EP0946740A1 - Neue konstruktionen und vektoren für gezielte und induzierbare gen-expression - Google Patents

Neue konstruktionen und vektoren für gezielte und induzierbare gen-expression

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Publication number
EP0946740A1
EP0946740A1 EP97913264A EP97913264A EP0946740A1 EP 0946740 A1 EP0946740 A1 EP 0946740A1 EP 97913264 A EP97913264 A EP 97913264A EP 97913264 A EP97913264 A EP 97913264A EP 0946740 A1 EP0946740 A1 EP 0946740A1
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EP
European Patent Office
Prior art keywords
promoter
region
recombinant vector
gene
recombinant
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP97913264A
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English (en)
French (fr)
Inventor
Abderrahim Mahfoudi
Patrick Benoit
Didier Branellec
Patrice Denefle
Nicolas Duverger
Laurence Berthou
Johan Auwerx
Bart Staels
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Aventis Pharma SA
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Rhone Poulenc Rorer SA
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Application filed by Rhone Poulenc Rorer SA filed Critical Rhone Poulenc Rorer SA
Publication of EP0946740A1 publication Critical patent/EP0946740A1/de
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • 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
    • C07K14/745—Blood coagulation or fibrinolysis factors
    • C07K14/755—Factors VIII, e.g. factor VIII C (AHF), factor VIII Ag (VWF)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/42—Vector systems having a special element relevant for transcription being an intron or intervening sequence for splicing and/or stability of RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/80—Vector systems having a special element relevant for transcription from vertebrates
    • C12N2830/85—Vector systems having a special element relevant for transcription from vertebrates mammalian

Definitions

  • the present invention relates to the field of biology, and in particular to the field of regulation of gene expression. It describes in particular new constructions and new vectors allowing a targeted and inducible expression of genes.
  • the present invention can be used in many fields, and in particular for the production of recombinant proteins, for the creation of transgenic animal models, for the creation of cell lines, for the development of screening tests, or even in gene therapy. and cellular.
  • the ability to control and direct gene expression is a very important issue in the development of biotechnology. In vitro, it improves the conditions for the production of recombinant proteins, for example by decoupling the cell growth phase and the production phase. Still in vitro, it also makes it possible to create cell lines capable of producing certain molecules at selected times. Thus, it is conceivable to construct cell lines producing, in a regulated manner, proteins transcomplementing defective viral genomes. Still in vitro, a regulated expression system allows the development of screening tests for molecules acting on the control of gene expression. The control of gene expression is also very important for ex vivo or in vivo therapeutic approaches, in which the possibility of selectively controlling the production of a therapeutic molecule is essential. In fact, depending on the applications, depending on the gene to be transferred, it is important to be able to target certain tissues or only certain parts of an organism in order to concentrate the therapeutic effect and limit dissemination and side effects.
  • This targeting can be carried out using vectors having a specific cell specificity.
  • Another approach is to use expression signals specific for certain cell types.
  • specific promoters have been described in the literature, such as the promoter of the genes coding for pyruvate kinase, villin, GFAP, the promoter of the intestinal fatty acid binding protein, the promoter ⁇ -actin from smooth muscle cells, or the promoter of the human albumin gene for example.
  • these promoters have a certain tissue specificity, they are not regulable and therefore offer limited possibilities of control.
  • application WO96 / 01313 describes a gene expression system regulated by tetracyc ne.
  • the invention now describes new constructs allowing targeted and regulated expression of genes
  • the invention describes in particular recombinant vectors allowing expression of inducible and hepatospecific genes
  • the invention also describes new promoter constructs having improved regulatory levels La
  • the present invention thus provides a particularly efficient means for targeting the expression of genes in hepatic cells, in vivo or in vitro, and for regulating this expression.
  • apoAII apolipoprotein Ail Apolipoprotein Ail
  • HDL high density lipoproteins
  • ApoAII is mainly synthesized in the liver, although contradictory results suggest a synthesis also in the intestine
  • the human apoAII gene has been cloned and sequenced (Tsao et al, J Biol Chem 260 (1985) 15222)
  • the promoter region extends sure approximately 1 kb upstream of the transcription initiation codon.
  • nucleotide -903 to -680 level includes regulatory elements located at the nucleotide -903 to -680 level, as well as additional multiple sites located at the intermediate (nucleotide -573 to -255) and proximal (-126 to -33) region.
  • Optimal expression is obtained when nuclear factors are linked to the proximal and distal regulatory elements of the promoter.
  • sequence of the promoter of the human apoAII gene from residue - 911 to +29 is shown in the sequence SEQ ID No. 1.
  • Fibrates often used as hypolipidemic agents, belong to the chemical family of peroxisome proliferators, insofar as they induce hepatomegaly linked to the proliferation of peroxisomes in rodents Their action is mediated by activated receptors (PPAR "Peroxisome Proliferator Activated Receptor"), a group of 4 nuclear receptors distinct ( ⁇ , ⁇ , ⁇ , ⁇ ).
  • PPAR Peroxisome Proliferator Activated Receptor
  • PPARs belong to the nuclear hormone receptor superfamily which binds to specific response elements called PPRE ("Peroxisome Proliferator Response Element")
  • PPREs have been identified in many genes coding for enzymes involved in the ⁇ -oxidation pathway , which have been shown to be inducible by fibrates
  • the applicant has now developed a system of expression of hepatospecific genes inducible by fibrates, usable in vitro and in vivo. More particularly, the applicant has constructed for the first time a vector having a tropism for the liver allowing expression of genes selectively in the liver or liver cells, and inducibly by fibrates. The Applicant has also constructed new promoters derived from the promoter of the human apoAII gene, having improved inducibility and strength properties
  • a first object of the invention resides in a recombinant vector for the inducible and hepatospecific expression of a characterized molecule in that it comprises, an expression cassette consisting of a nucleic acid encoding said molecule placed under the control of the promoter of the gene for human apolipoprotein Ail.
  • the recombinant vector is a viral vector derived from adenoviruses, comprising, inserted into its genome, said expression cassette.
  • the hepatospecific nature of the viruses of the invention means that these viruses allow the expression of a gene very selectively in hepatic cells, in vitro, ex vivo or in vivo. Low non-specific expression in other tissues or cell types can be tolerated, as long as a majority expression is observed in liver cells (preferably more than 80% of cells expressing the transgene are liver cells. more preferably, more than 90%)
  • the virus according to the invention does not induce any detectable expression in the intestine and therefore offers a particularly high selectivity. This is very important for toxic gene transfer and expression approaches, for which a very high level of selectivity is necessary.
  • the inducible systems described in the prior art have a medium inducibility, a factor of about five.
  • the results presented in the examples demonstrate that the adenovirus of the invention is inducible by a factor of about ten.
  • the level of inducibility is also very important for obtaining control over the quantity of molecules delivered in vivo. This is particularly sensitive in the case of immunogenic molecules or molecules capable of generating inflammatory responses. This is also particularly interesting for the expression of molecules whose biological efficiency involves high concentrations.
  • Another characteristic The particularly remarkable vector of the invention resides in the high levels of expression obtained.
  • inducible systems generally have, on the other hand, medium or even low levels of expression.
  • the system of the invention makes it possible to obtain expression levels in vivo comparable to those described for the strongest constitutive promoters.
  • the system of the invention therefore combines for the first time remarkable properties of selectivity, inducibility, and strength.
  • One of the aspects of the invention therefore resides in the use of the promoter of the human apoAII gene.
  • Another aspect of the invention lies in the construction of vectors derived from adenoviruses.
  • the vectors according to the invention combine remarkable properties of gene transfer, safety, tissue specificity, inducibility and strength.
  • the promoter used in the viruses of the invention comprises the regulatory elements of the promoter of the apoAII gene.
  • the promoter comprises the residues -91 1 to +29 of the apoAII gene (sequence SEQ ID ⁇ ° 1).
  • the promoter can be used.
  • the promoter understands the site of initiation of the transcription of the apoAII gene (numbered +1 on SEQ ID No. 1).
  • this promoter does not contain the first intron of the apoAII gene, which begins at nucleotide +38.
  • the fragment used has a 3 ′ end comprised between the residues +5 and +35, more preferably +10 and +30 of the apoAII gene.
  • the 5 'end it is preferable, in order to obtain high levels of expression in the liver, to retain at least part of the hepatic factor binding site.
  • This site is located at nucleotides -903 to -680. Therefore, advantageously, the fragment used has a 5 ′ end located upstream of the nucleotide -903. This end can be located for example in the region -950 to -910. Furthermore, for reasons of capacity cloning, it is advantageous to use a promoter region of reduced size. Therefore, it is preferred to use a fragment whose 5 'end is located in the region -925 to -910.
  • the promoter comprises the regulatory elements located at the nucleotides -903 to -680 (or - 903 to -720) and -126 to -33, but not the intermediate elements located at the nucleotides -573 to -255.
  • the promoter used advantageously comprises a deletion in the region between the residues -710 and -150.
  • the promoter can advantageously consist of a variant of the sequence SEQ ID No. 1 obtained by deletion of residues 708-210.
  • the promoter used comprises a deletion in the region between the residues -670 and -210.
  • the promoter can advantageously consist of a variant of the sequence SEQ ID No. 1 obtained by deletion of the residues; 653-210.
  • the adenoviruses according to the invention comprise as promoter a variant of the promoter of the apolipoprotein Ail gene comprising a repetition of J motifs. Multiplication of the J region advantageously also makes it possible to increase the character inducible by promoter fibrates.
  • Region J consists of the sequence TCAACCTTTACCCTGGTAG (SEQ ID No. 2, underlined on SEQ ID No. 1) It is localized in the Ail promoter at the level of nucleotides -734 to -716 of the promoter.
  • the Applicant has now constructed recombinant viruses comprising promoters modified at the level of the J region. These viruses have particularly advantageous properties for the transfer and expression of genes, in vitro and in vivo.
  • the promoter comprises from 2 to 5 J units. Even more preferably, it comprises 3 J units.
  • the repetition of J units can be positioned 5 ′ of the promoter, 3 ′ of the promoter, or inserted into the promoter sequence, preferably at the level of the native J sequence.
  • the multiplication of patterns J can advantageously be combined with a deletion as described above. This makes it possible to obtain a promoter having properties which are further improved in terms of power and of gene expression control.
  • the remarkable capacities of the vectors of the invention derive from the promoter used, as from the choice of the vector.
  • the demonstration of the functionality of the promoters in an adenoviral context in vivo has indeed made it possible to produce these very vectors. efficient.
  • Adenoviruses are linear double-stranded DNA viruses around 36 (kilobases) kb in size. There are different serotypes, the structure and properties of which vary somewhat, but which have a comparable genetic organization. More particularly, the recombinant adenoviruses can be of human or animal origin. As regards adenoviruses of human origin, mention may preferably be made of those classified in group C, in particular adenoviruses of type 2 (Ad2), 5 (Ad5), 7 (Ad7) or 12 (Ad12).
  • adenoviruses of animal origin mention may preferably be made of adenoviruses of canine origin, and in particular all the strains of the adenovirus CAV2 [Manhattan strain or A26 / 61 (ATCC VR-800) for example].
  • Other adenoviruses of animal origin are cited in particular in application WO94 / 26914 incorporated herein by reference.
  • the adenovirus genome includes in particular a repeated inverted sequence (ITR) at each end, an encapsidation sequence (Psi), early genes and late genes.
  • ITR inverted sequence
  • Psi encapsidation sequence
  • the main early genes are contained in the E1, E2, E3 and E4 regions. Among these, the genes contained in the E1 region in particular are necessary for viral propagation.
  • the main late genes are contained in regions L1 to L5.
  • the genome of the Ad5 adenovirus has been fully sequenced and is accessible on the database (see in particular Genebank M73260). Likewise, parts or even all of other adenoviral genomes (Ad2, Ad7, Ad12, etc.) have also been sequenced.
  • adenovirus For their use as recombinant vectors, various constructs derived from adenoviruses have been prepared, incorporating different therapeutic genes. In each of these constructions, the adenovirus was modified so as to render it incapable of replication in the infected cell. Thus, the constructions described in the prior art are deleted adenoviruses from the E1 region, essential for viral replication, at the level of which heterologous DNA sequences are inserted (Levrero et al., Gene 101 (1991) 195; Gosh-Choudhury et al., Gene 50 (1986) 161). Furthermore, to improve the properties of the vector, it has been proposed to create other deletions or modifications in the genome of the adenovirus.
  • thermosensitive point mutation was introduced into the mutant ts125, making it possible to inactivate the DNA binding protein of 72kDa (DBP) (Van der Vliet et al., 1975).
  • DBP 72kDa
  • Other vectors include a deletion of another region essential for viral replication and / or spread, the E4 region.
  • the E4 region is in fact involved in the regulation of the expression of late genes, in the stability of late nuclear RNA, in the extinction of the expression of proteins of the host cell and in the efficiency of replication of l 'Viral DNA.
  • Adenoviral vectors in which the E1 and E4 regions are deleted therefore have very reduced transcription background and expression of viral genes.
  • the recombinant adenovirus is a human adenovirus of group C. More preferably, it is an adenovirus Ad2 or Ad5.
  • the recombinant adenovirus used in the context of the invention comprises a deletion in the E1 region of its genome. Even more particularly, it comprises a deletion of the regions E1 a and E1 b. As a specific example, mention may be made of deletions affecting the nucleotides
  • the recombinant adenovirus used in the context of the invention further comprises a deletion in the E4 region of its genome. More particularly, the deletion in the E4 region affects all of the open phases. As a specific example, the deletions 33466-35535 or 33093-35535 can be cited. Other types of deletions in the E4 region are described in applications WO95 / 02697 and WO96 / 22378, incorporated herein by reference.
  • the expression cassette can be inserted at different sites of the recombinant genome. It can be inserted at the E1, E3 or E4 region, replacing the deleted or surplus sequences. It can also be inserted at any other site, apart from the sequences necessary in cis for the production of viruses (ITR sequences and packaging sequence).
  • Recombinant adenoviruses are produced in an packaging line, that is to say a cell line capable of complementing in trans one or more of the deficient functions in the recombinant adenoviral genome.
  • a packaging line that is to say a cell line capable of complementing in trans one or more of the deficient functions in the recombinant adenoviral genome.
  • One of these lines is for example the line 293 in which a part of the adenovirus genome has been integrated. More specifically, line 293 is a cell line human embryonic kidney containing the left end (approximately 11 -12%) of the genome of the adenovirus serotype 5 (Ad5), comprising the left ITR, the encapsidation region, the E1 region, including E1 a and E1 b , the region coding for the protein pIX and part of the region coding for the protein plVa2.
  • This line is capable of trans-complementing recombinant adenoviruses defective for the E1 region, that is to say devoid of all or part of the E1 region, and of producing viral stocks having high titers.
  • This line is also capable of producing, at permissive temperature (32 ° C.), stocks of virus further comprising the thermosensitive E2 mutation.
  • Other cell lines capable of complementing the E1 region have been described, based in particular on human lung carcinoma cells A549 (WO94 / 28152) or on human retinoblasts (Hum. Gen. Ther. (1996) 215). Furthermore, lines capable of transcomplementing several functions of the adenovirus have also been described.
  • Recombinant adenoviruses are usually produced by the introduction of viral DNA into the packaging line, followed by lysis of the cells after approximately 2 or 3 days (the kinetics of the adenoviral cycle being 24 to 36 hours).
  • the viral DNA introduced may be the complete recombinant viral genome, optionally constructed in a bacterium (WO96 / 25506) or in a yeast (WO95 / 03400), transfected in the cells. It can also be a recombinant virus used to infect the packaging line.
  • the viral DNA can also be introduced in the form of fragments each carrying a part of the recombinant viral genome and a zone of homology making it possible, after introduction into the packaging cell, to reconstitute the recombinant viral genome by homologous recombination between the different fragments .
  • the recombinant viral particles are isolated by centrifugation in a cesium chloride gradient.
  • the recombinant vector having a tropism for the liver can also be constructed from a non-viral vector of the plasmid type, in particular as described in applications WO96 / 26270 and PCT / FR96 / 01414.
  • the vectors of the invention allow the regulated, high-level and hepatospecific production of molecules of interest.
  • the molecule of interest is advantageously a therapeutic molecule. It can be a protein or a nucleic acid (tRNA,
  • the therapeutic molecule is a protein secreted into the circulation.
  • a protein secreted into the circulation By way of example, mention may be made of enzymes, blood derivatives, hormones, interleukin lymphokines, interferons, TNF, etc. (FR 9203120), growth factors, neurotransmitters or their synthetic precursors or enzymes, trophic factors : BDNF, CNTF, NGF, IGF, GMF, aFGF, bFGF, NT3, NT5, etc; apolipoproteins: ApoAl, ApoAIV, ApoE, etc.
  • the expression cassette advantageously comprises an appropriate signal sequence. It may in particular be the natural signal sequence of the secreted protein, if it is functional in a hepatic cell. It can also be any suitable heterologous sequence. By way of example, mention may be made of the signal sequence of apolipoprotein A1.
  • the cassette generally comprises a region situated at 3 ′ which specifies a signal for termination of transcription and for polyadenylation. We use for example the polyA site of SV40 virus. It is understood that the choice of these signals falls within the general competence of a person skilled in the art.
  • the invention also relates to a pharmaceutical composition comprising a vector as described above.
  • the pharmaceutical compositions of the invention can be formulated for topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular, transdermal, etc. administration.
  • the pharmaceutical composition contains pharmaceutically acceptable vehicles for an injectable formulation.
  • pharmaceutically acceptable vehicles for an injectable formulation may in particular be saline solutions (monosodium phosphate, disodium phosphate, sodium chloride, potassium, calcium or magnesium, etc., or mixtures of such salts), sterile, isotonic, or dry compositions, in particular lyophilized, which, by addition, as appropriate, of sterilized water or physiological saline, allow the constitution of injectable solutes.
  • Other excipients can be used such as for example a hydrogel. This hydrogel can be prepared from any biocompatible and non-cytotoxic polymer (homo or hetero). Such polymers have for example been described in application WO93 / 08845.
  • the doses of virus used for the injection can be adapted according to various parameters, and in particular according to the mode of administration used, the pathology concerned, the gene to be expressed, or even the duration of the treatment sought.
  • the recombinant adenoviruses according to the invention are formulated and administered in the form of doses of between 10 4 and 10 1 4 pfu, and preferably 10 ⁇ to 10 1 0 pfu.
  • the term pfu (“plaque forming unit”) corresponds to the infectious power of an adenovirus solution, and is determined by infection of an appropriate cell culture, and measures, generally after 15 days, the number of plaques of infected cells.
  • the vectors in particular adenovirus
  • the vectors can also be used for the creation of animal models of hepatic pathologies.
  • the invention also relates to any cell modified by a vector (in particular an adenovirus) as described above.
  • a vector in particular an adenovirus
  • These cells can be used for the production of recombinant proteins in vitro. They can also be intended for implantation in an organism, according to the methodology described in application WO95 / 14785. These cells are preferably liver cells.
  • the subject of the present invention is also a process for the production of recombinant proteins comprising the infection or the transfection of a cell population with a vector, a recombinant adenovirus or the corresponding viral genome comprising an expression cassette coding for a desired protein, culturing said recombinant cell population, and recovering said protein produced.
  • cells of hepatic origin are used. These can be established lines or primary cultures.
  • the invention also relates to novel variants of the human apolipoprotein Ail gene promoter having improved expression characteristics.
  • These variants according to the invention comprise in particular a repetition of J motifs as described above.
  • these variants advantageously comprise a deletion in the region between the residues -710 and -150 of the native promoter.
  • the invention also relates to hepatospecific and inducible promoters derived from the promoter of the human apolipoprotein Ail gene comprising a regulatory region composed of one or more J motifs of the apolipoprotein Ail promoter and a hepatospecific promoter region derived from another promoter.
  • the hepatospecific promoter region is derived from a hepatospecific promoter other than the promoter of the human gene for apolipoprotein Ail.
  • it is composed of a promoter chosen chosen from the promoter of serum albumin, the promoter of apolipoprotein A1, the promoter of apolipoprotein Cs.
  • the promoter region used preferably consists of the region necessary and sufficient for hepatic expression (minimum promoter).
  • This region generally includes the TATA box, and can be prepared according to conventional molecular biology techniques, as indicated in the cited references.
  • the first 209 base pairs of the promoter of the factor X gene are sufficient to confer hepatic expression (JBC cited above).
  • fragments -20 to -23; -54 to -57 and -66 to -77 of the fibrinogen gamma chain promoter constitute a minimal promoter allowing hepatospecific expression.
  • regions J preferably 1 to 5
  • these promoters can carry additional regulatory sequences of the "enhancer" type, making it possible to improve the levels of expression.
  • the hepatospecific promoter region can also be composed of a ubiquitous promoter coupled to an enhancer element conferring hepatospecific expression.
  • the enhancer element conferring the hepatospecific character can be chosen from the enhancer of apolipoproteins E / Cl (J. Biol. Chem., 268 (1993) 8221 -8229 and J. Biol. Chem, 270 (1995) 22577-2255), the albumin enhancer (Gene therapy, 3 (1996) 802-810), the transthyretin enhancer (Mol. Cell Biol. 15 (1995) 1364-1376), the enhancer of hepatitis B virus (Biol. Chem.
  • the ubiquitous promoter can be any nonspecific promoter of a tissue. It may in particular be a viral promoter or a domestic promoter ("housekeeping").
  • the viral promoters there may be mentioned more particularly the SV40 promoter (Mol Cell Biol 1982; 2: 1044-1051); RSV LTR, Rous sarcoma virus long terminal repeat (PNAS USA, 1982; 79: 6777-6781); CMV-IE, human cytomegalovirus (Gene 1986; 45: 101-105); MoMLV LTR, Moloney murine leukemia virus (Gene Therapy 1996; 3: 806-810) and the promoter of the HSV-TK gene, Thymidine Kinase (Nucleic Acid Res 1980; 8: 5949-5964).
  • SV40 promoter Mol Cell Biol 1982; 2: 1044-1051
  • RSV LTR Rous sarcoma virus long terminal repeat
  • CMV-IE human cytomegalovirus
  • MoMLV LTR Moloney murine leukemia virus
  • Thymidine Kinase Nucleic Acid Res 1980; 8: 5949-5964.
  • the promoter of the Human EF-1 alpha genes (Gene 1993, 134: 307-308), Chicken Beta Actin (Nucleic Acids Res 1983; 11: 8287-8301), POL II promoter, mouse RNA polymerase II (Mol Cell Biol 1987; 7: 2012-2018); PGK, Phosphoglycerate Kinase (Gene 1987; 61: 291-298); Histone H4 (Mol Cell Biol 1985; 5: 380-398), HMG, Hydroxymethylglutaryl CoA: human reductase (Mol Cell Biol 1987; 7: 1881 -1893), HK2, Hexokinase II of rats (J. Biol. Chem. 1995; 270: 16918-16925) and PRP, Prion (Virus genes 1992; 6: 343-356). Any other ubiquitous promoter known to those skilled in the art can also be used.
  • the hepatospecific promoter region can be obtained by coupling, according to conventional molecular biology techniques, all or a functional part of a ubiquitous promoter with the above enhancer element.
  • the oligonucleotides corresponding to the J sites containing the bases -737 to -715 of the human apoA-ll promoter can be cloned into the BamHI / GglII sites of plC20H (Gene 1984; 32: 481 -485), digested with HindIII, and subclones 5 ′ of the ubiquitous promoter chosen, for example of the promoter of Thymidine Kinase (TK) in the plasmid pBLCAT4 (Nucl Acid Res 1987; 15: 5490), to generate a vector containing the J sites and a promoter ubiquitous in front of a gene of interest.
  • TK Thymidine Kinase
  • the hepatic enhancer can be added either 5 'to the promoter or 3' to the polyadenylation site. These variants are particularly advantageous because they combine the properties of expression force, tissue specificity, and inducibility. These different variants can be used for the expression of genes of interest, in vitro and in vivo as indicated above and illustrated in the examples.
  • the invention also relates to recombinant vectors comprising an expression cassette composed of a gene of interest under the control of a promoter as described above.
  • the invention also relates to a composition comprising a recombinant vector as described above and a PPAR activator, for simultaneous or spread over time use.
  • the recombinant vector is advantageously a recombinant adenovirus as defined above, and the activator of PPAR is advantageously an activator of PPAR ⁇ .
  • the activators of PPAR ⁇ it is possible to use more particularly fibrates as well as any compound increasing the expression of transcription factors which bind to the J sites.
  • fib r ates As preferred examples of fib r ates, mention may be made, for example, of fibric acid and its analogs such as in particular gemfibrozil (Atherosclerosis 114 (1) (1995) 61), bezafibrate (Hepatology 21 (1995) 1025) , ciprofibrate (BCE & M 9 (4) (1995) 825), clofibrate (Drug Safety 11 (1994) 301), fenofibrate (Fenofibrate Monograph, Oxford Clinical Communications, 1995), clinofibrate (Kidney International. 44 (6) (1993) 1352), pirinixic acid (Wy-14,643) or 5,8,1 1, 14-eicosatetranoic acid (ETYA).
  • gemfibrozil Artherosclerosis 114 (1) (1995) 61
  • bezafibrate Hepatology 21 (1995) 1025)
  • ciprofibrate BCE & M 9 (4) (1995) 825
  • compositions according to the invention may comprise several activators of PPAR in combination, and in particular a fibrate or a fibrate analog associated with a retinoid.
  • the vector and the activator can be used simultaneously or spaced over time.
  • they can be packaged separately.
  • the vector and the activator are packaged separately and used spaced over time.
  • the vector is advantageously used first, then, in a second step, the activator from PPAR.
  • the term used designates bringing said vector or activator into contact with cells, in vitro, ex vivo or in vivo.
  • the contact can be carried out by incubation of a cell population as mentioned above with the vector (for example from 0.01 to 1000 ⁇ g of vector per 10 6 cells, or of virus at a Multiplicity of Infection (MOI) from 0.1 to 1000), followed by incubation with the activator (generally, in a concentration range between 10 ⁇ 3 mM and 10 mM, preferably between 10 ⁇ M and 500 ⁇ M ).
  • the activator generally comprises the administration of the vector (under the conditions described above) followed by the administration of the activator.
  • the activator can be administered orally, for example in food (for animals in particular) or in the form of capsules (for humans).
  • the daily doses administered to animals are of the order of 0.01 to 1% (weight / weight), preferably 0.2 to 0.5% (weight / weight).
  • a typical daily dose in mice for example is 50 mg.
  • a typical daily dose of fenobibrate in humans varies between 100 and 300 mg, preferably around 200 mg, which corresponds to a plasma concentration of approximately 15 ⁇ g / ml (Vidal, 1996).
  • repeated administrations / incubations of vector and / or activator can be performed.
  • Figure 1 Structure of the apoAII promoter and deleted forms.
  • Figure 2 Region J duplication strategy.
  • Figure 3 Structure of apoAII promoters including region 5 'J duplication, possibly combined with internal deletions.
  • Figure 4 Structure of the apoAII promoters comprising a duplication of the J region internally, possibly combined with internal deletions.
  • Figure 5 Representation of the recombinant adenovirus.
  • Figure 6 Inducibility and strength of the recombinant adenovirus in vivo.
  • the pBR322, pUC and phage plasmids of the M13 series are of commercial origin (Bethesda Research Laboratories).
  • the DNA fragments can be separated according to their size by electrophoresis in agarose or acrylamide gels, extracted with phenol or with a phenol / chloroform mixture, precipitated with ethanol and then incubated in the presence of the DNA ligase from phage T4 (Bioiabs) according to the supplier's recommendations.
  • the filling of the protruding 5 ′ ends can be carried out by the Klenow fragment of DNA Polymerase I of E. coli (Bioiabs) according to the supplier's specifications.
  • the destruction of the protruding 3 ′ ends is carried out in the presence of the DNA polymerase of phage T4 (Bioiabs) used according to the manufacturer's recommendations.
  • the destruction of the protruding 5 ′ ends is carried out by gentle treatment with nuclease S1.
  • PCR Polymerase-catalyzed Chain Reaction, Saiki RK et al., Science 230 (1985) 1350-1354; Mullis KB and Faloona FA, Meth. Enzym. 155 (1987) 335-350]
  • DNA thermal cycler Perkin Elmer Cetus
  • Verification of the nucleotide sequences can be carried out by the method developed by Sanger et al. [Proc. Natl. Acad. Sci. USA, 74 (1977) 5463-5467] using the kit distributed by Amersham.
  • Example 1 Cloning of the promoter of the human apolipoprotein Ail gene.
  • This example describes the cloning of the promoter gu human apoAII II gene It is understood that any other technique can be used to recloner this promoter. Furthermore, it is also possible, from the cloned fragments, to prepare shorter conventional 5 ′ and / or 3 ′ versions according to conventional molecular biology techniques.
  • the promoter of human apophpoprotein Ail was cloned by PCR from human genomic DNA Primers ATC GAA GCT TCT GAT ATC TAT TTA ACT GAT (SEQ ID No. 3) and CGT CTC TGT CCT TGG TGT CTG GAT CCA TCG (SEQ ID No. 4) which introduce, for the first, a HindIII site 5 'to the promoter, and for the second, a BamHI site at 3', made it possible to clone the promoter from the position -911 to the position + 29
  • the promoter sequence was confirmed by sequencing (SEQ ID No. 1) and the HindIII-BamHI fragment introduced into the vector pBLCAT ⁇ for verification of the transcriptional activity
  • a fragment comprising the apoAII promoter of residues -91 1 / + 160 was also obtained from the genomic library, then cloned into the vector pBLCAT ⁇ .
  • This example describes the construction of variants of the apoAII promoter comprising an internal deletion, in the region between the regulatory elements located at the nucleotides -903 to -720, and -126 to -33. These variants were constructed from the fragments -911 / + 29 and -911 / + 160 described in example 1.
  • the region -210 to +29 or -210 to +160 was obtained by PCR using as primer a oligonucleotide -210 / - 198 of sequence 5'-GACTCTAGATGTACCCCCTTA-3 '(SEQ ID No. 5) and an oligonucleotide internal to the CAT gene.
  • the fragment obtained was cloned into a plasmid pBLCAT5 to generate the plasmids -210 / + 160AII-CAT and - 210 / + 29AII-CAT.
  • the distal region -91 1 to -653 (Nl) was obtained by digestion of the fragment -911 to +29 using the enzyme alul, then cloning of the fragment obtained in the plasmids -210 / + 160AII-CAT and -210 / + 29AII- CAT.
  • the distal region -911 to -708 (N-J) was obtained by PCR using as an primer an oligonucleotide -708 / -722 of sequence 5'- GGAAGCTGCAGAGGCTTCTACCAG-3 '(SEQ ID No. 6). The fragment obtained was then cloned into plasmids -210 / + 160AII-CAT and -210 / + 29AII-CAT.
  • This example describes the construction of Ail Promoter variants in which the J region has been repeated.
  • oligonucleotides corresponding to site J. were synthesized using a DNA synthesizer.
  • Oligo 1 5'-gatcctTCAACCTTTACCCTGGTAGa-3 '(SEQ ID No. 7)
  • Oligo 2 5'-gatctCTACCAGGGTAAAGGTTGAag-3' (SEQ ID No. 8)
  • oligonucleotides reconstitute, at the 3 'end, a BamHI site and at the 5' end, a BglII site. These oligonucleotides were hybridized together and the fragment obtained was cloned at the BamHI-BglII sites in the vector plC20H ( Figure 2). The resulting plC20H-J plasmids were analyzed to determine the number of copies of J sites inserted, as well as their respective orientation.
  • the inserts contained in the above plasmids were excised in the form of HindIII fragments and clones in 5 ′ of the apoAII promoter (Example 1) or variants described in Example 2.1, at a HindIII site.
  • the structure of the resulting promoters is given in FIG. 3.
  • the inserts contained in the above plasmids were excised in the form of appropriate restriction fragments, then cloned into the apoAII promoter (Example 1) or into the variants described in Example 2.1, at a corresponding site located between the native J region and the regulatory region -126-33 of the native promoter.
  • the structure of the resulting promoters is given in FIG. 4.
  • the number of copies of region J in the final construct is determined by the choice of the plasmid.
  • Example 3 Study of the functionality of the variants of the apoAII promoter.
  • the functionality of the promoters was studied by transfection into a hepatic cell line, the HepG2 cells.
  • a control experiment was carried out in a non-hepatic cell line, the Hela cells.
  • PBK-CMV-PPAR ⁇ PPAR ⁇ expression plasmid
  • PBK-CMV PPAR ⁇ empty plasmid
  • CMV- ⁇ -Gal a ⁇ -Gal expression plasmid
  • the promoters according to the invention are strong, highly inducible, and specific for hepatic cells.
  • the promoters phA-ll N-I (J3); phA-11 'N-I (J3); phA-ll N-J (J3), phA-ll 'N- J (J3), phA-ll N-1 (J3 ⁇ ); phA-ll 'Nl (J3 ⁇ ), phA-ll NJ (J3 ⁇ ) and phA-ll' NJ (J3 ⁇ ) have the advantage of being small, compared to the native apoAII promoter This therefore advantageously allows, in a vector of gene transfer and / or expression, to have a higher cloning capacity
  • Example 4 Construction of inducible and hepatospecific adenoviruses.
  • adenoviruses This example describes the construction of inducible and hepatospecific adenoviruses, and giving very high expression levels These adenoviruses are useful for the expression of genes in vitro, ex vivo or in vivo
  • the adenoviruses described were constructed from the serotype Ad5 It is understood that any other serotype can be used, and in particular the serotypes Ad2, Ad7, Ad12 and CAV2.
  • the adenoviruses were constructed by homologous recombination, in a packaging line, between a shuttle vector bringing the left part of the viral genome and the DNA of a linearized adenovirus, bringing the right part of the viral genome
  • the shuttle vector constructed carries an expression cassette consisting of an apoAII promoter and a nucleic acid coding for a secreted molecule apolipoprotein Al (apoAl) This vector also brings the left part of the viral genome, that is to say the left ITR and a region allowing recombination.
  • the shuttle vectors used for the construction of the adenovirus were constructed from a plasmid pC05 (WO96 / 22378), a plasmid plC20H-Alb-U-AI-SV40 which contains the first intro of apoAl and The apoAl cDNA under the control of a promoter of albumin from Rat, and of a plasmid pBLAIICAT ⁇ which contains an apoAII promoter as described in example 1 or a variant according to example 2.
  • the 5 'primer introduces a NotI site upstream of this sequence and the 3' primer introduces a half site Nrul.
  • the PCR fragment obtained is treated with klenow to obtain blunt ends and cloned in the vector plC20H cleaved by Smal and Nrul, in the orientation which regenerates a Nrul site.
  • the resulting plasmid is called plC20H-SV40.
  • a PCR fragment is amplified from pXL2336 using the primers GGG ATC CGC TGG CTG CTT AGA GAC TGC (SEQ ID No. 11) and GGC GGC CGC CGG GAA GGG GGG CGG CGG (SEQ ID No. 12) respectively BamHI upstream of the first ApoAl exon and a NotI site downstream of the ApoAI coding sequence.
  • the PCR fragment is cloned into pCRI1 (Invitrogen) and its sequence verified.
  • the BamHI / NotI fragment containing the cDNA and the first intron of apoA-1 is then cloned at the same sites of the plasmid plC20H-SV40 to generate the plasmid plC20H-AI-SV40.
  • the oligonucleotides CAC GTG CTT GTT CTT TTT GCA GAA GCT CAG AAT AAA CGC TCA ACT GTG GC (SEQ ID n ° 13) and CGT GGC CAC AGT TGA GCG TTT ATT CTG AGC TTC TGC AAA AAG AAC AAG CA (SEQ ID n ° 14) are then hybridized with each other and cloned at the Dsal site of plC20H-AI-SV40 so that the Pmll site created during this cloning is on the side of the intron and the Dsal site is regenerated at other end.
  • These oligonucleotides make it possible to introduce a fragment of the 5 'untranslated part of the messenger RNA of ⁇ Globin.
  • the plasmid obtained is called plC20H-U-AI-SV40.
  • the oligonucleotides CGT GGC AGG CAG CAG GAC GCA CCT CCT TCT CGC AGT CTC TAA GCA GCC TTC GAA GCA TG (SEQ ID n ° 15) and CTT CGA AGG CTG CTT AGA GAC TGC GAG AAG GAG GTG CGT CCT GCT GCC TGC CA (SEQ ID n ° 16) are hybrid between them which creates a cohesive end Sphl and a cohesive end Hpall.
  • This fragment is introduced into a three-partner ligation with an Hpall / Dralll fragment (476/1518) of the plasmid plC20HAIb-U-AI-SV40 containing the cDNA of ApoAl and a Dralll / Sphl fragment (1518/239) of the same plasmid. .
  • the resulting plasmid is called pXL2699.
  • This construction creates a BstBI site compatible with ClaI upstream of the cDNA + intron fragment of apoAl.
  • the BstBI / SalI fragment of pXL2699, containing the ApoAl cDNA is cloned into the plasmid pCO5 cleaved by ClaI and SalI.
  • the resulting plasmid is called pCO5-U-AI-SV40.
  • the apoAII promoter selected (complete promoter or variants) is excised from the corresponding plasmid pBLAIICAT5 in the form of a Hindlll-BamHI fragment, cloned after treatment with Klenov at the EcoRV site of the plasmid pC05- U-AI-SV40 to generate the shuttle vectors pXLPromAII /HAVE.
  • the following vectors are thus obtained:
  • the adenoviruses were produced by homologous recombination, after co-transfection, into the appropriate cells, of two DNA fragments, one bringing the left part of the genome of the recombinant virus (shuttle vector described in example 4.1., Having a deletion in the E1 region), the other bringing the right part of the genome of the recombinant virus (possibly having a deletion in the E4 and / or E3 region).
  • the Ad-AII / AI adenovirus was obtained by homologous in vivo recombination between the DNA of the Ad-RSV- ⁇ Gal virus and the pXL AII / AI shuttle vector, according to the following protocol: the pXL AII / AI shuttle vector linearized by BstXI and the DNA of the Ad-RSV- ⁇ Gal virus linearized by the enzyme Clal, were co-transfected in line 293 in the presence of calcium phosphate, to allow homologous recombination. The recombinant adenoviruses generated were then selected by plaque purification.
  • the DNA of the recombinant adenovirus was amplified in the cell line 293, which which leads to a culture supernatant containing the unpurified recombinant defective adenovirus having a titer of approximately 10 10 pfu / ml.
  • the viral particles are then purified by centrifugation on a cesium chloride gradient according to known techniques (see in particular Graham et al., Virology 52 (1973) 456), or by chromatography (FR96 08164).
  • the Ad-AII / AI adenovirus can be stored at -80 ° C in 20% glycerol.
  • the structure of the recombinant genome is presented in Figure 5.
  • the IGRP2 cells (Yeh et al., J. Virol 70 (1996) 559), capable of transcomplementing the E1 and E4 functions of the adenovirus, are cotransfected with 5 mg of the shuttle vector digested with BstXI, and 10 mg of the DNA of the virus providing the functional deletion of the E4 region (for example Ad2dl808, Ad5dl1004, Ad5dl1007 or Ad5dl1014) digested with the enzyme ClaI. After the appearance of the cytopathic effect, the viruses are purified by at least two consecutive cycles of spreading in solid for the formation of plaques on IGRP2.
  • the ranges corresponding to the infection of the virus sought are then amplified by consecutive infection cycles.
  • High titer stocks are prepared by purification on a cesium chloride gradient. Viruses are stored at -80 ° C according to conventional techniques of those skilled in the art.
  • This example describes the functional properties of the adenoviruses of the invention, after administration in vivo.
  • Ad viruses Ad viruses 5 x 10 9 pfu of the Ad viruses (ApoAllprom-ApoAI-SV40 and Ad (RSV-ApoAI-bGH) were injected respectively into 8 and 3 C57bl6 mice.
  • Ad Ad
  • Plasma concentrations of human apoA-1 and HDL-cholesterol were measured weekly. The results obtained are presented in FIG. 6.
  • the plasma apoA-1 levels are below the detection threshold (10 mg / dl) for the mice injected with the Ad virus (ApoAllprom-ApoAI-SV40), 81 ⁇ 0.17 mg / dl for mice injected with Ad virus (ApoAllprom-ApoAI-SV40) treated with fenofibrate and 84 115 mg / dl for mice injected with Ad virus (RSV-ApoAI-bGH) which shows an induction of at least 8 times of the apoA-ll promoter under these conditions.
  • Ad virus Ad virus
  • RSV-ApoAI-bGH Ad virus
  • the level of HDL-cholesterol is not modified for mice injected with the Ad virus (ApoAllprom-ApoAI-SV40) (52 ⁇ 2 mg / dl): identical level to untreated animals.
  • the level of HDL-cholesterol is increased on day 7 up to 88,114 mg / dl and 121,122 mg / dl for respectively for the mice injected with the Ad virus (RSV-ApoAI-bGH) and the mice injected with the virus Ad (ApoAllprom-ApoAI-SV40) treated with fenofibrate (Figure 6).
  • SEQ ID No. 1 Sequence of the human apoAII promoter (-911 +29).
  • -734 -716 ⁇ TCTTATTCA CCTCTTTTCC TGCCAGAGCC CTCCATTGGG AGGGGACGGG -711 CGGAAGCTGT TTTCTGAATT TGTTTTACTG GGGGTAGGGT ATGTTCAGTG
  • ATCAGCATCC AGGTCATTCT GGGCTCTCCT GTTTTCTCCC CGTCTCATTA -611
  • ACCCTCATTC CCCTAACCCC CATAGCCCTC
  • AACCCTGTCC CTGATTTCAA -511 TTCCTTTCTC CTTTCTTCTG CTCCCCAATA TCTCTCTGCC AAGTTGCAGT

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US10610606B2 (en) 2018-02-01 2020-04-07 Homology Medicines, Inc. Adeno-associated virus compositions for PAH gene transfer and methods of use thereof
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