EP0862644A2 - Produit de recombinaison d'acide nucleique pour therapie genique, production et utilisation de ce produit pour le traitement de maladies cardiaques - Google Patents

Produit de recombinaison d'acide nucleique pour therapie genique, production et utilisation de ce produit pour le traitement de maladies cardiaques

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Publication number
EP0862644A2
EP0862644A2 EP96945991A EP96945991A EP0862644A2 EP 0862644 A2 EP0862644 A2 EP 0862644A2 EP 96945991 A EP96945991 A EP 96945991A EP 96945991 A EP96945991 A EP 96945991A EP 0862644 A2 EP0862644 A2 EP 0862644A2
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Prior art keywords
nucleic acid
acid construct
gene
construct according
heart
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English (en)
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Wolfgang-M. Franz
Thomas Rothmann
H.A. Katus
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Franz Wolfgang-M Dr
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Franz Wolfgang-M Dr
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Publication of EP0862644A2 publication Critical patent/EP0862644A2/fr
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    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/24—Drugs for disorders of the endocrine system of the sex hormones
    • A61P5/28—Antiandrogens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/24—Drugs for disorders of the endocrine system of the sex hormones
    • A61P5/36—Antigestagens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/38—Drugs for disorders of the endocrine system of the suprarenal hormones
    • A61P5/42—Drugs for disorders of the endocrine system of the suprarenal hormones for decreasing, blocking or antagonising the activity of mineralocorticosteroids
    • 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
    • C07K14/46—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
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4707—Muscular dystrophy
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70571—Receptors; Cell surface antigens; Cell surface determinants for neuromediators, e.g. serotonin receptor, dopamine receptor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C12N9/0073—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with NADH or NADPH as one donor, and incorporation of one atom of oxygen 1.14.13
    • C12N9/0075—Nitric-oxide synthase (1.14.13.39)
    • 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

Definitions

  • the present invention relates to a gene therapeutic nucleic acid construct containing a regulatory nucleic acid sequence from the 5 'end of the myosin light chain 2 gene (MLC-2) of the heart, which is functionally linked to a nucleic acid that is necessary for a therapeutically active gene product antisense nucleic acid or encoded for a ribozyme, as well as a method for its production and the use for gene therapy treatment of heart diseases.
  • MLC-2 myosin light chain 2 gene
  • the clinical picture of cardiomyopathy encompasses a group of cardiac muscle diseases which show up both in contractile and in electrophysiological disorders and ultimately lead to severe heart failure and / or sudden, electrophysiological cardiac death.
  • the search for monogenetic causes in familial forms of dilated and hypertrophic cardiomyopathy is currently the subject of numerous scientific studies. In this way, genetic causes of heart muscle diseases have recently been elucidated at the molecular level. For example, the so-called Duchenne muscular dystrophy (DMD) also causes cardiomyopathy.
  • DMD is an inherited disorder caused by mutations and deletions in the dystrophin gene.
  • the dystrophin gene is on the X chromosome localized and is expressed in healthy people in cardiac muscle cells. It was also found that with chronic congestive heart failure (CHF) the myocardium contains 50% less ß-adrenergic receptor than healthy myocardium.
  • CHF chronic congestive heart failure
  • somatic gene transfer is a promising method for treating genetically caused heart muscle diseases.
  • somatic gene transfer e.g. B. the gene transfer by injection of DNA, the liposome-assisted gene transfer or the gene transfer by means of retroviral, adenoviral or adeno-associated vectors.
  • Essential prerequisites for successful gene therapy are a high transmission rate, stable gene expression and, above all, tissue specificity.
  • WO94 / 11506 shows the successful gene transfer and the successful expression of a gene coding for the ⁇ -galactosidase under the control of the CMV promoter both in smooth muscle cells of the coronary vessels and in cardiac muscle cells.
  • cardiac muscle-specific expression could not be achieved.
  • the description generally refers to the cardiac muscle-specific troponin C (cTNC) promoter, it does not, however, show cardiac-specific in vivo expression.
  • the object of the present invention was therefore to find a nucleic acid construct which has a high transmission rate, a stable gene expression and above all a specificity for cardiac muscle cells for the gene therapy of heart diseases.
  • the present invention therefore relates to a gene-therapeutic nucleic acid construct containing a regulatory nucleic acid sequence from the 5 'end of the myosin light chain 2 gene (MLC-2) of the heart, preferably the heart of a mammal, in particular of humans or of a rodent, especially from the rat, which is functionally linked to a nucleic acid that codes for a therapeutically effective gene product, for an antisense nucleic acid or for a ribozyme.
  • MLC-2 myosin light chain 2 gene
  • a regulatory nucleic acid sequence in the sense of the present invention is generally understood to be the nucleic acid sequence located upstream from the transcription start point (+1) of the MLC-2 gene, which is the transcription of a downstream nucleic acid sequence connected to this sequence at the 3 'end, in particular with regard to the correct transcription start which controls the transcription rate and / or the myocardial tissue specificity, ie the regulatory nucleic acid sequence is functionally linked to the downstream nucleic acid sequence.
  • the sequence from about +18 to -19 to about -800 based on the start of transcription of the MLC-2 gene of the heart is particularly preferred (see Fig.
  • Another preferred embodiment is also a sequence from about +18 to -19 to about -1600 and in particular from about +18 to -19 to about -1800, especially from about +18 to -19 to about -2100 or from about + 18 to -19 to approximately -2700 based on the transcription start point of the MLC-2 gene of the heart (see Fig. 10).
  • the regulatory nucleic acid sequence mainly contains one or more regulatory elements selected from TATA-Box, HF-la, HF-lb, HF-2, HF-3, E-Box, MLE1 and / or CSS sequence, especially selected from TATA-Box, HF-la, HF-lb, HF-2, HF-3, E-Box and / or MLE1.
  • the TATA box is approximately between -198 and -19, the HF-1 element, a conserved 28 base long sequence, approximately between -72 and -45 and in particular the HF-la element approximately between - 57 and -65 and the HF-lb element approximately between -45 and -56, the HF-2 element approximately between -123 and -134, the HF-3 element approximately between -186 and -198, the E-Box element approximately between -72 and -77, the MLE1 element approximately between -165 and -176 and the CSS-like element approximately between -1723 and -1686 related to the transcription start point of the MLC-2 gene (see Fig. 10).
  • the regulatory sequences TATA box, HF-lb element, HF-la element, E-box element, HF-2 element, MLE1 element and HF-3 element are in this order within the first 200 Bases upstream from the gene's transcription start point (see Fig. 10).
  • the nucleic acid construct according to the invention contains the HF-la element, the HF-lb element, the MLE1 element and the HF-3 element, preferably together with the E-Box element, in particular together with the E-Box Element and / or HF-2 element. In any case, it is also preferred if the nucleic acid construct according to the invention additionally contains the heart-specific sequence CSS.
  • a gene therapeutic nucleic acid construct in the sense of this invention is understood to mean a nucleic acid construct with a nucleic acid sequence, which is in particular a DNA or RNA sequence, preferably a single-stranded or double-stranded, especially a double-stranded DNA sequence, the nucleic acid construct being a medicament for gene therapy treatment of heart diseases, in particular for the treatment of heart failure, dilated or hypertrophic cardiomyopathy, dystrophinopathy, vascular diseases, hypertension, arteriosclerosis, stenosis or restenosis of the blood vessels can advantageously be used.
  • the nucleic acid construct according to the invention is preferably combined with a virus vector and / or with liposomes, preferably with an adenovirus vector, especially with a replication-deficient adenovirus vector, or with an adeno-associated virus vector, especially with an adeno-associated virus vector, which consists exclusively of two inverted terminal Repeat sequences (ITR) exist, ligated.
  • a particularly preferred embodiment of the present invention is the genetic engineering connection of the nucleic acid construct according to the invention with an adenovirus vector, especially with a replication-deficient adenovirus vector.
  • An adenovirus vector and particularly a replication-deficient adenovirus vector are particularly preferred for the following reasons:
  • the human adenovirus belongs to the class of double-stranded DNA viruses with a genome of approximately 36 kilobase pairs (Kb).
  • the viral DNA codes for about 2700 different gene products, a distinction being made between early ("late genes”) and late (“late genes”) gene products with regard to the adenoviral replication cycle.
  • the "early genes” are divided into four transcriptional units, E1 to E4.
  • the late gene products code for the capsid proteins.
  • At least 42 different adenoviruses and the AF subgroups can be differentiated immunologically, all of which are suitable for the present invention.
  • the transcription of the viral genes presupposes the expression of the El region, which codes for a transactivator of the adenoviral gene expression.
  • the El gene region is generally replaced by a foreign gene with its own promoter or replaced by the nucleic acid construct according to the invention.
  • the exchange of the El gene region which is a prerequisite for the expression of the downstream adenoviral genes, results in an adenovirus that is not capable of replication. These viruses can then only multiply in a cell line that replaces the missing El genes.
  • Replication-deficient adenoviruses are therefore generally formed by homologous recombination in the so-called 293 cell line (human embryonic kidney cell line), which has a copy of the El region stably integrated in the genome.
  • 293 cell line human embryonic kidney cell line
  • a nucleic acid sequence for example that for a therapeutically active gene product according to the present invention or for a marker, for example ⁇ -galactosidase / ⁇ -Gal
  • the homologous recombination then takes place, for example, between the plasmids pAd.mlc-2 / ß-Gal and an El-deficient adenoviral genome such as, for.
  • viral plaques are harvested.
  • the replication-deficient viruses generated in this way are then used in high titers (for example IO 9 to 10 11 "plaque forming units” or plaque forming units) for infection of the cell culture or for somatic gene therapy.
  • the precise location of insertion of the foreign DNA into the adenoviral genome is not critical. It is Z. B. also possible to clone the foreign DNA in the place of the deleted E3 gene (Karlsson, S. et al. EMBO J. 1986, 5, 2377-2385). However, the El region or parts is preferred of which, e.g. B. the E1A or ElB region (see e.g. WO95 / 00655) replaced by the foreign DNA, especially if the E3 region is also deleted.
  • the present invention is not limited to the adenoviral vector system, but adeno-associated virus vectors are also particularly suitable in combination with the nucleic acid construct according to the invention for the following reasons:
  • the AAV virus belongs to the parvovirus family. These are characterized by an icosahedral, non-encapsulated capsid with a diameter of 18-30 nm, which contains a linear, single-stranded DNA of about 5 kb. A co-infection of the host cell with helper viruses is required for an efficient multiplication of AAV. Suitable aids are, for example, adenoviruses (Ad5 or Ad2), herpes viruses and vaccinia viruses (Muzyczka, N. (1992) Curr. Top. Microbiol. Immunol. 158, 97-129).
  • AAV In the absence of a helper virus, AAV goes into a latency state, whereby the virus genome is able to integrate stably into the host cell genome.
  • the ability of AAV to integrate into the host genome makes it particularly interesting as a transduction vector for mammalian cells.
  • the two approximately 145 bp long inverted terminal repetition sequences (ITR: inverted terminal repeats; generally see W095 / 23867) are sufficient for the vector functions. They carry the signals necessary for replication, packaging and integration into the host cell genome.
  • ITR inverted terminal repeats
  • a cell-free lysate which contains adenoviruses in addition to the recombinant AAV particles.
  • the adenoviruses can advantageously be removed by heating to 56 ° C. or by banding in a cesium chloride gradient. With this cotransfection method, rAAV titers are 10 5 -10 6 IE / ml achievable. Contamination by wild-type viruses is below the detection limit if the packaging plasmid and the vector plasmid have no overlapping sequences (Samulski, RJ (1989) J. Virol. 63, 3822-3828).
  • the transfer of foreign genes into somatic body cells can be carried out by AAV in resting, differentiated cells, which is particularly advantageous for the gene therapy of the heart. Long-term gene expression in vivo can also be ensured by the integration capability mentioned, which in turn is particularly advantageous. Another advantage of AAV is that the virus is not pathogenic to humans and is relatively stable in vivo.
  • the nucleic acid construct according to the invention is cloned into the AAV vector or parts thereof according to methods known to the person skilled in the art, such as, for. B. in W095 / 23867, by Chiorini, J.A. et al. (1995) Human Gene Therapy 6, 1531-1541 or Kotin, R.M. (1994) Human Gene Therapy 5, 793-801.
  • a further advantageous combination in the sense of the present invention is the complexation of the nucleic acid constructs according to the invention with liposomes, since this enables a very high transfection efficiency, in particular of cardiac muscle cells, to be achieved (Feigner, PL et al. (1987) Proc. Natl. Acad. Sei. USA 84 , 7413-7417).
  • lipofection small unilamellar vesicles are made from cationic lipids by ultrasound treatment of the liposome suspension.
  • the DNA is bound ionically on the surface of the liposomes in such a ratio that a positive net charge remains and the plasmid DNA is 100% complexed by the liposomes.
  • lipid mixtures DOTMA (1,2-dioleyloxypropyl-3-trimethylammonium bromide) and DOPE (dioleoylphosphatidylethanolamine) have meanwhile been synthesized and numerous new lipid formulations have been synthesized and tested for their efficiency in transfecting different cell lines (Behr, JP et al. (1989 ) Proc. Natl. Acad. Sei USA 86, 6982-6986; Feigner, JH et 97/17937 PC17DE96 / 02181
  • Suitable therapeutic gene products are, for example, dystrophin, the ⁇ -adrenergic receptor, nitrogen monoxide synthase or any other gene product that e.g. complements a monogenetic error, prevents or reduces electrophysiological disorders or can mitigate or cure other heart-specific diseases.
  • the gene coding for the therapeutic gene product contains one or more non-coding sequences, including intron sequences, preferably between the promoter and the start codon of the transgene, and / or a polyA sequence, especially on the 3 ' -End of the transgene, for example the endogenous polyA sequence of the respective gene, preferably an SV40 virus polyA sequence, since this can stabilize the mRNA in the heart muscle cell (Jackson, RJ (1993) Cell 74, 9-14 and Palmiter , RD et al. (1991) Proc. Natl. Acad. Sci. USA 88, 478-482).
  • the nucleic acid functionally linked to the regulatory nucleic acid of the MLC-2 gene can be not only a nucleic acid which codes for a therapeutically active gene product, but also a nucleic acid which is responsible for an "antisense” nucleic acid, preferably an “antisense” oligonucleotide, in particular an “antisense” DNA oligonucleotide or encoded for a ribozyme.
  • an antisense oligonucleotides and by ribozymes, the expression of genes in the heart can be specifically reduced or can be prevented, whereby a variety of Herz ⁇ specific diseases, such as. B.
  • Another object of the present invention is also a method for producing the nucleic acid construct according to the invention, wherein the regulatory nucleic acid sequence described in more detail above is functionally linked to a nucleic acid which codes for a therapeutically active gene product, for an antisense nucleic acid or for a ribozyme.
  • the regulatory nucleic acid mentioned and the nucleic acid which codes for a therapeutically active gene product, for an antisense nucleic acid or for a ribozyme are cloned either simultaneously or in succession into one of the virus vectors described in more detail above.
  • the method according to the invention is carried out according to genetic engineering methods which are generally known to the person skilled in the art (see, for example, Maniatis et al. (1982) Molecular cloning, A laboratory manual. Cold Spring Harbor Laboratory New York).
  • the protein or nucleic acid sequences of the therapeutically active gene products are available, for example, from the EMBL gene bank or any other publicly accessible gene bank.
  • the sequence of the rat heart MLC-2 gene is from Henderson, SA et al. (1989), known supra and the regulatory nucleic acid sequence of the MLC-2 gene can be found in Fig. 10. Based on these sequences and that described in Henderson, SA et al.
  • Another object of the present invention relates to a method in which the nucleic acid construct according to the invention with liposomes, such as e.g. described in more detail in DE 44 11 402, is complexed.
  • Another object of the present invention is the use of the nucleic acid construct according to the invention for the gene therapy treatment of a heart disease or for the manufacture of a medicament for the gene therapy treatment of a heart disease, the heart disease preferably being heart failure, dilated or hypertrophic cardiomyopathy, dystrophinopathy, vascular diseases, High blood pressure, arteriosclerosis, stenosis and / or restenosis of the blood vessels. It is particularly advantageous if the nucleic acid construct according to the invention acts essentially in the heart chamber (ventricle).
  • Another object of the present invention is therefore also a medicament containing a nucleic acid construct according to the invention and optionally a pharmaceutical carrier, for example a physiological buffer solution, preferably with a pH of approximately 6.0 to approximately 8.0, especially approximately 6.8 to about 7.8, especially about 7.4 and / or an osmolarity of from about 200 to about 400 milliosmols per liter (mosm / L), preferably from about 290 to about 310 mosm / L.
  • the pharmaceutical carrier can also suitable stabilizers, such as. B. nuclease inhibitors, preferably complexing agents such as EDTA and / or other auxiliaries known to those skilled in the art.
  • the application of the nucleic acid constructs according to the invention optionally in combination with the virus vectors or liposomes described above is generally carried out intravenously (iv), for. B. with the help of a catheter.
  • iv intravenously
  • PCGT Percutaneous Coronary Gene Transfer
  • the application of the nucleic acid constructs according to the invention, especially in the form of recombinant adenoviruses with the aid of a balloon catheter such as. B. Feldman et al. (Feldman, LJ et al. (1994) JACC 235A, 906-34), preferred, since in this way the transfection can be limited not only to the heart but also to the injection site within the heart.
  • the nucleic acid construct according to the invention shows a high transmission rate in the gene therapy treatment of heart diseases, is stable and expressible in the transfected cells and, above all, does not lose its specificity for cardiac muscle cells. This is so surprising because, for. B. the smmhc promoter loses its specificity for neonatal and adult smooth muscle cells (see Example 6 below) and a preferred mlc-2 promoter of the nucleic acid construct according to the invention, which does not contain the heart-specific sequence CSS, retains its specificity, in particular in connection with an adenovirus vector.
  • the mlc-2 promoter-controlled expression in cardiomyocytes, in particular in the ventricle is significantly higher than, for example, the mlc-2 promoter-controlled expression in vascular muscle cells, above all that the difference in Expression is about one to about three, especially about three to about six, especially about three to about four powers of ten.
  • the mlc-2 promoter restricted the expression of the luciferase to the heart much more than the C-mhc promoter (see Example 10 below). It is also particularly advantageous that with the nucleic acid construct according to the invention the heart-specific expression after in vivo application is limited to the heart chamber (ventricle) (see Example 11 below), since it is thereby possible, for example, to increase the contraction force of the ventricle.
  • Fig. 1 shows a schematic representation of the constructed plasmids pAd-Luc, pAd-rsvLuc, pAd-mlcLuc and pAd-smmhcLuc.
  • BamHI, Kpnl and Hindlll denote the restriction enzyme interfaces of the corresponding enzymes.
  • Fig. 2 shows the recombinant adenoviruses obtained by homologous recombination, which are derived from the adenovirus del324, the luciferase gene having been cloned into the former El region.
  • Expression of the luciferase gene is either by the smmhc promoter (Ad-smmhcLuc), which is specific for the smooth vascular muscles, the mlc-2v promoter (Ad-mlcLuc) for the heart muscle-specific expression, by the RSV promoter (Ad- rsvLuc) as a positive control or controlled by no promoter (Ad-Luc) as a negative control.
  • Ad-smmhcLuc the mlc-2v promoter
  • Ad-rsvLuc the RSV promoter
  • Fig. 4A-C show schematic representations of the luciferase activities of Ad-Luc, Ad-rsvLuc and Ad-mlcLuc in various primary cell tissues.
  • the thin line on each column represents the mean standard deviation of the experiments.
  • Fig. 5A-C show the schematic representations of the luciferase activity of Ad-Luc, Ad-rsvLuc and Ad-mlcLuc in different tissues after injection of the recombinant adenoviruses into the ventricle of neonatal rats.
  • the thin line on each column represents the mean standard deviation of the experiments.
  • Fig. 6A and B show the histological detection of the ⁇ -galactosidase activity in the myocardium after intracavitary injection of the recombinant adenovirus AD.RSVßgal.
  • Fig. 6A shows a photograph of a histological section through the apex (injection site).
  • Fig. 6B shows the photograph of a histological section through the left ventricle. The bar corresponds to 100 ⁇ m.
  • Fig. 7A-C show the detection of adenoviral DNA in 12 different tissues after intracavitary injection of the recombinant adenoviruses Ad-Luc, Ad-rsvLuc and Ad-mlcLuc.
  • Fig. 7A shows a photograph of a 2.4% agarose gel with the specific 860 bp PCR product, which was generated by amplification of decreasing amounts of Addel324 DNA.
  • 100 ng of rat genomic DNA mixed with Addel324 DNA were used as the sample: lane 1:10 pg; Lane 2: 1 pg; Lane 3: 100 fg; Lane 4: 10 fg; Lane 5: 1 fg; Lane 6; 0.1 fg; Lane 7: no viral DNA.
  • M corresponds to a DNA marker (100 bp ladder).
  • FIG. 7B shows a photograph of a 2.4% agarose gel with the specific 860 bp PCR product, amplified from 100 ng of genomic DNA which is isolated from the specified tissues after intracavitary injection of Ad-Luc, Ad-rsvLuc and Ad-mlcLuc had been.
  • a PCR approach with 100 ng genomic DNA of the rat mixed with 1 pg Addel324 DNA served as a positive control, a approach without Addel324 DNA as a negative control.
  • Fig. 7C shows a Southern blot of the Ad-mlcLuc infected animal according to Fig. 7B.
  • the ⁇ 2 P-labeled 860 bp PCR product from a control batch was used as the probe.
  • Fig. 8A and B show the luciferase activities of the recombinant adenoviruses Ad-o-mhcLuc (Fig. 8A) and Ad-mlcLuc (Fig. 8B) after intracavitary injection into the left main chamber of neonatal rats in different tissues.
  • Fig. 9A-C show the luciferase activities of the recombinant adenoviruses Ad-rsvLuc, Ad-mlcLuc, Ad-O-mhcLuc and Ad-Luc in the atrium (Fig. 9A) and in the ventricle (Fig. 9B).
  • the relationship between the activities in the atrium and in the ventricle is shown in Fig. 9C.
  • the columns show the median of four experiments, the dots representing the results for the respective test animals and the ratio of the luciferase activity in the ventricle to the atrium.
  • 10A-C show the nucleic acid sequence of a 2216 base pair long promoter of the rat MLC-2v gene upstream from the transcription start point (+1).
  • the cloning sequence for the BamHI restriction endonuclease is at position 158-163 and for Kpnl at position 189-194. From position 189-2405 is the 2216 base pair promoter of the MLC-2v gene.
  • the CSS-like sequence is at position 682-724, the HF-3 element at position 2207-2219, the MLE1 element at position 2229-2241, the HF-2 element at position 2271-2289, the E-Box element at position 2328-2333, the HF-la element at position 2340-2348, the HF-lb element at position 2349-2361 and the transcription start (+1) at position 2406.
  • the luciferase coding sequence begins at position 2461.
  • At position 1660-2406 is the 746 base pair long regulatory sequence of the plasmid pAd-mlcLuc (see Example 1).
  • the plasmids pAD-Luc, pAD-rsvLuc, pAd-mlcLuc, pAd-smmhcLuc (Fig. 1) and pAdctmhcLuc are derivatives of the plasmid pAd.RSVßgal (Stradtford-Perricaudet, LD, J. (1992) Clin. Invest.
  • the HindiII / Kpnl fragment of the plasmid pSVOAL which codes for the luciferase gene, was subcloned 5 'into the HindiII / Kpnl cloning interfaces of the vector pBluescriptSK (Stratagene) and the plasmid pBluescript-Luc was thereby generated (Wet, JR et al. (1987) Mol. Cell. Biol. 7, 725-735).
  • the BamHI / Kpnl luciferase fragment of the subclone pBluescript-Luc was then cloned into the BamHI / Kpnl interfaces of the plasmid pAD.RSV-ßgal, and the plasmid pAd-Luc was thereby generated.
  • the BamHI / HindiII RSV fragment (587 bp) of the plasmid pAD.RSV- ⁇ gal was cloned into the BamHI / VietnameseII interfaces of the subclone pBluescript-Luc and the plasmid pBluescript-RSV-Luc was thereby generated.
  • the BamHI / Kpnl RSV-Luciferase fragment of the plasmid pBluescript-RSV-Luc was then cloned into the BamHI / Kpnl interfaces of pAd.RSV-ßgal and the plasmid pAd-rsvLuc was thereby generated.
  • the BamHI / Kpnl mlc-luciferase fragment (746 base pair long "myosin light chain” -2v promoter according to Fig. 10 and 1.8 kb luciferase gene) from the plasmid pMLCIl ⁇ s' was directly into the BamHI / Kpnl interfaces of the plasmid pAd-RSVßgal cloned (Henderson, SA et al. (1989) J. Biol. Chem. 264, 18142-18148).
  • the mlc-2 / luciferase fusion construct was cut out at the restriction enzyme sites Kpnl, the overhanging ends were filled in a so-called "Klenow reaction” and PvuII linkers were ligated at both ends.
  • the 4.0 kb long mlc-2 / luciferase DNA fragment was then inserted into the PvuII site at the 3 'end of the 1.3 m.u. in analogy to the recombinant plasmid pAd.RSVßgal. Region of the adenovirus type 5 (Ad 5) genome coupled.
  • the 1.2 kb BamHI / Hindlll smmhc fragment (rabbit "smooth muscle myosin heavy chain" promoter / -1225 / -4) from the plasmid pRBSMHC-1225ßgal (Kallmeier, RC et al. (1995) J. Biol. Chem. 270, 30949-30957) and cloned in the BamHI / HindIII opened subclone pBluescript-Luc in front of the luciferase gene and the subclone pl.2smmhcBluescript-Luc was thereby constructed.
  • the BamHI / Kpnl smmhc luciferase fragment of this subclone was then cloned into the BamHI / Kpnl sites of the plasmid pAd-RSVßgal and the plasmid pAd-smmhcLuc was generated.
  • the recombinant adenoviruses were generated by standard methods by homologous recombination between the plasmids pAd-Luc, pAd-rsvLuc, pAd-mlcLuc, pAd-smmhcLuc and pAd-ctmhcLuc and the genomic DNA of adenovirus del324 (Ad5) in 293 cells in vivo (Thimmappaya , B. et al. (1982) Cell 31, 543-551 and Stradtford-Perricaudet, LD et al. (1992), supra and Graham, FL et al. (1977) J. Gen. Virol. 36, 59-74 ).
  • the recombinant adenoviruses have a deletion in the E3 region and the transgenes Luc, RSV-Luc, mlcLuc, pAd-smmhcLuc and pAd-O-mhcLuc replace the El region.
  • the day before the transfection 2 ⁇ 10 6 293 cells were plated out in a small cell culture dish.
  • 5 ⁇ g of the large Clal fragment of the genomic DNA from Addel324 were co-transfected into 293 cells together with 5 ⁇ g of the AatII linearized plasmids pAd-Luc, pAd-RSV-Luc, pAd-mlcLuc, pAd-s ⁇ unhcLuc and pAd- ⁇ mhcLuc according to the calcium phosphate method .
  • the positive viral clones were again subjected to a single plaque cleaning before they were propagated in 293 cells for large-scale processing and were purified by means of two cesium chloride density gradient centrifugation (Stradtford-Perricaudet, LD, 1992, supra). Finally the viruses were treated against TD buffer (137 mM NaCl, 5 mM KCl, 0.7 mM Na 2 HP0 4 , 0.5 mM CaCl 2 , 1 mM MgCl 2 , 10% (v / v) glycerol, 25 mM Tris -HCl, pH 7.4) dialyzed and frozen at -72 ° C.
  • TD buffer 137 mM NaCl, 5 mM KCl, 0.7 mM Na 2 HP0 4 , 0.5 mM CaCl 2 , 1 mM MgCl 2 , 10% (v / v) glycerol, 25 mM Tris -HCl, pH 7.
  • the "plaque assay” was carried out using 293 cells. All recombinant adenoviruses had a titer of approximately 10 11 "plaque forming units" (pfu) / ml.
  • the DNA of the viral stock solutions was isolated and examined for the correct integration of the inserts by analysis using restriction endonucleases and PCR. Furthermore, the viral stock solutions were examined by means of PCR on the wild type Ad-5, whereby no contamination was detectable in 50 ng of the adenoviral DNA (Zang, WW et al. (1995) BioTechniques 18, 444-447).
  • the cells were harvested 48 hours after infection.
  • the luciferase activity in protein extracts was then determined according to established protocols using the Lumat LB 9501 transilluminometer (Bertold, Wildbad) (Ausubel, F.M. et al. (1989) Current Protocols in Molecular Biology. Greene and Wiley, New York).
  • the protein concentration of the lysates was determined according to Bradford (1976) (BioRad, Kunststoff). Luciferase activity was converted to pg luciferase per ⁇ g protein (Krougliak, V. & Graham, FL (1995) Hum. Gene Ther. 6, 1575-1586 and Franz, WM et al. (1993) Circ. Res. 73, 629- 638).
  • the rats were decapitated 5 days after injection. Twelve different tissues (intercostal muscle, heart, thymus, lungs, diaphragm, abdominal muscle, liver, stomach, spleen, kidneys, quadriceps femoris, brain) were removed and immediately frozen in liquid nitrogen. The tissue samples were then weighed in 200 ⁇ l lysis buffer (1% (v / v) Triton X-100, 1 mM DTT, 100 mM potassium phosphate pH 7.8) was taken up, digested in a glass homogenizer and centrifuged for 15 minutes at 4 ° C. in a refrigerated centrifuge.
  • lysis buffer 1% (v / v) Triton X-100, 1 mM DTT, 100 mM potassium phosphate pH 7.8
  • luciferase The supernatant was used for the determination of luciferase (Acsadi, G. et al. (1994) Hum. Mol. Gen. 3, 579-584 and Ausubel, FM (1989), supra).
  • the substrates luciferin and ATP were added and the light emission, which is proportional to the luciferase activity, was measured photometrically at 560 nm in a transilluminometer. Luciferase activity was reported in "relative light units" (RLU) / mg wet tissue weight after subtracting the background activity determined for the various tissues in uninfected animals.
  • RLU relative light units
  • the hearts of neonatal rats were frozen in nitrogen-cooled isopentane and stored at -70 ° C.
  • the heart tissue was embedded in 0. CT (Tissue Tek, Miles, USA) freezing medium and 10 ⁇ m tissue sections were made with a cryostat (Frigocut 2800 E, Leica).
  • the genomic DNA was extracted from the sediments of the tissue homogenates of the neonatal rats infected with adenoviruses using the QIAamp tissue kit (from Quiagen, Hilden) according to the manufacturer's instructions. Two of the animals infected with Ad-Luc, Ad-RSV-Luc and Ad-mlcLuc were examined for the tissue distribution of the injected viruses by PCR (polymerase chain reaction) to detect the adenoviral DNA (Zhang, WW (1995) BioTechniques 18, 444- 447).
  • genomic DNA 100 ng was used as a sample together with 40 ng of the oligonucleotides E2B-1 and E2B-2 and 1.25 U Taq polymerase from Promega in a reaction volume of 25 ⁇ l. Gel electrophoresis of the specific PCR product gave an 860 bp band.
  • the sensitivity of the PCR was determined in preliminary tests. For this purpose, 100 ng of genomic DNA from an uninfected rat were mixed with decreasing amounts of Addel324 DNA and used as a sample in a PCR reaction. In order to increase the sensitivity of the detection of the PCR, the PCR products were transferred to a GeneScreenPlus nylon membrane (NEN, Boston, Massachusetts) by capillary blot and then detected by Southern blot hybridization (Ausubel, FM et al. (1989), supra). The adenoviral 860 bp DNA fragment of the positive control amplified by PCR was used as the probe.
  • the PCR product was purified from the gel and radioactively labeled with ⁇ 2 p by "random hexanucleotide prime" and used as a sample for the hybridization. In this way, the sensitivity of the PCR detection could be improved by a factor of 10 to 100.
  • A10- (rat smooth muscle cell line), H9c2- ((rat myoblast cell line) and HeLa- (human Cervical carcinoma cell line) Cells were complemented in "Dulbecco's modified Eagle's medium” (DMEM), 293 cells in MEM, with 10% fetal calf serum (FCS), 100 U / ml penicillin, 0.1 ⁇ g / ml streptomycin and 2 mM L - Cultivated glutamine.
  • DMEM Dulbecco's modified Eagle's medium
  • FCS fetal calf serum
  • lxlO 5 cells from the established cell lines H9c2, AlO and HeLa were plated in triplicate on "12 well" culture dishes.
  • the cells were incubated in 0.2 ml of the respective serum-free medium which contained the recombinant adenoviruses Ad-Luc, Ad-RSVLuc, Ad-mlcLuc and Ad-smmhcLuc in a "multiplicity of infection" (moi) of 10 (10 viruses / Cell). After 1 hour of incubation at 37 ° C. with slight oscillation, 2 ml of the respective completed medium were added every 15 minutes. All infection experiments were repeated 4 times. Three days after the infections, the luciferase activities were measured as described above.
  • moi multiplicity of infection
  • Fig. 3 The results of the experiments are shown schematically in Fig. 3. It can be seen that the luciferase activity of the adenovirus Ad-mlcLuc is lower than the negative control with the promoterless adenovirus Ad-Luc in all cell lines examined.
  • Ad-smmhcLuc shows increased activity in the HeLa cell line and Ad-rsvLuc shows the highest luciferase activity as a positive control in all cell lines examined.
  • Fig. 4 The results of the experiments are shown schematically in Fig. 4. It can be seen that only in neonatal cardiomyocytes is the luciferase activity of the recombinant adenovirus Ad-mlcLuc higher than the negative control with the adenovirus Ad-Luc, but less than the positive control with the adenovirus Ad-rsvLuc, but 300-900 times higher than in smooth ones Vascular muscle cells. It can also be seen that the luciferase activity of Ad-mlcLuc is 129 times higher than that of Ad-smmhcLuc. It follows that the mlc-2 promoter is active in neonatal cardiomyocytes, while the expected activity of the smmhc promoter was absent in neonatal and adult smooth muscle cells.
  • luciferase activity was determined in twelve different tissues (intercostal muscle, heart, thymus, lungs, diaphragm, abdominal muscle, liver, stomach, spleen, kidney, brain and quadriceps femoris).
  • the determined luciferase activity in RLU / mg tissue is summarized in Fig. 5.
  • Adenovirus AD-mlcLuc which carries the myocardium-specific mlc-2v promoter, showed luciferase activity that was restricted to the myocardium (Fig. 5c).
  • Ad-rsvLuc The injection of the positive control Ad-rsvLuc showed the highest luciferase activity in the intercostal muscle, in the heart and a strong luciferase activity in the lungs, thymus and diaphragm (Fig. 5b).
  • Ad-Luc low luciferase activity was measured in the intercostal muscle, heart, thymus and diaphragm (Fig. 5a).
  • the ad-mlcLuc-induced luciferase activity was 17 times higher than that of Ad-Luc, while in all other tissues the luciferase activity of Ad-Luc and Ad-mlcLuc was comparable. This showed that Ad-mlcLuc is specifically active in the heart.
  • the distribution of infected cardiac muscle cells after injection of adenoviruses into the ventricle of neonatal rats was additionally checked in experiments using the injection of the recombinant adenovirus Ad-rsvßgal.
  • the recombinant adenovirus Ad-rsvßgal expresses the ⁇ -galactosidase as a reporter gene under the control of the "Rous Sarcoma Virus" (rsv) promoter.
  • the animal was sectioned five days after the injection and the expression of the ⁇ -galactosidase was determined after staining of the transgene. In the histological sections, infected cells can be recognized by the nucleus turning blue.
  • Ad-mlcLuc The luciferase activity achieved by Ad-mlcLuc was 0.05% of the luciferase activity of Ad-rsvLuc.
  • the genomic DNA from 12 tissues (intercostal muscle, heart, thymus, lung, diaphragm, abdominal muscle, liver, stomach, spleen, kidney, brain, Quadriceps femoris) and the presence of the adenoviral DNA in these tissues was determined by the PCR.
  • adenoviral DNA The sensitivity of the detection of adenoviral DNA was determined in preliminary experiments by mixing 100 ng of genomic DNA from uninfected rats with decreasing amounts of adenoviral DNA Addel324 (from 10 pg to 0.1 fg) and then analyzing them by means of PCR. It was found that 10 fg of the adenoviral DNA Addel324 could still be detected in 100 ng of genomic DNA from uninfected animals. This corresponds to 0.017 adenoviral genomes per cell (Fig. 7A). In animals infected with adenovirus, the viral DNA was regularly detected in the intercostal muscle, heart, thymus, lungs, diaphragm and liver (Fig. 4B).
  • FIG. 4C shows a representative Southern blot for an Ad mlcLuc injected animal.
  • the experiments described show that the gene expression of the recombinant adenovirus Ad-mlcLuc is due to the hermus-specific mlc-2v promoter and not to a locally increased virus concentration.
  • the recombinant adenovirus Ad-mhcLuc is more active in the kidney, spleen, liver, diaphragm, lung and intercostal muscle than Ad-mlcLuc. It follows that the mlc-2 promoter in the adenoviral vector system limits the expression of luciferase to the heart much more than the o-mhc promoter and in addition the mlc-2 promoter is 3-4 times more active in the heart than the otmhc promoter.

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Abstract

L'invention concerne un produit de recombinaison d'acide nucléique pour thérapie génique contenant une séquence d'acide nucléique régulatrice de l'extrémité 5' du gène des chaînes légères de myosine 2 (MLC-2) du coeur qui est reliée de façon fonctionnelle à un acide nucléique lequel code un produit génique thérapeutiquement efficace, un acide nucléique antisens ou un ribozyme. L'invention concerne également un procédé de production et l'utilisation de ce produit dans le traitement par thérapie génique de maladies cardiaques.
EP96945991A 1995-11-17 1996-11-14 Produit de recombinaison d'acide nucleique pour therapie genique, production et utilisation de ce produit pour le traitement de maladies cardiaques Withdrawn EP0862644A2 (fr)

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US6752987B1 (en) 1995-02-28 2004-06-22 The Regents Of The University Of California Adenovirus encoding human adenylylcyclase (AC) VI
EP0934422B1 (fr) * 1996-09-05 2006-12-06 The Regents of the University of California Therapie genique utile pour traiter l'insuffisance cardiaque globale
DE19725186C2 (de) * 1997-06-13 2000-06-15 Medigene Ag Herz- und Skelettmuskel-spezifische Nukleinsäure, ihre Herstellung und Verwendung
WO1999036538A1 (fr) * 1998-01-13 1999-07-22 Massachusetts Institute Of Technology Renforcement du chronotropisme cardiaque
US6776987B1 (en) 1998-01-13 2004-08-17 Massachusetts Institute Of Technology Enhancement of cardiac chronotropy
WO2000077201A1 (fr) 1999-06-15 2000-12-21 Astrazeneca Ab Proteine-3 inhibiteur d'apoptose (iap-3), designee sous le nom de livine
EP1255822A2 (fr) 1999-12-27 2002-11-13 The Regents Of The University Of California Therapie genique destinee a une insuffisance cardiaque congestive
DE10014690A1 (de) * 2000-03-24 2001-10-18 Franz Wolfgang M Verfahren zur Isolierung in vito differenzierter Körperzellen
WO2008126083A2 (fr) * 2007-04-11 2008-10-23 Technion Research & Development Foundation Ltd. Procédés d'identification et de sélection de cellules dérivées de cellules souches embryonnaires humaines

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WO1994011506A1 (fr) * 1992-11-18 1994-05-26 Arch Development Corporation Transfert de genes au moyen d'un adenovirus au muscle lisse cardiaque et vasculaire
ES2249761T3 (es) * 1993-06-24 2006-04-01 Advec Inc. Vectores de adenovirus para terapia genica.
US6080569A (en) * 1993-06-24 2000-06-27 Merck & Co., Inc. Adenovirus vectors generated from helper viruses and helper-dependent vectors
PL179877B1 (pl) * 1993-07-13 2000-11-30 Rhone Poulenc Rorer Sa Zdefektowany replikacyjnie adenowirus, linia komórkowa do infekowania zdefektowanym adenowirusem oraz srodek farmaceutyczny PL PL PL PL PL PL PL PL PL PL
US5602301A (en) * 1993-11-16 1997-02-11 Indiana University Foundation Non-human mammal having a graft and methods of delivering protein to myocardial tissue
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