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WO1996015245A1 - Procede d'inhibition de la proliferation cellulaire pathologique non neoplasique - Google Patents

Procede d'inhibition de la proliferation cellulaire pathologique non neoplasique Download PDF

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WO1996015245A1
WO1996015245A1 PCT/US1995/015191 US9515191W WO9615245A1 WO 1996015245 A1 WO1996015245 A1 WO 1996015245A1 US 9515191 W US9515191 W US 9515191W WO 9615245 A1 WO9615245 A1 WO 9615245A1
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cells
cell
smooth muscle
cell cycle
expression
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PCT/US1995/015191
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English (en)
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Jeffrey M. Leiden
Eliav Barr
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Arch Development Corporation
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Priority to AU45020/96A priority Critical patent/AU4502096A/en
Publication of WO1996015245A1 publication Critical patent/WO1996015245A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—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
    • C07K14/4736—Retinoblastoma protein
    • 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
    • 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
    • C07K14/4746—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used p53
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/82—Translation products from oncogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/022—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from an adenovirus

Definitions

  • the present invention relates to a process of inhibiting non- neoplastic pathological cell proliferation in a cell in vivo.
  • a process of the present invention involves transducing a cell with an eukaryotic expression vector comprising a polynucleotide that encodes a dominant-negative cell cycle regulatory protein.
  • Somatic gene therapy can be defined as the ability to program the expression of foreign genes in non-germ line (i.e., non-sperm and egg) cells of an animal.
  • Methods of somatic gene therapy can be divided into two categories, ex vivo gene therapy involving the removal of cells from a host organism, transfection of a foreign gene into those cells, and re-implantation or transplantation of the transformed or transgenic cells back into a recipient host.
  • ex vivo gene therapy involves transfection of a foreign gene directly into cells of a recipient host without the need for prior removal of those cells from the host.
  • somatic gene therapy for human subjects is dependent upon a number of factors.
  • Several possible strategies to introduce genes into tissues of the body have been employed in the past (Stratford-Perricaudet et al.. 1990; Rosenfeld et al.. 1992; Wolfe et al.. 1992). Procedures to introduce foreign genes into cells include direct transfection (Davis et al. 1986) and retroviral gene transfer (Dichek et al.. 1991: Wilson et al..
  • adenovirus-mediated gene transfer has been investigated as a means of somatic gene therapy into eukaryotic cells and into whole animals (van Doren et al.. 1984a; van Doren et al.. 1984b; Ghosh-Choudhury and Graham. 1987: Stratford-Perricaudet et 3 , 1990: Rosenfeld et al.. 1991: Rosenfeld et al.. 1992).
  • a problem with adenovirus mediated gene transfer is the low level of gene product expression in target cells and a resultant lack of a functional effect.
  • adenovirus-mediated gene transfer has been used to treat ornithine transcarbamylase (OTC) deficiency in newborn mice
  • OTC ornithine transcarbamylase
  • the expression of the ornithine transcarbamylase enzyme in the virus infected mice was typically at or below expression levels in normal mice with the result that the defect was only partially corrected (Stratford-Perricaudet et al.. 1990).
  • adenovirus-mediated gene transfer would be applicable to treatment of a disease requiring an overexpression of a gene product.
  • adenovirus mediated transfer of the gene for cystic fibrosis transmembrane conductance regulator (CFTR) into the pulmonary epithelium of cotton rats has been attempted, although it has not been possible to assess the biological activity of the transferred gene because there was no physiologic effect of gene transfer despite expression of the CFTR protein in lung airway cells (Rosenfeld et al.. 1992). Still further, lung expression of 1-antitrypsin protein was not associated with a physiologic effect ( Rosenfeld et al.. 1991). Taken together, those data do not demonstrate that adenovirus can transfer genes into cells and direct the expression of sufficient protein to achieve a physiologically relevant effect.
  • CFTR cystic fibrosis transmembrane conductance regulator
  • Somatic gene therapy can be used in the treatment of a number of pathological conditions characterized by unregulated or pathological cellular proliferation.
  • Somatic gene therapy can also be used to directly study the molecular mechanisms regulating abnormal or pathological cell proliferation such as occurs in metastatic cancer cells or in injured vascular smooth muscle cells.
  • the present invention provides a process of inhibiting non- neoplastic pathological cellular proliferation in a cell in vivo.
  • the process comprises transducing the cell with an eukaryotic expression vector including a polynucleotide that encodes an inhibitory cell cycle regulatory protein.
  • the vector drives expression of the polynucleotide in the cell.
  • the expression vector is replication-defective adenovirus type 5.
  • the adenovirus lacks the early gene region E1 or the early gene regions E1 and E3.
  • the cell whose proliferation is being inhibited is a vascular smooth muscle cell.
  • transducing is accomplished by infusing the expression vector into an artery that contains the vascular smooth muscle cell.
  • the inhibitory cell cycle regulatory protein is p53 or p21. In another embodiment, the inhibitory protein is a dominant-negative cell cycle regulatory protein such as a non- phosphorilatable form of Rb-1.
  • the expression vector preferably further includes an enhancer-promoter other than an adenovirus enhancer- promoter, where the enhancer-promoter's operatively linked to the polynucleotide that encodes the regulatory protein.
  • the enhancer-promoter includes the CMV promoter, an SV40 early promoter, a RSV promoter, the elongation factor promoter (EF1 ⁇ ), or a MCK enhancer or 4 ⁇ 2 heavy chain.
  • the enhancer-promoter is specific for vascular smooth muscle such as an endothelin promoter or a smooth muscle -actin promoter.
  • the present invention provides a process of inhibiting pathological proliferation of vascular smooth muscle cells.
  • the process includes the step of increasing the level of p21 in the cells.
  • the level of p21 is increased by increasing the expression of p21 in the cells.
  • p21 expression is increased by transforming those cells with an expression vector that contains a polynucleotide that encodes p21 operatively linked to a promoter that drives expression of p21 in the cell.
  • a preferred expression vector is a replication-defective adenoviral vector.
  • An especially preferred adenoviral vector is designated Adp21.
  • the expression of p21 in vascular smooth muscle cells can be increased in those cells either in vivo or in vitro. Where the cells are located in vivo, cells can be transformed with a suitable expression vector using direct in vivo or ex vivo transformation procedures.
  • the expression vector is injected into a blood vessel containing the cell. Intraarterial injection is preferred.
  • a process of the present invention can be used to inhibit any pathological proliferation of vascular smooth muscle cells.
  • the process is used to inhibit the pathological proliferation following arterial injury or during restenosis.
  • the present invention provides a process of treating a vascular proliferative disorder in an animal in need of such treatment comprising administering to the animal an effective amount of an expression vector that contains a polynucleotide that encodes p21 operatively linked to a promoter that drives expression of p21 in vascular smooth muscle.
  • the present invention addresses one or more shortcomings in the prior art through the provision of a process for inhibiting non- neoplastic pathological cell proliferation without necessarily causing cell death.
  • an eukaryotic expression vector construct is used to deliver a gene to the proliferating cell and thus affect expression of that gene's product.
  • the gene product is a dominant-negative cell cycle regulatory protein. Expression of the gene product thereby alters function of those cells and inhibits the pathological proliferation.
  • a process of the present invention can be used to inhibit proliferation of a cell situated in vivo in a living organism.
  • the present invention provides a process of inhibiting non- neoplastic pathological cellular proliferation in a cell in vivo, the process comprising transducing the cell with an eukaryotic expression vector comprising a polynucleotide that encodes an inhibitory cell cycle regulatory protein, the vector driving expression of the polynucleotide in the cell.
  • non-neoplastic pathological cellular proliferation means unregulated, uncontrolled or abnormally controlled cell proliferation. That phrase is meant to incorporate all non-neoplastic types of cellular proliferation other than the normal or physiological type of proliferation that a particular cell type undergoes.
  • Exemplary non-neoplastic pathological cellular proliferations are fibroblast proliferation in keloid formation after surgery, prostate epithelial cell proliferation in benign prostatic hypertrophy, uterine smooth muscle and fibroblast proliferation in uterine fibroids, colonic epithelial and connective tissue cell proliferation in benign colonic polyps, benign neuromas, skin epithelial cell proliferation in hyperkeratotic skin diseases and vascular smooth muscle proliferation following vascular injury.
  • Exemplary vascular injuries are restenosis following balloon angioplasty of coronary arteries, restenosis following balloon angioplasty of peripheral arteries (e.g., renal, femorals, carotids), restenosis following stenting of the coronary arteries, coronary artery bypass graft restenosis and occlusion, peripheral artery bypass graft stenosis, restenosis of arterial-venous shunts in renal dialysis patients, primary pulmonary hypertension, accelerated atherosclerosis following heart transplantation and glomeruloproliferative disorders.
  • peripheral arteries e.g., renal, femorals, carotids
  • a process of the present invention is used to inhibit vascular smooth muscle proliferation during restenosis.
  • a preferred cell therefore, is a vascular smooth muscle cell.
  • the arterial wall is a complex multicellular structure that plays important roles in inflammation, coagulation, and the regulation of blood flow.
  • Vascular smooth muscle cells are located predominantly in the arterial tunica media and are important regulators of vascular tone and blood pressure. These cells are normally maintained in a non- proliferative state in vivo.
  • Arterial injury results in the migration of vascular smooth muscle cells into the intimal layer of the arterial wall where they proliferate and elaborate extracellular matrix components. This neointimal smooth muscle cell proliferative response has been implicated as important in the pathogenesis of atherosclerosis (Forrester et al., i99i; Ji2_eLa 1990). Page missing at the time of publication
  • p21 also known as Cip 1 (CDK- interacting protein), Sdi1 (senescent cell-derived inhibitor), and WAF1 (wild-type p53-activated fragment)
  • Cip 1 CDK- interacting protein
  • Sdi1 senescent cell-derived inhibitor
  • WAF1 wild-type p53-activated fragment
  • p21 monomers can associate with active cyclin D/CDK complexes in proliferating fibroblasts
  • over-expression of p21 in such fibroblasts has been shown to potently inhibit the in vitro kinase activity of these cyclin/CDK complexes and to arrest these cells in the G1 phase of the cell cycle.
  • p21 may inhibit cell cycle progression by inhibiting the cyclin D/CDK dependent phosphorylation of Rb.
  • this effect has not been demonstrated directly in mammalian cells.
  • p21 In addition to its ability to inhibit the kinase activities of cyclin/CDK complexes, p21 has also been reported to bind to and inhibit the activity of the DNA polymerase oc co-factor, PCNA. Recent studies have suggested that different regions of the p21 protein are required for its CDK and PCNA inhibitory activities. Thus, p21 appears to inhibit cell cycle progression by at least two independent molecular mechanisms.
  • adenovirus-mediated over- expression of p21 inhibits VSMC proliferation in response to serum stimulation in vitro. This effect is associated with a complete inhibition of Rb phosphorylation and with the formation of p21/PCNA complexes in the cells programmed to overexpress p21.
  • Localized infection of the arterial wall at the time of balloon angioplasty with a replication- defective adenovirus encoding p21 markedly inhibited restenosis in the rat carotid artery model of vascular injury.
  • Eukaryotic expression vectors are well known in the art ( Sambrook et al.. 1989).
  • a preferred expression vector construct is an adenovirus vector construct.
  • the use of adenovirus as a vector for cell transfection is well known in the art.
  • Adenovirus vector-mediated cell transfection has been reported for various cells (Stratford-Perricaudet. et al.. 1992).
  • An adenovirus vector of the present invention is replication defective.
  • a virus is rendered replication defective by deletion of the viral early gene region 1 (E1).
  • An adenovirus lacking an E1 region is competent to replicate only in cells, such as human 293 cells, which express adenovirus early gene region 1 genes from their cellular genome.
  • an adenovirus vector used in the present invention is lacking both the E1 and the E3 early gene regions. Techniques for preparing replication defective adenoviruses are well known in the art (See, e.g. McGrorv et a , 1988, and Guzman et al.. 1982).
  • an adenovirus vector can be of any of the 42 different known serotypes or subgroups A-F.
  • Adenovirus type 5 of subgroup C is the preferred starting material for production of a replication-defective adenovirus vector.
  • An adenovirus is engineered to contain a coding DNA sequence for use as a vector.
  • a recombinant adenovirus has been described by Gluzman et al., 1982.
  • Individual DNA sequences such as cDNAs that encode a gene product are inserted into the adenovirus to create a vector construct.
  • a coding sequence for a gene product is introduced or incorporated into an adenovirus at the position from which the E1 coding sequences have been removed.
  • the position of insertion within the adenovirus sequences is not critical to the present invention.
  • a coding sequence can also be inserted in lieu of the deleted E3 region in E3 replacement vectors as described previously by Karlsson et al. (1986).
  • the E1 region of adenovirus is replaced by the coding DNA sequence or gene.
  • the resulting adenovirus vector is co-transfected into 293 cells together with a plasmid carrying a complete adenovirus genome to propagate the adenovirus.
  • An exemplary such plasmid is pJM17.
  • Co- transfection is performed in accordance with standard procedures well known in the art.
  • 293 cells are cultured in Dulbecco's modified Eagle's medium containing fetal calf serum. Confluent cultures are split the day before calcium phosphate cotransfection of plasmids.
  • the cells are shocked (e.g., a 15% glycerol shock) to boost transfection efficiency and the cells are overlaid with agar in DMEM containing fetal calf serum, penicillin, streptomycin sulfate, and other antibiotics or antifungal agents as needed. Monolayers are incubated until viral plaques appear (about 5-15 days).
  • shocked e.g., a 15% glycerol shock
  • plaques are picked, suspended in medium containing fetal calf serum, and used to infect a new monolayer of 293 cells.
  • viral lysates are subjected to a freeze/thaw cycle and designated as primary stocks.
  • the presence of recombinant virus is verified by preparation of viral DNA from infected 293 cells, restriction analysis, and Southern blotting. Secondary stocks are subsequently generated by infecting 293 cells with primary virus stock at a multiplicity of infection of 0.01 and incubation until lysis.
  • Recombinant adenovirus vectors can be propagated on, e.g., human 293 cells, or in other cell lines that are permissive for conditional replication-defective adenovirus infection, e.g., those which express adenovirus E1 gene products "in trans" so as to complement the defect in a conditional replication-defective vector. Further, the cells can be propagated either on plastic dishes or in suspension culture, to obtain virus stocks thereof.
  • a coding sequence can comprise introns and exons so long as the coding sequence comprises at least one open reading frame for transcription, translation and expression of that polypeptide.
  • a coding sequence can comprise a gene, a split gene or a cDNA molecule.
  • the coding sequence comprises a split gene (contains one or more introns)
  • a cell transformed or transfected with a DNA molecule containing that split gene must have means for removing those introns and splicing together the exons in the RNA transcript from that DNA molecule if expression of that gene product is desired.
  • a coding sequence in an adenovirus vector can code for any dominant-negative cell cycle regulatory protein.
  • the phrase "dominant negative cell cycle regulatory protein” means a protein that acts to inhibit cell proliferation or arrest cell growth at any of the cell cycle stages (e.g., S, GQ. or G- j ).
  • Such dominant-negative cell cycle regulatory proteins are well known in the art. Exemplary and preferred such proteins are the p53 gene product, the p21 gene product and the retinoblastoma gene product (Rb).
  • the classical tumor suppressor genes, p53 and Rb have each been shown to play important roles in regulating cell cycle progression in a number of mammalian cell types (Simons et al.. 1992; Morishita et AL 1993; Barr and Leiden. 1994; Hollingsworth et al.. 1993; Perrv and Levine. 1993; Helin and Harlow. 1993; Friend. 1994). Moreover, recent evidence suggests that p53-dependent G-i arrest of cell cycle progression is mediated at least in part through Rb or Rb-like proteins ( Slebos et al.. 1994).
  • Rb or p53 either by mutation or by viral oncoproteins results in unregulated proliferation and tumorigenesis in both animals and humans (Lee et al.. 1988: Lee et al.. 1987; Friend et al., 1987; Williams et al.. 1994; Malkin et al.. 1990; Livingstone et al.. 1992: Donehower et al.. 1992).
  • Rb plays a critical role in regulating the proliferation of vascular smooth muscle cells both in response to growth factor stimulation in vitro, and to injury in vivo and indicate significant parallels between the molecular mechanisms underlying carcinogenesis and those leading to human vascular proliferative disorders such as atherosclerosis and restenosis.
  • in vivo gene transfer of dominant-negative cell cycle regulatory proteins into a number of different cell types are likely be useful for the treatment of a v -iety of human diseases associat ⁇ with uncontrolled cellular proliferation.
  • the retinoblastoma gene product (Rb) is an important inhibitor of cell cycle progression in many mammalian cell types (Hollingsworth et aL. 1993; Perry and Levine. 1993; Helin and Harlow. 1993; Friend. 1994).
  • Rb is unphosphorylated, and, in that state, binds to and inactivates a set of cellular transcription factors including E2F and Elf-1 that are important for cell cycle progression (Chen et al.. 1989; DeCaprio et al.. 1992; Kovesdi et al., 1986; Wang et al., 1993; Buchkovich et al.. 1989; Mihara ⁇ LaL.
  • Rb becomes rapidly phosphorylated causing disruption of the Rb/E2F and Rb/Elf-1 complexes (Chen et al.. 1989; DeCaprio et al.. 1992; Kovesdi et aladmi 1986; Wang et al.. 1993; Buchkovich et al.. 1989; Mihara et al.. 1989: Bandara et al.. 1991: Huang et al.. 1991: Kaeline et al.. 1991).
  • the release of these Rb-associated transcription factors is associated with progression through the G-
  • Rb in regulating normal cell cycle progression has been underscored by the finding that mutations and deletions of Rb are associated with abnormal cell cycle progression and malignancies in both mice and humans (Lee et al.. 1988; Lee et al.. 1987; Friend et aL, 1987).
  • Rb viral oncogenes including the SV40 large T antigen, adenovirus E1A and human papillomavirus E7 proteins bind to Rb, thereby competitively disrupting the Rb- transcription factor complexes and leading to unregulated cell proliferation
  • the polynucleotide sequences of p21, p53 and Rb are well known in the art.
  • a coding sequence of an adenovirus vector construct is preferably operatively linked to an enhancer-promoter other than an adenovirus enhancer-promoter.
  • a promoter is a region of a DNA molecule typically within about 100 nucleotide pairs in front of (upstream of) the point at which transcription begins (i.e., a transcription start site). That region typically contains several types of DNA sequence elements that are located in similar relative positions in different genes.
  • promoter includes what is referred to in the art as an upstream promoter region, a promoter region or a promoter of a generalized eukaryotic RNA Polymerase II transcription unit.
  • An enhancer provides specificity of time, location and expression level for a particular encoding region (e.g., gene).
  • a major function of an enhancer is to increase the level of transcription of a coding sequence in a cell that contains one or more transcription factors that bind to that enhancer.
  • an enhancer can function when located at variable distances from transcription start sites so long as a promoter is present.
  • the phrase "enhancer-promoter” means a composite unit that contains both enhancer and promoter elements.
  • An enhancer-promoter is operatively linked to a coding sequence that encodes at least one gene product.
  • the phrase "operatively linked” means that an enhancer-promoter is connected to a coding sequence in such a way that the transcription of that coding sequence is controlled and regulated by that enhancer-promoter.
  • Means for operatively linking an enhancer-promoter to a coding sequence are well known in the art. As is also well known in the art, the precise orientation and location relative to a coding sequence whose transcription is controlled, is dependent inter alia upon the specific nature of the enhancer-promoter.
  • a TATA box minimal promoter is typically located from about 25 to about 30 base pairs upstream of a transcription initiation site and an upstream promoter element is typically located from about 100 to about 200 base pairs upstream of a transcription initiation site.
  • an enhancer can be located downstream from the initiation site and can be at a considerable distance from that site.
  • An enhancer-promoter used in a vector construct of the present invention can be any enhancer-promoter that drives expression in a target cell.
  • the human cytomegalovirus (CMV) immediate early gene promoter has been used to result in high-level expression of a gene.
  • CMV human cytomegalovirus
  • the use of other viral or mammalian cellular promoters which are well-known in the art is also suitable to achieve expression of the gene product provided that the levels of expression are sufficient to achieve a physiologic effect.
  • enhancer-promoters are the CMV promoter, the Rous sarcoma virus (RSV) promoter, the EF1 ⁇ promoter, the muscle-specific creatine kinase (MCK) enhancer or the 4F2 heavy chain enhancer (Zambetti et al.. 1992; Yi et al.. 1991 and Sternberg et aL. 1988).
  • an enhancer-promoter with well-known properties, the level and pattem of gene product expression can be optimized. For example, selection of an enhancer-promoter that is active specifically in vascular smooth muscle permits tissue-specific expression of the gene product.
  • a vascular smooth muscle specific enhancer- promoter is an endothelin promoter (See e.g., Lee et al.. 1990 and Bloch et al.. 1989) or a smooth muscle -actin promoter (See e.g.,
  • a coding sequence of an adenovirus vector construct is operatively linked to a transcription terminating region.
  • RNA polymerase transcribes an encoding DNA sequence through a site where polyadenylation occurs.
  • DNA sequences located a few hundred base pairs downstream of the polyadenylation site serve to terminate transcription.
  • Those DNA sequences are referred to herein as transcription-termination regions. Those regions are required for efficient polyadenylation of transcribed messenger RNA (mRNA).
  • a preferred transcription-terminating region used in an adenovirus vector construct of the present invention preferably comprises a polyadenylation signal of SV40 bovine growth hormone 3 or the protamine gene.
  • Transducing is accomplished by delivering the expression vector to the cell or cells whose pathological proliferation is to be inhibited. Delivering is accomplished by infusing the vector into an artery that perfuses the target cell. In this way, delivery is localized to the target tissues.
  • Targeted delivery of adenovirus vector containing a coding sequence for ⁇ -galactosidase ( ⁇ -gal) to cardiac muscle cells was accomplished by infusing that construct via a catheter placed into the left coronary artery or coronary sinus ostium (See Examples 1 , 2, and 5 hereinafter).
  • Targeted delivery of adenovirus vector containing a coding sequence for Rb or p21to vascular smooth muscle cells was accomplished by infusing that construct via a catheter placed into an artery (See Examples 4 and 5 hereinafter).
  • adenovirus vector employed in replication defective, it is not capable of replicating in the cells that are ultimately infected. Moreover, it has been found that the genomic integration frequency of adenovirus is usually fairly low. Thus, where continued treatment is required it may be necessary to reintroduce the virus every 6 months to a year. In these circumstances, it may therefore be necessary to conduct long term therapy, where expression levels are monitored at selected intervals.
  • An adenovirus vector construct is typically delivered in the form of a pharmacological composition that comprises a physiologically acceptable carrier and the adenovirus vector construct.
  • An effective expression-inducing amount of an adenovirus vector construct is delivered.
  • the term "effective expression-inducing amount” means that number of virus vector particles necessary to effectuate expression of a gene product encoded by a coding sequence contained in that vector. Means for determining an effective expression-inducing amount of an adenovirus vector construct are well known in the art.
  • An effective expression-inducing amount is typically from about 10 7 plaque forming units (pfu) to about 10 15 pfu, preferably from about 10 8 pfu to about 10 4 pfu and, more preferably, from about 10 9 to about 10 12 pfu.
  • a specific dose level for any particular subject depends upon a variety of factors including the infectivity of the adenovirus vector, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, and the severity of the particular disease undergoing therapy.
  • adenovirus is a virus that infects humans
  • certain individuals that have developed antibodies to certain adenovirus proteins.
  • an immunological reaction is believed to be a possibility
  • Such a test can be performed in a variety of accepted manners, for example, through a simple skin test or through a test of the circulating blood levels of adenovirus-neutralizing antibodies.
  • This example describes the use of recombinant replication defective adenoviruses in the preparation of virus vector constructs comprising a coding DNA sequence.
  • adenovirus (Guzman et al.. 1982) containing distinct cDNAs (AdCMV-cDNA) were prepared in accordance with standard techniques well known in the art.
  • E.coli ⁇ -galactosidase cDNA carrying the SV40 T antigen nuclear targeting signal (Bonnerot et al.. 1987) was inserted into pAdCMV to create a distinct construct comprising the cytomegalovirus (CMV) promoter, the ⁇ -Gal cDNA and a polyadenylation signal from either the SV40 virus or the mouse protamine gene, and flanked by adenovirus type 5 sequences.
  • CMV cytomegalovirus
  • E1 and E3 region of adenovirus were deleted and the E1 region was replaced by the ⁇ -Gal encoding sequence.
  • AdCMV ⁇ -gal The resulting plasmid, designated AdCMV ⁇ -gal, was cotransfected into 293 cells. Co-transfection was performed as follows: 293 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 2% fetal calf serum. Confluent dishes were split to non-confluent flasks the day before cotransfection with pAdCMV ⁇ -gal. Monolayers were incubated until the appearance of viral plaques.
  • DMEM Dulbecco's modified Eagle's medium
  • plaques were picked, suspended in DMEM containing 2% fetal calf serum and used to infect a new monolayer of 293 cells. When greater than 90% of the cells showed infection, viral lysates were subjected to a freeze/thaw cycle and were designated as primary stocks. Recombinant virus with the correct structure was verified by preparation of viral DNA from productively-infected 293 cells, restriction analysis, and Southern blotting. Secondary stocks were subsequently generated by infecting 293 cells with primary virus stock and incubation until lysis.
  • the large scale production of recombinant adenovirus was performed in 293 cells. Infected cells were lysed 48 hours post- infection. Virus-containing extracts were centrifuged to remove debris before precipitation of the virus. Virus was collected by centrifugation, resuspended in isotonic medium, purified, and sterilized.
  • CsCI 1 g/ml
  • vascular smooth muscle cells were placed in serum free medium (50% DMEM, 50% Ham's F- 12, 292 mg/ml l-glutamine, 5 mg/ml insulin, 5 mg/ml transferrin, 5 ng/ml selenious acid) for 96 hours and then stimulated by incubation in 45% DMEM, 45% Ham's F-12, 10% FCS.
  • Cell lysates were prepared as described previously (Chen et al.. 1989: DeCaprio et al.. 1992; JSQYS ⁇ LaL, 1986; Wang et al.. 1993; Buchkovich et al.. 1989; Mihara et al..
  • AdEFIHA ⁇ Rb a replication-defective adenovirus vector, AdEFIHA ⁇ Rb, that encodes a non- phosphorylatable, constitutively active form of human Rb (hRb) containing a 10 amino acid N-terminal epitope tag from the influenza hemagglutinin molecule (HA).
  • HA influenza hemagglutinin molecule
  • This mutant form of Rb (HA ⁇ Rb) has been reported previously to inhibit E2F- and E If- 1 -dependent transcription in P19 (Hamel et al.. 1992) and T cells (Chen et al.. 1989; DeCa p rio et al.. 1992; Kovesdi et al., 1986; Wang et al.. 1993; Buchkovich et al.. 1989; Mihara et al.. 1989;
  • AdBAc.lacZ a replication-defective adenovirus containing the bacterial lacZ gene under the control of the chicken ⁇ -actin promoter and cytomegalovirus enhancer (Kozarsky et aL 1993).
  • quiescent rat aortic smooth muscle cells were infected with 20 pfu/cell of AdEFIHA ⁇ Rb and then stimulated to proliferate by incubation in 10% FCS.
  • Control cultures were infected with Ad ⁇ Ac.lacZ or were left uninfected prior to serum stimulation.
  • Serum stimulation caused the rapid proliferation of the uninfected or Ad ⁇ Ac.lacZ- infected vascular smooth muscle cells. During the first 48 hours after stimulation, these cells underwent approximately 3 doublings.
  • infection with AdEFIHA ⁇ Rb caused a greater than 90% reduction in smooth muscle cell proliferation.
  • Ad ⁇ Ac.lacZ and AdEFIHA ⁇ Rb- infected cells were more than 97% viable at the end of the experiment as determined by trypan blue exclusion.
  • the lack of proliferation seen following AdEFIHA ⁇ Rb infection represented cell cycle arrest as opposed to cell killing in these experiments.
  • Rb has been shown to block cell cycle progression at the G- j /S transition (Goodrich et aL 1991).
  • AdEFHA ⁇ Rb-infected vascular smooth muscle cells were arrested prior to the S phase of the cell cycle, 3 Hthymidine incorporation was measured in these cells following serum stimulation. Serum stimulation of both the uninfected and Ad ⁇ Ac.lacZ- infected vascular smooth muscle cells was associated with significant increases in 3 H- thymidine incorporation during the first 24 to 48 hours after stimulation.
  • the rat carotid artery injury model represents a well characterized, reproducible vascular proliferative disorder that is dependent on smooth muscle cell migration and proliferation (Simons et al.. 1992; Morishita et aL 1993; Barr and Leiden. 1994; Clowes and Reidy. 1983).
  • Balloon angioplasty of the porcine femoral artery produces a neointimal lesion that has been used as a model of human vascular proliferative disease.
  • both the size and structural organization of this vessel closely resemble those of the human coronary arteries (Prescott et aL 1991: Reitman et al.. 1982; Weiner et aL, 1985: Ohno et al.. 1994).
  • RNA and a commercially available kit Perkin Elmer, Norwalk, CT
  • reverse transcriptase reverse transcriptase
  • PCR was performed as described previously (Barr et aL 1994) using primers specific for the HA ⁇ Rb cDNA [AAGCTTCCCGGGG AATTCACC ATGGGGTACCCATACG ATGTTCCAG ATTACG (sense)(SEQ ID NO:1) and
  • ATAGCATTATCAACCTTGGTACTGG (antisense)(SEQ ID NO:2)] or the mouse ⁇ -actin cDNA [GTGACGAGGCCCAGAGCAAGAG (sense)(SEQ ID NO:3) and AGGGGCCGGACTCATCGTACTC (antisense)(SEQ ID NO:4)].
  • Southern blot analysis was performed using a radiolabeled probe corresponding to bp 1 to 392 of the HA ⁇ Rb cDNA (Barr et al.. 1994).
  • HA ⁇ Rb RNA was detected in the AdEFIHA ⁇ Rb-infected but not in the control AdBglll-infected or contralateral uninfected carotid arteries.
  • rat carotid arteries were subjected to balloon angioplasty and immediately infected with either 2 X 10 9 pfu of AdEF1 HA ⁇ Rb or a control AdBglll virus.
  • a third set of arteries was treated with vehicle (HEPES-buffered saline) alone.
  • Two assays were used to measure smooth muscle cell proliferation in vivo.
  • AdEFIHA ⁇ Rb- infected and control arteries were stained for 5'-bromodeoxyuridine (BrdU) (Simons et aL 1992; Morishita et al..
  • Carotid arteries of adult rats were injured by balloon angioplasty. Immediately following balloon injury, arteries were infected with 2 X 1p 9 pfu of either AdBglll or AdEFIHA ⁇ Rb, or were left uninfected. Animals received subcutaneous injections of 25 mg/kg of 5'- bromodeoxyuridine at 12 hour intervals starting 24 hours following injury for a total of 4 doses. Carotid arteries were fixed in situ by intravascular administration of 4% paraformaldehyde, paraffin- embedded, and sectioned.
  • Deparaffinized 5 mm sections were treated with 3% H 2 ⁇ 2 in methanol and permeabilized by incubation in 0.4% pepsin and 3.3 M HCI. Treated sections were blocked in 1.5% horse serum, incubated with a 1:100 dilution of an -BrdU mAb (Becton- Dickinson, San Jose, CA). The sections were then incubated with a 1:200 dilution of biotinylated horse -mouse Ig antiserum, followed by avidin-conjugated horseradish peroxidase (Vectastain Elite ABC kit, Vector laboratories, Burlingame, CA). Sections were then treated with diaminobenzidine (DAB) and counterstained with Hematoxylin and Eosin. Statistical analyses were performed using Sigmaplot (Jandel Scientific, Corte Madera, CA).
  • Neointimal and medial boundaries were determined by digital planimetry of tissue sections using the MOCHA program (Jandel Scientific, Corte Madera, CA) on a Gateway 486 computer. The neointimal and medial cross-sectional areas were measured from six sections of each artery and the mean of these six determinations was used to calculate the neointimal to medial cross-sectional ratio for each animal.
  • Uninfected and control AdBglll-infected arteries had l/M area ratios of 1.4 0.1 and 1.2 0.1 , respectively.
  • the AdEF1 HA ⁇ Rb-infected arteries demonstrated a 42% reduction in the l/M ratio as compared to the AdBglll-infected controls and a 50% reduction as compared to uninfected control arteries.
  • overexpression of HA ⁇ Rb following adenovirus-mediated in vivo gene transfer at the time of injury resulted in significant reductions in both vascular smooth muscle cell proliferation and restenosis in the rat carotid artery model of balloon angioplasty.
  • arteries harvested 20 days after injury were stained with an -von Willebrand Factor mAb (Hruban et al.. 1987).
  • Rat carotid arteries were harvested 21 days after balloon injury and infection with AdEFIHA ⁇ Rb and embedded in paraffin.
  • 5 mm sections were stained with a commercially available -vWF mAb (Dako, Santa Barbara, CA) (Hruban et al.. 1987) followed by a biotinylated goat anti-mouse IgG using an automatic immunostainer (Ventana Immunosystems, Arlington, AZ).
  • Slides were developed with avidin- conjugated alkaline phosphatase and fast red-naphthol (Ventana Immunosystems) according to the manufacturer's instructions.
  • AdEF1 HA ⁇ Rb-infected arteries demonstrated a 47% reduction in the l/M area ratio as compared to the AdBglll-infected controls.
  • AdEF1 HA ⁇ Rb infection significantly reduces neointimal formation in two different animal models of restenosis.
  • AdEFIHA ⁇ Rb infection did not result in increased vascular inflammation or cell necrosis as compared to vehicle-treated or AdBglll- treated control arteries.
  • routine serum chemistries including electrolytes, liver function tests, complete blood counts, and clotting parameters were ail normal in rats 21 days following intra-arterial infusions of AdEFIHA ⁇ Rb.
  • Pigs receiving AdEFIHA ⁇ Rb demonstrated a mild reduction in serum phosphate as compared to saline control- treated animals. The mechanism of this reduction remains unclear.
  • HSV TK Herpes Simplex Virus thymidine kinase
  • cytostatic therapy using Rb gene transfer has the advantage of arresting cell cycle progression without causing cell necrosis or inflammation in the vessel wall.
  • adenovirus-mediated gene transfer can be performed using percutaneous catheter-based techniques. The data further demonstrate the safety, efficacy and feasibility of this approach in two different animal models of restenosis.
  • Adp2l and AdlacZ are E1- and E3-deleted replication-defective adenovirus vectors derived from Ad5 sub360.
  • Adp21 encodes the human p21 cDNA under the transcriptional control of the human elongation factor-1 ⁇ (EF1 ⁇ ) gene promoter and the human 4F2 heavy chain gene transcriptional enhancer.
  • AdlacZ contains the bacterial lacZ gene under the transcriptional control of the chicken ⁇ -actin gene promoter and the cytomegalovirus transcriptional enhancer. Both viruses were prepared and grown as high titer stocks in 293 cells. All virus stocks were purified by centrifugation in discontinuous CsCI gradients and dialyzed against a Hepes-buffered saline solution.
  • VSMCs Isolation and infection of primary rat aortic VSMCs were performed as described in Example 1. For measurements of cell proliferation, DNA synthesis, and cell cycle analysis passage 3, VSMCs were incubated in serum-free medium for 48 hours prior to infection with either Adp21 or AdlacZ. This protocol resulted in 70- 90% of the cultured cells accumulating in GO + G1 of the cell cycle as assessed by propidium iodide staining and FACS analysis.
  • Adp21- or AdlacZ-infected cells were stimulated to proliferate by incubation in growth medium (45% DMEM, 45% Hams F-12, and 10% FBS) (GibcoBRL, Grand Island, NY).
  • VSMCs were pulse-labeled for 4 hours in growth medium containing methyl-3H thymidine (1uCi/ml, 2 Ci/mmol; Amersham, Arlington Heights, IL). Each experiment was performed in triplicate in 6- we 11 tissue culture plates (Falcon, Franklin Lakes, NJ).
  • Adp21- infected VSMCs expressed markedly elevated levels of p21 as compared to both uninfected and AdlacZ-infected control cells. High levels of p21 expression were observed within 24 hours of infection and the levels of p21 expression increased further between 24 and 48 hours after infection. Of note, in some experiments, both uninfected and AdlacZ-infected cell lysates displayed low levels of expression of a 21 kD protein that co-migrated with in vitro translated human p21.
  • AdlacZ-infected cells proliferated rapidly during the first 48 hours following serum stimulation, undergoing approximately two doublings. In contrast, infection with Adp21 resulted in dose-dependent reductions in VSMC proliferation. At both the 24 and 48 hour time points, each of the Adp21 -infected cell cultures (10, 20 and 40 PFU per cell) demonstrated significantly less proliferation than the control AdlacZ-infected cells (P ⁇ 0.02).
  • VSMCs were fixed overnight at 4%C with 75% ethanol and stained for 30 minutes at room temperature with propidium iodide (50 mg/ml). Cells were analyzed with a Becton Dickinson FACScan and
  • p21 can bind to and inhibit the activity of the DNA polymerase cofactor, PCNA. Inhibition of PCNA-dependent DNA synthesis may therefore reflect a second mechanism by which p21 inhibits cell cycle progression.
  • PCNA DNA polymerase cofactor
  • Recent studies have demonstrated that different regions of the p21 protein are involved in binding to CDKs and PCNA thereby suggesting that p21 may inhibit cell cycle progression by at least two distinct molecular mechanisms.
  • quiescent rat aortic VSMCs were infected with 40 PFU per cell of Adp21 or AdlacZ and then stimulated to proliferate for 24 hours by incubation in medium containing 10% FCS. Lysates prepared from these infected cells were immunoprecipitated with an -p21 antibody and the immunoprecipitates subjected to immunoblot analyses with either -p21 or -PCNA antibodies.
  • p21 was immunoprecipitated from the Adp21 -infected but not from the AdlacZ- infected VSMCs. More importantly, PCNA was co-immunoprecipitated with p21 from Adp21 -infected but not from AdlacZ-infected VSMC lysates. Thus, p21 forms complexes with PCNA following Adp21 infection of VSMCs.
  • the rat carotid artery balloon injury model represents a well- characterized, highly reproducible vascular proliferative disorder that is dependent on VSMC proliferation and migration. Previous studies using this model have demonstrated that medial VSMC proliferation begins within 2 days of arterial injury, reaching a peak within 4 days. By 20 days after balloon angioplasty, nearly all of the injured arteries develop a stable neointimal lesion and demonstrate no evidence of VSMC proliferation in either the neointima or the media of the vessel wall.
  • rat carotid arteries were subjected to balloon angioplasty and immediately infected with 2 X 10 9 PFU of either
  • Adp21 or AdlacZ This protocol results in the transduction of more than 70% of the medial VSMC in the injured arterial segment.
  • injured arterial segments were harvested 4 days after infection and crude lysates prepared from these arteries were assayed for p21 expression by immunoblot analysis.
  • cell lysates were prepared and 100 ⁇ g total protein from each sample was fractionated by SDS-PAGE in 15% gels and subjected to immunoblot analysis using a rabbit polyclonal -p21 antibody (Pharmingen, San Diego, CA) (1:1000 dilution).
  • a peroxidase-labeled, goat -rabbit antiserum (1:3000 dilution) (GibcoBRL) was used in conjunction with the ECL chemiluminescence system (Amersham) for detection of bound primary antibody.
  • the beads were washed 3 times with Tween lysis buffer and the immunoprecipitated proteins were released by boiling in SDS-PAGE loading buffer, fractionated by electrophoresis in 15% denaturing SDS polyacrylamide gels and subjected to immunoblot analyses using a polyclonal rabbit - p21 antibody (1:1000 dilution) (Pharmingen) or a mouse -PCNA mAb (1:100 dilution) (Santa Cruz, Santa Cruz, CA) as described above.
  • passage 3 VSMCs cells were incubated in serum free medium for 96 hours, infected with adenovirus as described above and, 24 hours after infection, were stimulated to proliferate by incubation in growth medium.
  • Cell lysates were prepared and protein corresponding to 5 X 10 5 cells from each sample was fractionated by electrophoresis in 7.5% SDS polyacrylamide gels and subjected to immunoblot analysis using an -Rb monoclonal antibody (1:200 dilution) (Pharmingen) and a peroxidase-labeled, goat -mouse IgG antiserum (1 :2000 dilution) (Gibco/BRL) in conjunction with ECL chemiluminescence system (Amersham).
  • Rat carotid arteries were harvested 20 days after balloon injury and adenovirus infection. 5 ⁇ m sections from paraffin-embedded arteries were stained with hematoxylin and eosin and the neointimal and medial boundaries were determined on coded slides by an investigator blinded to the experimental conditions. Areas and ratios were determined by digital planimetry of tissue sections using the
  • Image Pro-Plus image analysis system (Fryer Co, Chicago, IL). The l/M ratios were measured from 6 sections of each artery subjected to balloon angioplasty and adenovirus infection. The mean of these determinations was used to calculate the l/M cross-sectional ratios for each animal.
  • Coronary artery disease (CAD) due to atherosclerosis is the leading cause of morbidity and mortality in the United States.
  • PTCA remains a mainstay of therapy for symptomatic CAD, with more than 400,000 procedures expected in 1995 in the United States alone. While the procedure is initially successful in relieving arterial stenoses in the vast majority of patients, clinically significant restenosis continues to complicate the procedure in up to 40% of cases.
  • HSV-tk herpes simplex virus thymidine kinase gene
  • Biopolymer-mediated delivery of antisense oligonucleotides directed to c-myb or PCNA and cdc2 have also both been reported to inhibit neointimal hyperplasia in the rat carotid artery injury model.
  • antisense oligonucleotides may cause degradation of multiple RNA species and have important non-specific effects on intracellular and cell surface proteins.
  • antisense oligonucleotides may be subject to significant batch-to-batch variability and to date it is been impossible to efficiently deliver antisense oligonucleotides to the vascular wall using non-viral, catheter mediated approaches.
  • adenovirus mediated over-expression of a non-phosphorylatable constitutively active form of Rb has also been shown to significantly reduce restenosis in both the rat carotid and pig iliofemoral artery models of balloon angioplasty.
  • a cytostatic gene therapy approach using catheter-mediated delivery of Adp21 as described herein is a clinically applicable, effective and non-toxic treatment for vascular proliferative disorders.
  • the degree of inhibition of restenosis achieved by Adp21 gene transfer in the present studies (46%) was comparable to that achieved with over-expression of HA ⁇ Rb, as well as with HSV-tk gene transfer with systemic ganciclovir therapy, and the intraluminal delivery of antisense oligonucleotides.
  • administration of Adp21 was not associated with significant inflammatory responses and clinical toxicity in the present study.
  • Adp21 appears to inhibit two proliferative pathways (cyclin/CDKs and PCNA) makes it a more potent cytostatic agent than AdHA ⁇ Rb.
  • the more pleiotropic activities of Adp21 may result in more undesirable side effects in vivo.
  • Co-administration of Adp21 and AdHA ⁇ Rb can be more efficacious than the administration of either virus alone.
  • cyclin/CDK kinases are important regulators of proliferation in a wide variety of cell types, adenovirus mediated over-expression of p21 is likely useful for the treatment of other human diseases associated with deregulated cell proliferation.
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)

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Abstract

La présente invention se rapporte à la mise en oeuvre d'un transfert de gène afin d'inhiber la prolifération cellulaire pathologique non néoplasique. Le procédé consiste à administrer à une cellule proliférative un vecteur d'expression eucaryotique comprenant une séquence polynucléotidique codant pour une protéine de régulation négative dominante du cycle cellulaire. L'administration est réalisée par injection d'un vecteur d'expression dans une artère qui irrigue ou renferme la cellule proliférative.
PCT/US1995/015191 1994-11-11 1995-11-13 Procede d'inhibition de la proliferation cellulaire pathologique non neoplasique WO1996015245A1 (fr)

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WO1996025507A3 (fr) * 1995-02-17 1996-11-07 Us Health Procedes de preparation et d'utilisation de vecteurs adenoviraux recombines
WO1996027008A3 (fr) * 1995-02-28 1996-11-14 Max Planck Gesellschaft Agent utilise dans le traitement de tumeurs et d'autres hyperplasies
WO1998037190A1 (fr) * 1997-02-20 1998-08-27 Hepavec Aktiengesellschaft Für Gentherapie METHODE PERMETTANT DE PROVOQUER UNE MORT CELLULAIRE PROGRAMMEE DANS DES CELLULES MALIGNES PAR REDUCTION DU RAPPORT PROTEINES Rb/PROTEINES INDUISANT L'APOPTOSE
WO2000039293A3 (fr) * 1998-12-23 2000-11-09 Max Delbrueck Centrum Agent inhibant la neoformation tissulaire
JP2007504274A (ja) * 2003-05-10 2007-03-01 ペン、ジャオフイ 増殖性疾患を治療するためのアデノウイルスベクターとｐ５３遺伝子との組換え体遺伝子医薬

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996025507A3 (fr) * 1995-02-17 1996-11-07 Us Health Procedes de preparation et d'utilisation de vecteurs adenoviraux recombines
WO1996027008A3 (fr) * 1995-02-28 1996-11-14 Max Planck Gesellschaft Agent utilise dans le traitement de tumeurs et d'autres hyperplasies
WO1998037190A1 (fr) * 1997-02-20 1998-08-27 Hepavec Aktiengesellschaft Für Gentherapie METHODE PERMETTANT DE PROVOQUER UNE MORT CELLULAIRE PROGRAMMEE DANS DES CELLULES MALIGNES PAR REDUCTION DU RAPPORT PROTEINES Rb/PROTEINES INDUISANT L'APOPTOSE
WO2000039293A3 (fr) * 1998-12-23 2000-11-09 Max Delbrueck Centrum Agent inhibant la neoformation tissulaire
JP2007504274A (ja) * 2003-05-10 2007-03-01 ペン、ジャオフイ 増殖性疾患を治療するためのアデノウイルスベクターとｐ５３遺伝子との組換え体遺伝子医薬
JP4695086B2 (ja) * 2003-05-10 2011-06-08 ペン、ジャオフイ 増殖性疾患を治療するためのアデノウイルスベクターとｐ５３遺伝子との組換え体遺伝子医薬

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