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  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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  • C12N15/86—Viral vectors
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  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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  • 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
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  • C12N2710/00011—Details
  • C12N2710/10011—Adenoviridae
  • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
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  • 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

Definitions

• the enhancer region can be positioned in the same orientation or in the reverse orientation with respect to the direction of transcription of the promoter region.
• the enhancer region is chosen from the enhancer region of the immediate early cytomegalovirus gene (CMV-IE), the enhancer region of the red sarcoma virus LTR (LTR-RSV), the enhancer region SV40 virus, and the enhancer region of the EF1 ⁇ gene. More preferably, in the hybrid promoters of the invention, the enhancer region is the enhancer region of the immediate early gene of the cytomegalovirus (CMV-IE), preferably of the human cytomegalovirus (hCMV-IE).
• a particular enhancer region consists for example of the fragment -522 to -63 of the hCMV-IE gene, or of any fragment comprising at least part of it and exhibiting enhancer activity.
• a region comprising all or part of the promoter of the gene coding for ⁇ -actin of smooth muscle cells (SMact) or of the SM22 gene is advantageously used for implementing the invention.
• the promoter of these genes has been described for its specific character of smooth muscle cells (see in particular Ueyama H. et al., Mol. Cell. Biol, 4 (1984) 1073-1078; Solway J. et al., J. Biol. Chem., 270 (1995) 13460-13469).
• the enhancer region and the promoter region can be isolated by conventional techniques from nucleic acid libraries or from total cellular DNA, for example by amplification by means of specific probes. These fragments can also be synthesized artificially using the sequence information available in the prior art. When these fragments are obtained, they can be easily combined with one another by means of ligases and other restriction enzymes, to generate hybrid promoters of the invention. In addition, these fragments can be modified by digestion, mutation, insertion or addition of base pairs, either in order to facilitate their cloning, or in order to modify their functional properties.
• the fragments can be associated directly with one another or, on the contrary, spaced by base pairs having no significant influence on the activity of the hybrid promoter.
• the hybrid promoters of the invention thus have the capacity to express a nucleic acid of interest in a specific manner in smooth muscle cells.
• the "specific" character of the expression means that the promoter activity is significantly very higher in the smooth muscle cells. Although a non-specific expression can exist in other cells, the corresponding level of activity generally remains very low (negligible) compared to that observed in smooth muscle cells, generally at least a factor of 10 .
• the results presented in the examples show in this respect an expression differential which can reach a factor of 140, which testifies to the high selectivity of the promoters of the invention.
• the nucleic acid can code, for example, for a protein chosen from: proteins involved in the cell cycle, such as for example p21 or any other kinase inhibitor protein cyclin-dependent (cdk), the retinoblastoma (Rb) gene product, GAX, GAS-1, GAS-3, GAS-6, Gadd 45, Gadd 153, cyclins A, B and D.
• apoptosis such as for example p53, members of the family of apoptosis inducers such as Bas, Bcl-X s , Bad or any other antagonist of Bcl 2 and of Bcl-Xi.
• proteins capable of modifying the proliferation of smooth muscle cells such as for example an intracellular antibody or an ScFv inhibiting the activity of proteins involved in cell proliferation, such as for example the Ras protein, mapkinase, or receptors for tyrosine kinase or growth factors.
• proteins inducing angiogenesis such as for example members of the VEGF family, members of the FGF family and more particularly FGF1, FGF2, FGF4, FGF5, angiogenin, EGF, TGF ⁇ , TGF ⁇ , TNF ⁇ , Scatter Factor / HGF , members of the angiopoetins family, cytokines and in particular interleukins including IL-1, IL-2, IL-8, angiotensin-2, plasminogen activator (TPA), urokinase (uPA) , the molecules involved in the synthesis of active lipids (prostaglandins, Cox-1).
• proteins inducing angiogenesis such as for example members of the VEGF family, members of the FGF family and more particularly FGF1, FGF2, FGF4, FGF5, angiogenin, EGF, TGF ⁇ , TGF ⁇ , TNF ⁇ , Scatter Factor / HGF , members of the angiopoetins family, cytokines and in particular inter
• adenovirus viruses there may preferably be mentioned adenovirus viruses, retroviruses, herpes virus or recombinant adeno-associated viruses.
• the construction of this type of recombinant virus defective for replication has been widely described in the literature, as well as the infection properties of these vectors (see in particular S. Baeck and KL March (1998), Circul. Research vol. 82, pp 295-305), T. Shenk, BN Fields, DM Knipe, PM Howley et al (1996), Adenoviridae: the viruses and their replication (in virology).
• Pp 211-2148 EDS - Ravenspublishers / Philadelphia, P. Yeh and M. Perricaudet (1997), FASEB Vol. 11, pp 615-623.
• a particularly preferred recombinant virus for the implementation of the invention is a defective recombinant adenovirus.
• Adenoviruses are linear double-stranded DNA viruses around 36 (kilobases) kb in size. There are different serotypes, including the structure and properties vary somewhat, but 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 (Adl2).
• 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 El 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, Ad 12, 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 72kDa DNA binding protein (DBP) (Van der Vliet et al., 1975).
• Other vectors include a deletion from another region essential for replication and / or viral propagation, 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 noise and expression of viral genes.
• Such vectors have been described by example in applications WO94 / 28152, WO95 / 02697, WO96 / 22378).
• vectors carrying a modification in the IVa2 gene have also been described (WO96 / 10088).
• the recombinant adenovirus used in the context of the invention comprises a deletion in the E1 region of its genome. Even more particularly, it includes a deletion of the Ela and Elb regions. As a specific example, mention may be made of deletions affecting nucleotides 454-3328; 382-3446 or 357-4020 (with reference to the Ad5 genome).
• line 293 is a human embryonic kidney cell line containing the left end (approximately 11-12%) of the genome of the adenovirus serotype 5 (Ad5), comprising the left ITR, the packaging region , the El region, including Ela and Elb, the region coding for the pIX protein and part of the region coding for the pIVa2 protein.
• This line is capable of trans-complementing recombinant adenoviruses defective for the E 1 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.
• 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 recombinant viral particles are isolated by centrifugation in a cesium chloride gradient.
• An alternative method has been described in application FR96: 08164 incorporated herein by reference.
• the use of a hydrogel is particularly advantageous for the transfer of nucleic acids into the vascular walls, and in particular into the smooth muscle cells of the vascular walls.
• the doses used for the injection can be adapted according to different parameters, and in particular according to the mode of administration used, the aim pursued (labeling, pathology, screening, etc.), the gene to be expressed, or even the duration of the expression sought.
• the recombinant viruses according to the invention are formulated and administered in the form of doses of between 10 ⁇ and 10 ⁇ pfu, and preferably 10 ⁇ to 10 ⁇ pfu.
• pfu plaque forming unit
• plaque forming unit corresponds to the infectious power of a viral solution, and is determined by infection of an appropriate cell culture, and measurement of the number of plaques of infected cells. The techniques for determining the pfu titer of a viral solution are well documented in the literature.
• the cassettes, vectors or compositions of the invention can be incubated at conventional doses in the presence of the selected cell populations. These incubations can be carried out on culture dishes, flasks, fermenters, or any other device chosen.
• the invention also relates to any cell modified by a cassette or a vector (in particular an adenovirus) as described above.
• modified cell is understood to mean any cell containing a construct according to the invention. 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 human smooth muscle cells.
• the invention also relates to the use of a hybrid promoter as defined above for the specific expression of a nucleic acid in smooth muscle cells, in vitro, ex vivo or in vivo.
• Figure 2 Schematic representations of the plasmids whose expression cassette contains the hybrid promoter mSM22 ⁇ .
• Figure 3 Activities of hybrid promoters evaluated in transient transfections in vitro in rabbit smooth muscle cells in primary culture (rabbit SMC), in endothelial cells derived from human umbilical cord carcinoma (ECV304), in myoblasts of mice (C2C12) as well as in epithelial cells from a carcinoma of the human cervix (HeLa).
• the relative activity of each promoter is expressed as a percentage of the luciferase activity obtained with the plasmid pCMV-leadTK.
• Enh-X the enhancer sequence of hCMV-IE is cloned upstream of promoter X according to its normal orientation.
• hnE-X the enhancer sequence of hCMV-IE is cloned upstream of promoter X in the opposite orientation.
• Figure 4 Activities of hybrid promoters evaluated in transient transfections in vitro in mouse embryonic fibroblasts (NIH 3T3), in cells derived from a human ENT carcinoma (TU182), as well as in transformed human embryonic renal cells (293) . The relative activity of each promoter is expressed as a percentage of the luciferase activity obtained with the plasmid pCMV-leadTK.
• Enh-X the enhancer sequence of hCMV-IE is cloned upstream of promoter X according to its normal orientation.
• hnE-X the enhancer sequence of hCMV-IE is cloned upstream of promoter X in the opposite orientation.
• Figure 5 Activities of hybrid promoters evaluated in gene transfer in vivo in the cranial tibial muscle of C57BL6 mice. The relative activity of each promoter is expressed as a percentage of the luciferase activity obtained with the plasmid pCMV-leadTK.
• the plasmid pGL3 -Basic used for cloning the different promoter regions, is of commercial origin (Promega Corporation).
• the plasmids pCMV ⁇ (Clontech Laboratories Inc.) and pUC18 (Boehringer Mannheim) are also of commercial origin.
• the enzymatic amplification of DNA fragments by the PCR technique can be carried out using a DNA thermal cycler ⁇ (Perkin Elmer Cetus) according to the manufacturer's recommendations.
• the electroporation of plasmid DNA in Escherichia coli cells can be carried out using an electroporator (Bio-Rad) according to the manufacturer's recommendations.
• EXAMPLE 1 Construction of hybrid promoters and expression plasmids containing them.
• - Primer 6417 (5 'GATGGTCCCTACTTATGCTGCTA 3') (SEQ ID 1) starting at position -1034 (promoter region) of the human specific smooth muscle ⁇ -actin gene (Ueyama H. et al., Mol. Cell. Biol, 4 (1984) 1073-1078. Access Genbank D00618).
• - Primer 6418 (5 'CTTCCATCATACCAAACTACATA 3') (SEQ ID 2) at position 1974 of the sequence D00618 located inside the first intron of the hSMact gene.
• the reaction mixture comprises 1 mg of genomic DNA, 10 pmol of each of the two primers (6417 and 6418), 100 mM of each deoxyribonucleotide (dATP, dCTP, dGTP, dTTP), 2 mM MgC12 and 5 units of Taq DNA polymerase ( PerkinElmer).
• the reaction volume is made up to 50 ml adjusted to the optimal concentration of the ACP buffer recommended by Perkin Elmer.
• the PCR amplification is carried out in Micoamp TM tubes (Perkin Elmer) using a PTC-100 TM thermocycler (MJ Research, Inc.).
• This amplification consists of a denaturation step at 95 ° C for 2 min followed by 30 cycles including a denaturation step of 15 sec at 95 ° C, a hybridization step of 30 sec at 60 ° C and an extension step 1 min at 72 ° C. These thirty cycles are followed by an additional extension of 5 min then the ACP reactions are stored at 10 ° C.
• This fragment was digested with MluI and then was cloned into the plasmid phSMact previously digested with MluI and treated with alkaline phosphatase.
• two different plasmids were obtained: pXL3130 and pXL3131.
• the schematic representations of these plasmids are gathered in the figure ( Figure 1).
• These plasmids comprise, in the form of an Mlul-NDSl fragment, a hybrid promoter consisting of the enhancer of the promoter of the hCMV-IE gene and of the promoter of the hSM ⁇ -actin gene.
• This DNA was used as a template for PCR amplification using the following primers:
• this primer introduces an Xhol site (lowercase underlined) in position -436 of the promoter of the mouse SM22 alpha gene (Solway J. et al., J. Biol. Chem., 270 (1995) 13460-13469; Access Genbank L41161).
• this primer introduces a HindIII site at position +43 of mSM22 alpha.
• Plasmid pXL3152 and pXL3153 A DNA fragment corresponding to the enhancer region of the promoter of the hCMV-IE gene between the positions -522 and -63 relative to the transcription initiation site, was amplified by PCR using the plasmid pCMV ⁇ as template and oligonucleotides 8557 (5 'ATC GAC GCG TGC CCG TTA CAT AAC TTA CGG 3') and 8558 (5 'ATC GAC GCG TCC GCT CGA GCG TCA ATG GGG CGG AGT TG 3') (SEQ ID 6) as primers.
• This fragment was digested with MluI and then was cloned into the plasmid pmSM22 previously digested with MluI and treated with alkaline phosphatase.
• two different plasmids were obtained: pXL3152 and pXL3153.
• the schematic representations of these plasmids are collated in FIG. 2.
• These plasmids comprise, in the form of an Mlul-NDTl fragment, a hybrid promoter consisting of the enhancer of the promoter of the hCMV-IE gene and of the promoter of the mSM22 gene.
• the expression vector pCGN previously described by Tanaka et al. contains the CMV promoter (-522 / + 72) fused to the "leader" of the HSV tk gene (+ 51 / + 101) upstream of a sequence coding for the epitope hemagglutinin.
• Plasmid pCGN (10 ng) was used as a template for PCR amplification. The PCR reaction and the amplification were carried out under the same conditions as those used for hSMact and mSM22 (Examples 1.1 and 1.2).
• the PCR fragment thus obtained is purified and then phosphorylated using the polynucleotide kinase from phage T4 (New England Biolabs).
• the vector pGL3-Basic Promega was linearized with purified NcoL then treated with Klenow DNA polymerase (Boehringer Manheim) in order to fill the Ncol site.
• Klenow DNA polymerase Boehringer Manheim
• This vector is then dephosphorylated using alkaline phosphatase (Boehringer Manheim) and then used for the insertion of the phosphorylated PCR fragment.
• the guanosine (G) of primer 6719 makes it possible to restore the Ncol site only when the CMV-leader tk fragment is oriented with the 5 ′ part (primer 6718, position -522 of CMV) downstream of the HindIII site of pGL3 -Basic and its 3 ′ end (primer 6719, leader tk) is ligated to the Ncol site of pGL3-Basic (first luciferase ATG).
• the plasmid thus obtained is designated pCMV-leadTK.
• This example illustrates the tissue specificity properties of the hybrid promoters of the invention in vitro.
• SMC Rabbit smooth muscle cells
• DMEM TM medium (Life Technologies Inc.) supplemented with 20%) of fetal calf serum (SVF).
• ECV304 cells are cultured in 199 TM medium (Life Technologies Inc.) supplemented with 10%> of FCS.
• C2C12 myoblasts, HeLa cells, NIH 3T3 cells and TU 182 cells are cultured in DMEM TM medium supplemented with 10% of SVF.
• the 293 cells are cultured in MEM TM medium (Life Technologies Inc.) supplemented with pyruvate, non-essential amino acids and 10% FCS. All the cultures are carried out in an oven at 37 ° C., in a humid atmosphere and under a partial pressure of CO 2 of 5%.
• plasmid DNA 250 ng of plasmid of interest and 250 ng of pUC18
• DMEM TM medium 20 ⁇ l final
• the 20 ⁇ l of the DNA / lipid mixture are brought into contact with the cells, in the absence of FCS, for 2 hours.
• the culture medium is then supplemented with SVF so as to obtain the percentage of SVF required for the culture of each cell type.
• the relative activity in rabbit SMC is at least 5 times greater than that observed in other cell types: (i) from 5 to 20 times greater for the hSMact promoter (Table I), and (ii) 5 to 25 times higher for the mSM22 promoter (Table II).
• This example illustrates the tissue specificity properties of the hybrid promoters of the invention in vivo. 3.1. Gene transfer in skeletal muscle.
• the various plasmids were injected, intramuscularly, into the cranial tibial muscle of female C57BL6 mice aged 5 weeks. Each plasmid, diluted in a NaCl solution at 150 mM final, is injected at a rate of 10 ⁇ g per muscle. Three days after injection, the muscles are removed in 2 ml of Cell Culture Lysis Reagent TM buffer (Promega Corporation), and ground using a Diax homogenizer (Heidolph). The ground material is then centrifuged for 15 minutes at 4000 g, then the luciferase activity is evaluated using the Luciferase Assay System TM kit (Promega Corporation) according to the recommendations of the supplier.
• Cell Culture Lysis Reagent TM buffer Promega Corporation
• Diax homogenizer Heidolph
• the relative activities of the two specific promoters (hSMact and mSM22) as well as those of two of the hybrid promoters of the invention (Enh-hSMact and Enh- mSM22) were also evaluated in vivo after transfer of naked DNA into the cranial tibial muscle of mice.
• the results collated in FIG. 5 show that the activity of the Enh-hSMact promoter is 100 times lower than that of the CMV promoter.
• the activity of the Enh-mSM22 promoter is 17 times lower than that of the CMV promoter.
• EXAMPLE 4 Construction of recombinant adenoviruses expressing the GAX protein under the control of specific hybrid promoters.
• the purpose of this example is to describe an adenoviral vector carrying the gene coding for the protein GAX operatively linked to the hybrid promoter of the invention composed of the enhancer CMV and of the promoter SM ⁇ -actin (enh-hSMact).
• the human gax gene comprises 912 base pairs and codes for a transcription factor of 303 amino acids involved in stopping cell growth (growth-arrest-specific homeobox) and having a role in the proliferation of human smooth muscle cells.
• This homeodomain gene was originally isolated from the aorta and is expressed in particular in adult cardiovascular tissue (Gorski et al. 1993).
• Plasmid pXL3297 is an expression vector containing the human gax gene. It was digested with the enzymes HindIII and Avril in order to introduce the human gax gene into the previous plasmid pXL3282 also digested with the enzymes HindIII and Avril to give the plasmid pXL 3300.
• the expression cassette for the human gax gene is then introduced into a recombinant human adenovirus of serotype 5 (Ad5) deleted from the El and E3 regions by co-transfection and homologous recombination between the plasmid carrying the expression cassette for the gax gene and adenovirus, in packaging cells. These cells are preferably line 293.
• the production of a stock of adenovirus containing the expression cassette for the human gax gene results from the lysis of the cells. packaging 2 or 3 days after infection and isolation of the recombinant viral particles by cesium chloride gradient centrifugation.
• the viral particles are then used to study the expression of the human gax gene under the control of the promoter of the invention in smooth muscle cells.
• messenger RNAs is analyzed 24 hours after infection of the smooth muscle cells by dot blot and northern blot using an oligonucleotide whose sequence is present in the gax gene.
• Smooth muscle cells in the exponential growth phase are infected with an adenovirus containing the gene coding for the GAX protein under the control of the enh-hSMact promoter in the absence and in the presence of 125 ng of lipofectin (48-well plates).
• the adenoviruses (in variable dilutions) and the lipofectamine are incubated for 30 minutes at room temperature in a medium deprived of serum.
• the mixture or the virus alone is brought into contact with the cells for one hour at 37 ° C.
• the medium containing the virus is removed and the cells are incubated in DMEM medium containing 0.5% of FCS.

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