plane: coronal serial sections 30 μιη thick are made over the entire length of the median septum and in the anterior, median and posterior regions of the hippocampus. For the median septum, sections 180 μιη apart (1 section out of 6) are stained with cresyl violet (in order to evaluate the neuronal density) and immunolabelled with an anti-ChAT antibody (Biochem) (so as to identify the cholinergic neurons) . The immunohistochemical method is that of streptavidin-biotin peroxidase visualized with DAB. For the hippocampus, sections 180 μπι apart are stained according to the histochemical method for AChE (acetyl choline esterase) so as to detect the cholinergic innervation. The sections are mounted on glass slides.
Quantitative analysis The number of cholinergic neurons (ChAT-positive) , in the median septum is the parameter for evaluation of the effects of the adenovirus Ad-aFGF. The enumeration is carried out on a sample (1 section out of 6 over the entire length of the median septum) . For each section, the ChAT-positive neurons are counted separately on both sides of the septum. The cumulative results for all the sections are expressed by the ratio of the number of ChAT-positive neurons on the injured side over the number of ChAT-positive neurons on the uninjured side.
Behavioural analysis It is known that a bilateral lesion of the septo-hippocampal route leads to memory deficiency. The protective functional effects of the injection of adenovirus Ad-aFGF on this type of lesion were detected by analysis of the memory performances of the animals during 2 behavioural tests: the Morris swimming pool test (visuospatial reference memory) and the TMTT test (two-trials memory task; "short-term memory of a new environment") .
Example 6: Transfer in vivo of the aFGF gene by a recombinant adenovirus to rats with lesion of the nigro-striatal route This example describes the transfer of the aFGF gene in vivo by means of an adenoviral vector according to the invention. It shows, in an animal model of the lesion of the nigro-striatal route, that the vectors of the invention make it possible to induce the expression in vivo of thereapeutic quantities of aFGF .
In previously anaesthetized rats, the nigro-striatal route was injured at the level of the median mesencephalic bundle (MFB) by injection of the toxin 6-hydroxydopamine (60H-DA) . This chemical lesion by injection was unilateral along the following stereotaxic coordinates: AP: 0 and -1; ML: +1.6; V: -8.6 and -9 (the AP and ML coordinates are determined relative to the bregma, the V coordinate relative to the dura mater) . The incisive bar is fixed at the +5 mm level .
The recombinant adenovirus aFGF was injected 31 immediately after the lesion, into the black substance and the striatum, on the lesion side. More particularly, the injected adenovirus is the adenovirus Ad-aFGF prepared in Example 3.1., used in purified form (3.5 x 10s pfu/μΐ) , in a phosphate buffered saline solution (PBS) .
The injections were carried out with the aid of a cannula (external diameter 280 μχη) connected to a pump. The rate of injection is fixed at 0.5 μΐ/min, after which the cannula remains in place for an additional 4 minutes before being withdrawn. The injection volumes into the striatum and the black substance are 2 x 3 μΐ and 2 μΐ respectively. The adenovirus concentration injected is 3.5 x 106 pfu/μΐ.
For the injection into the black substance, the stereotaxic coordinates are the following: AP = -5.8; ML = +2; V = -7.5 (the AP and ML coordinates are determined relative to the bregma, the V coordinate relative to the dura mater) .
For the injections into the striatum, the stereotaxic coordinates are the following: AP = +0.5 and -0.5; ML = 3; V = -5.5 (the AP and ML coordinates are determined relative to the bregma, the V coordinate relative to the dura mater) .
The therapeutic effects of the administration of the adenovirus according to the invention were detected by three types of analysis: a histological and immunohistochemical analysis, a quantitative analysis 32 and a behavioural analysis.
Histological and immunohistochemical analysis Chemical lesion of the nigro-striatal route induces neuronal loss in the black substance as well as the dopaminergic denervation in the striatum (visualized in immunohistology by an anti-tyrosine hydroxylase, TH, antibody) .
Histological analysis of the injected brains is carried out 3 weeks after the intracerebral injection of the adenovirus Ad-aFGF under the conditions described in Example 6. The coronal serial sections 30 μχη thick are made in the black substance and the striatum. Sections 180 μπι apart (1 section out of 6) are stained with cresyl violet (so as to evaluate the neuronal density) and immunolabelled with an anti-tyrosine hydroxylase (TH) antibody (so as to detect the dopaminergic neurons in the black substance and their innervation in the striatum) .
Quantitative analysis The number of dopaminergic neurons (TH-positive) in the black substance is the parameter for evaluation of the effects of the adenovirus Ad-aFGF. The enumeration is carried out on a sample (1 section out of 6 over the entire length of the black substance) . For each section, the TH-positive neurons are counted separately on both sides of the black substance. The cumulative results for all the sections are expressed as a proportion: number of TH-positive neurons on the injured side relative to the number of TH-positive neurons on the uninjured side.
Behavioural analysis The protective functional effects of the injection of adenovirus Ad-aFGF on the lesion of the nigro-striatal route were detected by analysis of the sensorimotor performances of the animals in 2 behavioural tests: the tests of rotation induced by dopaminergic agonists (apomorphine, amphetamine and levodopa) , and the prehension (paw-reaching) test.
Example 7: Transfer in vivo of the aFGF gene by a recombinant adenovirus to rats with lesion of the perforating route This example describes the transfer of the aFGF gene in vivo by means of an adenoviral vector according to the invention. It shows, in an animal model of the lesion of the perforating route, that the vectors of the invention make it possible to induce the expression in vivo of therapeutic quantities of aFGF.
In previously anaesthetized rats, the entorhinohippocampal route (perforating route) was unilaterally sectioned with the aid of a surgical knife. The stereotaxic coordinates used to this end are, relative to the lambda: ΆΡ: +0.75; ML: +4.1 to 34 6.6; V: -7.7 (V coordinate determined relative to the dura mater) .
The recombinant adenovirus aFGF is injected immediately after the lesion, either at the level of the lesion, or at the level of the hippocampus and the entorhinal cortex. More particularly, the injected adenovirus is the adenovirus Ad-aFGF prepared in Example 3.1., used in purified form (3.5 x 10s pfu/μΐ) , in a phosphate buffered saline solution (PBS) .
The injections were carried out with the aid of a cannula (external diameter 280 μα.) connected to a pump. The rate of injection is fixed at 0.5 μΐ/min, after which the cannula remains in place for an additional 4 minutes before being withdrawn. The injection volumes into the hippocampus, the entorhinal cortex and the lesion site of the perforating route are 3 μΐ, 2 μΐ and 2 μΐ respectively. The adenovirus concentration injected is 3.5 x 10s pfu/μΐ.
The therapeutic effects of the administration of the adenovirus according to the invention can be detected by a behavioural analysis under the conditions of Example 5.
Parts of the description which are out of ambit of the following claims do not constitute part of the claimed invention.
Claims (17)
1. An implant comprising mammalian cells infected with a defective recombinant adenovirus, which lacks a region of its genome necessary for its replication in a host cell, containing a DNA sequence encoding acidic fibroblast growth factor (aFGF) and a secretion sequence 5' to and in the same reading frame as the DNA sequence encoding the aFGF and an extracellular matrix.
2. An implant according to Claim 1, wherein said cell is a human cell preferably selected from the group of fibroblast, myoblast, hepatocyte, endothelial cell, glial cell and keratynocyte.
3. An implant according to claim 1 or 2, wherein said extracellular matrix comprises a gelling compound preferably selected from the group of collagen, gelatin, glucosaminogylcan, fibronectin and lectins.
4. An implant according to any of claims 1 to 3, wherein the extracellular matrix comprises a support permitting anchorage of the infected cells.
5. * An implant according · to Claim 4, wherein the support is preferentially polytetrafluoroethylene fibres.
6. An implant according to any of claims 1 to 5, wherein the secretion sequence is functional in nerve cells, preferably said secretion sequence is a cytokine.
7. An implant according to any of claims 1 to 6, wherein the DNA sequence is a complementary DNA (cDNA) sequence. -36- 112993/2
8. An implant according to any of claims 1 to 6, wherein the DNA sequence is a genomic DNA (gDNA) sequence.
9. An implant according to any of claims 1 to 8, wherein said DNA sequence encodes the human 154, 140 or 134 amino acid aFGF protein molecule, preferably the 134 amino acid molecule.
10. An implant according to any of claims 1 to 9, wherein the DNA sequence is under the control of a signal permitting its expression in a nerve cell.
11. An implant according to any of claims 1 to 10, wherein the expression signal is selected from the group of viral promoters, preferably El A, MLP, CMV and RSV-LTR promoters.
12. An implant according to any of claims 1 to 11, wherein the cDNA sequence encoding human aFGF is preceded by a secretion sequence, under the control of the RSV-LTR promoter.
13. An implant according to any of claims 1 to 11, wherein the gDNA sequence encoding human aFGF is preceded by a secretion sequence, under the control of the RSV-LTR promoter.
14. An implant according to any of claims 1 to 13, wherein the DNA sequence encoding human aFGF is under the control of a promoter permitting selective or specific expression in nerve cells.
15. An implant according to any of claims 1 to 14, wherein said promoter is the neuron-specific enolase promoter or the GFAP promoter.
16. An implant according to any of claims 1 to 15, wherein said recombinant defective adenovirus comprises the ITRs and a sequence -37- 112993/2 permitting encapsulation, and in which the El gene and at least one of the E2, E4, or L1-L5 genes are nonfunctional.
17. An implant according to any of claims 1 to 16, wherein said recombinant defective adenovirus is a type Ad 2 or Ad 5 human adenovirus or a CAV-2 type canine adenovirus.
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| FR9403190A FR2717495B1 (en) | 1994-03-18 | 1994-03-18 | Recombinant viruses, preparation and use in gene therapy. |
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| EP (1) | EP0750675A1 (en) |
| JP (1) | JPH09510357A (en) |
| AU (1) | AU1896195A (en) |
| CA (1) | CA2184409A1 (en) |
| FR (1) | FR2717495B1 (en) |
| IL (1) | IL112993A (en) |
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| US5792453A (en) * | 1995-02-28 | 1998-08-11 | The Regents Of The University Of California | Gene transfer-mediated angiogenesis therapy |
| US6100242A (en) * | 1995-02-28 | 2000-08-08 | The Regents Of The University Of California | Gene therapies for enhancing cardiac function |
| SI20750A (en) * | 1999-04-26 | 2002-06-30 | Aventis Pharma S.A. | Use of a recombinant defective adenovirus comprising a nucleic acid encoding an angiogenic factor for treating pulmonary hypertension |
| FR2792531B1 (en) * | 1999-04-26 | 2003-01-31 | Aventis Pharma Sa | USE OF DEFECTIVE RECOMBINANT ADENOVIRUS COMPRISING A NUCLEIC ACID ENCODING AN ANGIOGENIC FACTOR FOR THE TREATMENT OF PULMONARY ARTERIAL HYPERTENSION |
| ATE393235T1 (en) * | 2000-11-27 | 2008-05-15 | Dnavec Research Inc | PARAMYXOVIRUS VECTOR ENCODING FIBROBLAST GROWTH FACTOR 2 (FGF2) AND USE THEREOF |
| US20070134204A1 (en) * | 2005-12-09 | 2007-06-14 | Henrich Cheng | Method for treating nerve injury and vector construct for the same |
| WO2009128474A1 (en) | 2008-04-16 | 2009-10-22 | 財団法人化学及血清療法研究所 | Method of producing thrombin-immobilized bioabsorbable sheet preparation |
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| IE914393A1 (en) * | 1990-12-18 | 1992-07-01 | Amgen Inc | Analogs of acidic fibroblast growth factor having enhanced¹stability and biological activity |
| WO1993008828A1 (en) * | 1991-11-08 | 1993-05-13 | Syntex-Synergen Neuroscience Joint Venture | Methods for the treatment of neuronal damage associated with ischemia, hypoxia or neurodegeneration |
| FR2688514A1 (en) * | 1992-03-16 | 1993-09-17 | Centre Nat Rech Scient | Defective recombinant adenoviruses expressing cytokines and antitumour drugs containing them |
| NZ256018A (en) * | 1992-09-25 | 1997-07-27 | Rhone Poulenc Rorer Sa | Recombinant adenoviral vectors and their use in directing expression and transcription of selected nucleotide sequences in cells of the central nervous system |
| FR2704234B1 (en) * | 1993-04-22 | 1995-07-21 | Centre Nat Rech Scient | RECOMBINANT VIRUSES, PREPARATION AND USE IN GENE THERAPY. |
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- 1995-03-02 EP EP95911370A patent/EP0750675A1/en not_active Withdrawn
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| WO1995025803A1 (en) | 1995-09-28 |
| ZA952242B (en) | 1996-01-09 |
| FR2717495B1 (en) | 1996-04-12 |
| EP0750675A1 (en) | 1997-01-02 |
| IL112993A0 (en) | 1995-06-29 |
| FR2717495A1 (en) | 1995-09-22 |
| AU1896195A (en) | 1995-10-09 |
| CA2184409A1 (en) | 1995-09-28 |
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