WO2007003229A1 - Tumor restricted oncolytic virus with improved cell transduction spectrum - Google Patents

Tumor restricted oncolytic virus with improved cell transduction spectrum Download PDF

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Publication number
WO2007003229A1
WO2007003229A1 PCT/EP2005/053112 EP2005053112W WO2007003229A1 WO 2007003229 A1 WO2007003229 A1 WO 2007003229A1 EP 2005053112 W EP2005053112 W EP 2005053112W WO 2007003229 A1 WO2007003229 A1 WO 2007003229A1
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nucleic acid
gene
target cell
target
functioning
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PCT/EP2005/053112
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French (fr)
Inventor
Thomas Wirth
Florian Kühnel
Stefan Kubicka
Lars Zender
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Medizinische Hochschule Hannover
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Priority to PCT/EP2005/053112 priority Critical patent/WO2007003229A1/en
Priority to PCT/EP2006/063770 priority patent/WO2007003612A1/en
Publication of WO2007003229A1 publication Critical patent/WO2007003229A1/en

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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
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A61K35/761—Adenovirus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C—CHEMISTRY; METALLURGY
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    • 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
    • C12N2710/10332—Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/80—Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
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    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/50—Vectors comprising as targeting moiety peptide derived from defined protein
    • C12N2810/60—Vectors comprising as targeting moiety peptide derived from defined protein from viruses
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/001—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination
    • C12N2830/005—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination repressible enhancer/promoter combination, e.g. KRAB
    • C—CHEMISTRY; METALLURGY
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    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
    • C12N2840/203—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES
    • C12N2840/206—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES having multiple IRES

Definitions

  • the present invention relates to a nucleic acid for transforming a target cell of a target organism, a corresponding virus and uses of both, particularly for the treatment of solid tumours.
  • Viral vectors have been employed for gene delivery into procaryotic and eucaryotic cells. Recently, they have gained an increased importance in gene therapy applications for somatic gene transfer, and in particular in cancer gene therapy. Traditionally, adenoviral and herpes simplex viral vectors are the most prominent and best known systems for gene transfer in mammalian and human cells. In gene therapy applications, vectors and especially viral vectors have been constructed that comprise a therapeutically active gene for transforming a target, defective cell into a no longer defective cell or into a dormant or apoptotic state.
  • a common problem of present viral vectors for gene therapy applications is that either the viral tropism is to narrow, such that not all target cells can effectively be transformed, and/or that the number of non-target cells of a treated organism, particularly a mammal, is unacceptably high, leading to severe site effects.
  • tumor cells frequently downregulate the CAR-receptor, which determinates the adenoviral tropism. This tumor cell property exacerbates the susceptibility of tumors to adenoviral gene therapy or hampers effective distribution of replicative oncolytic adenoviral vectors throughout the tumor tissue in the course of viral replication.
  • the efficacy of cancer therapy using either non-replicative or replicative adenoviral vector species is still an unsolved problem.
  • the system is not readily suitable for in vivo gene therapy applications, particularly for cancer therapy in mammals like a human.
  • the therapeutically active gene is down regulated in normal, non-degenerated cells to avoid potentially disruptive properties of the therapeutically active gene to such normal cells.
  • the system does not readily lend itself to in vivo gene therapy applications, as non-replicative vectors are not expected to exert a potent and longlasting antitumor-response, mainly due to the vector tropism limitations as described above.
  • the means should be easy to apply and should further allow the introduction of therapeutically active genes effective in the target cells and/or allow tumor cell- specific onset of viral replication and enhanced spreading of the mean within the target tissue.
  • a nucleic acid for transforming a target cell of a target organism comprising a nucleic acid replication element essential for replication of the nucleic acid, wherein said replication element is under control of a regulatory factor, to allow replication of the nucleic acid in the target cell and to minimise replication of the nucleic acid in a non-target cell, an adapter gene coding for an adapter protein comprising a protein transduction domain and a coupling domain for coupling to said nucleic acid.
  • an element i.e. a gene or a structural element of the nucleic acid, that is essential for replication of the nucleic acid is brought under the control of a regulatory factor such as to allow replication of the nucleic acid in the target cell and to minimize replication of the nucleic acid in a non-target cell.
  • the nucleic acid of the present invention is adapted for transforming a target cell of a target organism.
  • Target cells within the meaning of the present invention are preferably eucaryotic cells and particularly mammal cells, and most preferably cells lacking an element, e.g. a gene, essential or highly desirable for normal functioning of the respective cell.
  • Particularly preferred target cells are tumor cells, preferably within solid tumor types derived from various tissue origin.
  • the target cells are, in preferred uses of the nucleic acid of the invention parts of a living target organism.
  • transformation 11 means, for the purposes of the present invention, the process of introducing a heterologous nucleic acid into a cell, particularly a target cell, and at least transient expression of a gene of said heterologous nucleic acid.
  • Particularly preferred transformations are those wherein a gene of said heterologous nucleic acid, most preferably a gene whose expression finally leads to a therapeutic effect, is at least transiently expressed in a target cell.
  • protein transduction domain signifies a domain of 10 to 30 or more amino acid residues that is enriched in basic amino acids, especially in arginine and/or lysine and/or Histidine. Particularly preferred protein transduction domains are described in the above mentioned publication of K ⁇ hnel et al., Journal of Virology, 2004, under the names VP22, TaT, AntP, and also a stretch of at least 6 and preferably up to 10 consecutive arginine amino acids.
  • the nucleic acid according to the present invention comprises a replication element that is essential for the replication of the nucleic acid.
  • the replication element can be a gene encoding for a product (particularly a protein) that is necessary in the reproduction of said nucleic acid in a target cell.
  • the term "element” can also refer to a structural element of the nucleic acid indispensable for replication, particularly an origin of replication, a regulatorily active site, or a termination region.
  • the replication element of the nucleic acid is under control of a regulatory factor, so that replication of the nucleic acid is allowed or promoted in the target cell and minimized or inhibited in a non-target cell.
  • the regulatory factor can be encoded by the nucleic acid of the invention.
  • the regulatory factor can also be encoded by the genomic DNA of the target or non-target cell; in this case, the regulatory factor can be a trans-acting factor.
  • the regulatory factor can also be encoded by a helper nucleic acid; in this case, the regulatory factor can also be a trans-acting factor. Suitable regulatory factors are described below in detail.
  • the regulatory factor is part of a regulatory switch which reacts to a factor most preferably provided by the target cell, and either directly or indirectly influences the functioning of the replication element.
  • a direct influence can be exerted by directly promoting or enhancing the expression of the replication element, if said replication element is a gene, or, for example, by binding to the replication element in order to promote the replication of said nucleic acid, if said replication element is a structural element.
  • A.n indirect influence is preferably exerted by enhancing the expression or function of another factor, preferably a protein, that itself or via further factors enhances the functioning of the regulatory element.
  • An indirect influence can also preferably be exerted by suppressing a suppressor of said replication element, again either directly or through interceding further factors.
  • the replication element is a gene essential for replication of said nucleic acid in eucaryotic cells, most preferably a packaging or cell lysis gene.
  • the nucleic acid is one of a virus
  • the replication element is one of the genes essential for viral replication.
  • the nucleic acid is an adenovirus derived viral nucleic acid
  • the replication element is one of the early genes of the adenoviral reproduction cycle.
  • the * replication element is under control of transcriptionally active and/or repressive elements which can be 1) constitutively active, such as the CMV-promoter, the
  • RSV-promoter the HSV-tk promoter, the E1A promoter and the SV4O promoter as well as subtypes and artificial derivatives thereof, 2) promoters active in a tumor-specific environment, such as, but not limited to the hTERT promoter and the alpha fetoprotein promoter, 3) sites for DNA-binding factors attracting the regulatory protein encoded and controlled by the said regulatory switch and 4) sites binding the hypoxia inducible factor.
  • the adapter encoded by the nucleic acid of the present invention is designed to directly or indirectly couple to said nucleic acid and simultaneously display a protein transduction domain as described above.
  • the cell tropism of the nucleic acid is easily broadened so that even when treating living target organisms like mammals and humans, in principle all cell types accessible by protein transduction domains are prone to transformation by the nucleic acid according to the present invention.
  • the nucleic acid replication element under the control of the regulatory factor prevents unacceptable high replication and titres of said nucleic acid in non-target cells of the target organism which in turn limits incidence of the adapter protein within said cells and in the environment thereof.
  • the nucleic acid of the present invention therefore offers for the first time the possibility to infect permissive and hitherto non-permissive tumor cells in a receptor-independent manner and simultaneously balances the potentially harmful stress exerted on non-target cells of the target organism by restricting the replication to target cells.
  • nucleic acids according to the present invention further comprise a gene coding for a restriction nuclease, said restriction nuclease being heterologous to the target cell and/or to the target organism, and a restriction site specifically recognized by the heterologous restriction nuclease, wherein said restriction nuclease gene is under control of a regulatory factor, to allow functioning of the restriction nuclease in a non-target cell and to minimise functioning of the restriction nuclease in a target cell.
  • restriction nuclease encoded by the restriction nuclease gene preferably is harmless to the integrity of the non-target cell genome, and is further preferably a restriction endonuclease, a rare cutting restriction endonuclease, such as but not limited to intron-encoded ('homing') endonucleases or designed zincfinger endonucleases.
  • the restriction nuclease recognises a restriction site inserted in the nucleic acid of the present invention, said restriction site being alien or heterologous to the genomic DNA at least of a non-target cell of the target organism.
  • restriction nuclease encoded by the nucleic acid of the present invention is therefore capable of selectively digesting the nucleic acid of the present invention in non-target cells of the target organism, and preferably rendering it incapable of replication in the digested form, thus leading to irreversible destruction of said nucleic acid in non-target cells.
  • the restriction nuclease is under direct or indirect control of a regulatory factor to allow functioning of the restriction nuclease in a non-target cell and to minimize or inhibit functioning of the restriction nuclease in a target cell.
  • the control of the regulatory factor, functioning as part of a regulatory switch, on the restriction nuclease of the present invention can be exerted correspondingly to the control of the regulatory factor exerted on the nucleic acid replication element as described above.
  • the regulating factor controlling the nucleic acid replication element is also the regulating factor controlling the functioning of the restriction nuclease.
  • restriction nuclease can be controlled either by up- or downregulation of the restriction nuclease gene or by enhancing or inhibiting the function of the restriction nuclease protein itself.
  • the nucleic acid of the present invention therefore further minimizes potentially harmful influences of said nucleic acid to non-target cells by actively destroying the nucleic acid in such non-target cells.
  • the regulating factor is a nucleic acid binding, trans-acting factor produced by non-target cells, and preferably is the p53 gene product.
  • the regulating factor enhances expression of the restriction nuclease gene, and also enhances expression of a further regulator protein encoded by the nucleic acid of the present invention.
  • the further regulator protein suppresses expression of a replication gene essential for the reproduction of the nucleic acid of the present invention.
  • one regulating factor directly controls the expression of the restriction nuclease and indirectly J controls expression of the replication gene essential for reproduction of the nucleic acid of the invention.
  • nucleic acids of the present invention comprise a regulator gene coding for a regulator protein for i) allowing or enhancing the functioning of the replication element in a target cell and/or for preventing or repressing the functioning of the replication element in a non-target cell, ii) allowing or enhancing the functioning of the adapter protein in a target cell and/or for preventing or repressing the functioning of the adapter protein in a non-target cell, iii) allowing or enhancing the functioning of the restriction nuclease in a non-target cell and/or for preventing or repressing the functioning of the restriction nuclease in a target cell, and/or for iv) allowing or enhancing the functioning of a target gene in a target cell and/or for preventing or repressing the functioning of the target gene in a non-target cell.
  • Particularly preferred regulator proteins comprise a DNA binding moiety and a moiety for modification of transcription.
  • Particularly preferred DNA binding moieties are those of Gal4, LexA, TetR and r TetR (NCBI accession numbers NC001148, J01643 extending. X00694).
  • Transcription modification domains can be activator (enhancer) domains, particularly preferred those with acetyltransferase activity or those attracting another factor with histone acetyltransferase activity.
  • a particularly preferred activator domain is that of the Herpesvirus-VP16 protein (NCBI accession number: M57289).
  • Transcription modification domains can also be repressor domains, particularly preferred those derived from Kr ⁇ ppel-like zink- finger proteins, such as KIRAB (NCBI accession number: M67509) and can also be proteins that influence the methylation status of their target DNA, such as MeCP (NM004992), or they can be derived from factors which display Histone Deacetylase activity or attract other factors that have Histonedeacetylase activity, such as for example, but not exclusively, the SIN family (AF 038848) or NcoR (NM006311).
  • the regulator protein encoded by the regulated gene in case i) allows or enhances the functioning of the replication element in a target cell and/or prevents or represses the functioning of the replication element in a non-target cell.
  • the regulator protein can be the regulating factor described above, under " whose control the replication element is.
  • the regulator protein is responsive to an element, particularly preferred a metabolic product, of a target or non-target cell such as to allow and/or repress, respectively, the functioning of the replication element in a target and non-target cell as described just before.
  • the element the regulator protein is responsive to preferably allows to distinguish between target and non-target cells, and preferably is the p53 gene product.
  • nucleic acid can be provided with nucleic acid sequences coding for siRNAs when transcribed along with the messenger coding for the regulatory protein. This can be achieved by application of either intron-encoded siRNA or linking the shRNA coding nucleic acid downstream of the polyadenylation signal.
  • SiRNA can either applied as shRNA or MicroRNA.
  • Preferred shRNA sequences target and interfere with factors promoting replication of said nucleic acid such as, but not exclusively, target-cell factors like E2F, c-myc and/or HSP70, and/or viral factors like E1 A and several ORFs of the E4 locus.
  • the regulator protein can allow or enhance the functioning of the adapter protein in a target cell and/or prevent or repress the functioning of the adapter protein in a non-target cell.
  • the regulator protein can up- and downregulate the expression of the adapter protein gene, and/or it can also stimulate or repress the functioning of the adapter protein itself.
  • the regulator protein is adapted to allow or enhance the functioning of the restriction nuclease in a non-target cell and/or prevent or repress the function of the restriction nuclease in a target cell.
  • the influence of the regulator protein on the restriction nuclease can again be exerted by up- and downregulation of the corresponding gene and/or by stimulation or repression of the restriction nuclease itself, in a manner corresponding to that of cases i) to ii).
  • the regulator protein is adapted to allow or enhance the functioning of the target gene in a target cell and/or prevent or repress the functioning of the target gene in a non-target cell.
  • the influence of the regulator protein on the restriction nuclease can again be exerted by up- and downregulation of the corresponding gene and/or by stimulation or repression of the restriction nuclease itself, in a manner corresponding to that of cases i) - iii). This is particularly useful in the case that the target gene is a therapeutically active gene, preferably one which leads to induction of apoptosis.
  • the cases i) to iv) are not meant to be mutually exclusive, but can be combined on the same nucleic acid of the present invention. Also, the regulator protein does not have to be identical for all of these cases.
  • a particularly preferred nucleic acid according to the present invention is one wherein the function of regulator gene, the nucleic acid replication element, the adapter protein gene and the restriction nuclease gene are directly or indirectly linked to the status of the human p53 gene product within the target and non- target cells, respectively.
  • the p53 tumour suppressor gene is a gene upregulated following DNA damage, viral infection, hypoxia or other factors threatening the cellular and genomic integrity. Aberrant forms of the p53 gene product are frequently found in tumours, where the p53 gene product is e.g. functionally altered (lack of DNA-binding activity), bound in complexes together with oncogenic proteins or has decreased protein stability.
  • active ⁇ 53 serves as e. g.
  • the nucleic acid according to the present invention is therefore most suitable for the treatment of tumours, as its effects on non-tumour cells is kept in check by at least by one, preferably two, more preferably three and most preferably all four of the security measures implemented by the nucleic acid of the present invention (i. e. controlled regulator gene and subsequent nucleic acid replication, controlled functioning and expression of the adapter and/or the corresponding gene, controlled functioning and/or expression of the restriction nuclease and controlled functioning and/or expression of the regulator gene.
  • the security measures implemented by the nucleic acid of the present invention i. e. controlled regulator gene and subsequent nucleic acid replication, controlled functioning and expression of the adapter and/or the corresponding gene, controlled functioning and/or expression of the restriction nuclease and controlled functioning and/or expression of the regulator gene.
  • a particularly preferred nucleic acid according to the present invention is one wherein the function of regulator gene, the nucleic acid replication element, the adapter protein gene and the restriction nuclease gene are not linked to the status of neither p73 plus derived subforms nor p63 and derived subforms thereof, within the target and non-target cells, respectively. This is of particular importance as many ⁇ 53 altered tumor cells harbour normal p63/p73, a subgroup of proteins which are able to replace p53 function under certain circumstances, such as genotoxic stress.
  • the nucleic acid replication element, the adapter gene, the restriction nuclease gene and/or the regulator gene is therefore preferably under control of a normal human p53 gene product and independent of p73 and/or p63 and/or subtypes thereof.
  • the independence from p73 and p63 and their respective subtypes is preferably achieved by using doxorubicine, an adenovirus or an adenoviral vector for transduction of the target and non-target cells.
  • virus for transforming a target cell of a target organism comprising a nucleic acid according to the present invention.
  • Viruses are particularly capable of infecting eucaryotic, mammalian and human cells and have in the past been extensively studied for gene therapy applications.
  • a virus comprising a nucleic acid of the present invention is therefore particularly suited for gene therapeutic applications as attempted in the prior art and described above.
  • the virus of the present invention most suitably is one wherein the adapter protein encoded by the nucleic acid of the present invention is capable of coupling to an adenoviral fibreknob protein. It is most useful when the virus itself is derived from an adenovirus and comprises a fibreknob protein to allow cell infection. Adenoviruses and viruses derived therefrom are particularly well studied for gene therapeutic applications.
  • the virus of the present invention is preferably also suitable for coapplication with chemotherapy, e.g. Doxorubicin. DNA damaging agents lead to an upregulation of p53 und therefore support correct and sensitive function of the regulatory switch as described above according to the invention.
  • chemotherapy is applied prior to the application of the virus of the present invention as this leads to preactivated p53 in non-target cells and diminishes negative interference loops of the replication unit with the transcriptional activity of p53. It is therefore also preferred to manufacture a medicament for treatment of tumors, said medicament comprising a) a chemotherapeutic agent, and b) a virus according to the invention.
  • this virus not the entire gene for the adapter protein is included in the viral DNA. Instead, short nucleic acid sequences coding for protein transduction domains, corresponding to those used in the said adapter proteins, are directly inserted into the Hl-Loop region of the adenoviral fiberknob protein. In this way, the derived virus displaying the protein transduction, domain in the fiberknob protein can directly couple to target and/or non-target cells in a receptor-independent manner.
  • the viral tropism alteration is therefore not limited to sites of high replication. This property limits safety of the vector but enhances efficacy of viral spreading.
  • the protein transduction domains present in the Hl- Loop region are provided with stretches of several cystein residues.
  • the presence of cystein residues allow the virus to multimerize with each other to build concatamers or aggregates via disulfide bonds.
  • Viral aggregation preferably occurring in the oxidative environment of a solid tumor as a result of elevated apoptosis rate and nutrient deprivation, should hinder the viral progeny being washed out of the tumor by the blood flow. Therefore viral replication should be more concentrated to the tumor area.
  • the nucleic acid according to the present invention and the corresponding virus are preferably constructed to destroy connective tissue within solid tumors, mainly consisting of fibroblasts, which serves as physical backbone for the tumor as well as supplier with nutrients and survival factors. Furthermore, as fibroblasts are refractory to adenoviral infection, tumor connective tissue serves as physical barriers for effective expansion of adenoviral infection, thus greatly limiting therapeutic efficacy when using adenoviral vectors with natural tropism.
  • the nucleic acid according to the present invention and the corresponding virus therefore allow the vector to target and to destroy those preestablished barriers, subsequently allowing viral distribution throughout the entire tumor mass. Additionally, these vectors can also target tumor stem cells, as stem cells are known to be CAR-downregulated.
  • the nucleic acid according to the present invention and the corresponding virus preferably are a medicament, preferably for the treatment of a tumor, and are ⁇ used alone or in combination with a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier is one that does not or only to an acceptable extend damage the health of a target organism treated with the medicament of the present invention.
  • Particularly preferred carriers are water and physiological saline.
  • Figure 1 is a schematic illustration of the reproductive cycle of a virus according to the present invention.
  • Figure 2 is a set of three construction schemes for viruses according to the present invention.
  • Figure 3 is a construction scheme for a further virus according to the present invention.
  • a virus according to the present invention preferably is an adenoviral based virus.
  • Suitable viruses and their construction schemes can be found in the publications of K ⁇ hnel et al., Journal of Virology 2004 and Cancer Gene Therapy 2004, as cited above.
  • FIG. 1 is a schematic representation of the functioning of a virus according to the present invention.
  • the virus comprises a regulator gene, namely a repressor gene GAL4-KRAB, wherein the GAL4-domain is a specifically DNA binding domain and the KRAB-dornain is a repressor domain that primarily affects the basal transcription machinery.
  • the regulator gene is under the control of an artificial p53 dependent promoter prMin-RGC.
  • This promoter contains thirteen p53 binding sites derived from the ribosomal gene cluster, in combination with a minimal CMV-promoter providing a
  • the regulator protein GAL4-KRAB is therefore expressed in the presence of p53, as is indicated in the lower left portion of figure 1 representing a normal, mammalian cell.
  • the regulator protein GAL4-KFLAB is not expressed, as is indicated in the lower right portion of figure 1 which represents a degenerate, mammalian tumor cell.
  • the adenovirus of figure 1 further comprises an E1A gene as nucleic acid replication element under the control of a CMV-gal promoter.
  • E1A gene as nucleic acid replication element under the control of a CMV-gal promoter.
  • GAL4-KRAB repressor expression of the adenoviral E1A gene is downregulated and the gene is effectively silenced.
  • This is indicated in figure 1 by a crossed-out arrow indicating the absence of transcription starting from the CMV-gal promoter and by crossed-out icons of packaged adenoviruses.
  • GAL4-KRAB transcription of the E1A early adenoviral replication gene starts from the CMV-gal promoter, so that the E1A gene is effectively transcribed and the corresponding gene product is expressed.
  • a left-pointing arrow starting from the CMV-gal promoter, circular representations of the E1A gene product and by icons of packaged adenoviral vectors in the lower right half of Figure 1.
  • the regulator protein GAL4-KRAB acts as a frans-acting factor to prevent replication of the virus in the presence of normal p53 protein, while the absence of GAL4-KRAB allows reproduction of the adenovirus of the preferred embodiment in the absence of normal p53 protein.
  • GAL4-KRAB acts as a frans-acting factor to prevent replication of the virus in the presence of normal p53 protein
  • the absence of GAL4-KRAB allows reproduction of the adenovirus of the preferred embodiment in the absence of normal p53 protein.
  • FIG. 2 shows three construction schemes for viral nucleic acids of the present invention.
  • a CMV-gal promoter as described above is operably fused to an expression cassette com prising adenoviral early replication genes E1A and E1B and also the reporter ge ne EGFP.
  • an expression cassette comprising a gene for the restriction endonuclease I-SCE I and the regulator gene coding for the GAL4-KRAB fusion repressor protein.
  • a I-SCE I restriction endonuclease site is inserted to allow cleavage of the nucleic acid between the CMV-gal promoter and the nucleic acid replication element (the E1 A gene).
  • an expression cassette comprising the adenoviral early replication genes E1A and E1B is operably located. Between the E1Apr promoter and the E1A gene, a I-SCE I restriction endonuclease site is located to allow cleavage of the nucleic acid.
  • the nucleic acid further comprises a I-SCE I restriction endonuclease gene under the control of a p53 dependent promoter prMin-RGC.
  • a nucleic acid constructed according to this construction scheme functions like a nucleic acid constructed in accordance to the first construction scheme of figure 2 in that at the onset of I-SCE I expression, the nucleic acid is cleaved between the promoter responsible for expression of the nucleic acid replication element (here: the E1A gene), so that replication of the nucleic acid is prevented.
  • the promoter responsible for expression of the nucleic acid replication element here: the E1A gene
  • no I-SCE I restriction endonuclease enzyme is produced, so that normal reproduction of the nucleic acid via the adenoviral repl ⁇ cation pathway commences, which ultimately leads to cell death of the target cell.
  • the third construction scheme of figure 2 parallels the first construction scheme but lacks the EGFP reporter gene in the CMV-gal dependent expression cassette.
  • replication of the nucleic acid is prevented t>oth by I-SCE I cleavage between the CMV-gal promoter and the early adenoviral replication gene E1A in the presence of normal p53 protein.
  • replication is also prevented by p53 dependent expression of the GAL4-KRAB fusion repressor protein which silences the CMV-gal dependent expression cassette comprising the nucleic acid replication element essential for replication of "the nucleic acid (here: the adenoviral E1A and E1B genes).
  • FIG 3 shows a construction scheme of a nucleic acid according to the present invention comprising an adapter gene coding for an adapter protein comprising a protein transduction domain and a coupling domain for coupling to an adenoviral fibreknob protein.
  • the nucleic acid comprises, under the control of a cytomegalovirus promoter (CMV), a gene coding for a fusion protein as described in K ⁇ hnel et al., Journal of Virology 2004, as cited above.
  • CMV cytomegalovirus promoter
  • the protein transduction domain is VP22 domain
  • the protein transduction domain is Tatjs- 57 protein transduction domain.
  • the adapter protein comprising any of the protein transduction domains described above will be released from the target cell and will bind to adenoviruses, particularly to adenoviruses of the present invention comprising the nucleic acid of the present invention.
  • the target cell range is greatly expanded as described above.

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Abstract

The present invention relates to a nucleic acid for transforming a target cell of a target organism, a corresponding virus and uses of both, particularly for the treatment of solid tumours.

Description

Tumor restricted oncolytic virus with improved cell transduction spectrum
The present invention relates to a nucleic acid for transforming a target cell of a target organism, a corresponding virus and uses of both, particularly for the treatment of solid tumours.
Viral vectors have been employed for gene delivery into procaryotic and eucaryotic cells. Recently, they have gained an increased importance in gene therapy applications for somatic gene transfer, and in particular in cancer gene therapy. Traditionally, adenoviral and herpes simplex viral vectors are the most prominent and best known systems for gene transfer in mammalian and human cells. In gene therapy applications, vectors and especially viral vectors have been constructed that comprise a therapeutically active gene for transforming a target, defective cell into a no longer defective cell or into a dormant or apoptotic state.
However, a common problem of present viral vectors for gene therapy applications is that either the viral tropism is to narrow, such that not all target cells can effectively be transformed, and/or that the number of non-target cells of a treated organism, particularly a mammal, is unacceptably high, leading to severe site effects. In particular, it is well known that tumor cells frequently downregulate the CAR-receptor, which determinates the adenoviral tropism. This tumor cell property exacerbates the susceptibility of tumors to adenoviral gene therapy or hampers effective distribution of replicative oncolytic adenoviral vectors throughout the tumor tissue in the course of viral replication. As a consequence, the efficacy of cancer therapy using either non-replicative or replicative adenoviral vector species is still an unsolved problem.
In the past, several ways have been tried to overcome the above drawbacks. In one route, it has been tried to broaden the tropism or target cell range accessible via a given viral vector. For example, Kϋhnel et al., Journal of Virology 2004, 13743 - 13754 "Protein Transduction Domains Fused to Virus Receptors Improve Cellular Virus Uptake and Enhance Oncolysis by Tumor-Specific Replicating Vectors" describe the use of adapter proteins to facilitate coupling of an adenoviral vector via its fibreknob protein to mammalian cell types otherwise not accessible by adenoviral infection. The authors demonstrate that the system allows to overcome the natural tropism of adenoviral vectors and to infect previously non-permissive cells in a receptor-independent manner. On the other hand, the authors had to resort to the tumor-specifically replicating hTERT-Ad virus to limit adenovirus replication and for keeping viral titres in check. The system is not readily suitable for in vivo gene therapy applications, particularly for cancer therapy in mammals like a human.
Another, independent route has been described by Kϋhnel et al., Cancer Gene Therapy (2004), 28 - 40 "Tumor-specific adenoviral gene therapy: transcriptional repression of gene expression by utilizing p53-signal transduction pathways" and in DE 101 25 839 A1. In this route, expression of a therapeutically active gene from an adenoviral vector is internally downregulated by a repressor which in turn is expressed in the presence of a normal, non-deviative p53 gene product. This way, the expression of the therapeutically active gene could be limited to cells with non-normal p53 function, e. g. degenerated cells either lacking p53 gene product or those cells having a reduced stability p53 gene product or abnormally low levels of this protein. The therapeutically active gene is down regulated in normal, non-degenerated cells to avoid potentially disruptive properties of the therapeutically active gene to such normal cells. However, the system does not readily lend itself to in vivo gene therapy applications, as non-replicative vectors are not expected to exert a potent and longlasting antitumor-response, mainly due to the vector tropism limitations as described above.
It was the object of the present invention to provide means for transforming a target cell of a target organism, wherein said means should allow highly effective targeting of target cells independent of their CAR-receptor status, while avoiding potentially hazardous or disruptive side effects on the whole organism. The means should be easy to apply and should further allow the introduction of therapeutically active genes effective in the target cells and/or allow tumor cell- specific onset of viral replication and enhanced spreading of the mean within the target tissue.
According to the present invention there is provided a nucleic acid for transforming a target cell of a target organism, comprising a nucleic acid replication element essential for replication of the nucleic acid, wherein said replication element is under control of a regulatory factor, to allow replication of the nucleic acid in the target cell and to minimise replication of the nucleic acid in a non-target cell, an adapter gene coding for an adapter protein comprising a protein transduction domain and a coupling domain for coupling to said nucleic acid.
The inventors have now found that the potentially disruptive side effects of a broad vector spectrum can already be avoided by limiting the replication of said vector in non-target cells. Accordingly, in the nucleic acid according to the present invention, an element, i.e. a gene or a structural element of the nucleic acid, that is essential for replication of the nucleic acid is brought under the control of a regulatory factor such as to allow replication of the nucleic acid in the target cell and to minimize replication of the nucleic acid in a non-target cell.
The nucleic acid of the present invention is adapted for transforming a target cell of a target organism. Target cells within the meaning of the present invention are preferably eucaryotic cells and particularly mammal cells, and most preferably cells lacking an element, e.g. a gene, essential or highly desirable for normal functioning of the respective cell. Particularly preferred target cells are tumor cells, preferably within solid tumor types derived from various tissue origin. The target cells are, in preferred uses of the nucleic acid of the invention parts of a living target organism.
The term "transformation11 means, for the purposes of the present invention, the process of introducing a heterologous nucleic acid into a cell, particularly a target cell, and at least transient expression of a gene of said heterologous nucleic acid. Particularly preferred transformations are those wherein a gene of said heterologous nucleic acid, most preferably a gene whose expression finally leads to a therapeutic effect, is at least transiently expressed in a target cell.
The term "protein transduction domain" signifies a domain of 10 to 30 or more amino acid residues that is enriched in basic amino acids, especially in arginine and/or lysine and/or Histidine. Particularly preferred protein transduction domains are described in the above mentioned publication of Kϋhnel et al., Journal of Virology, 2004, under the names VP22, TaT, AntP, and also a stretch of at least 6 and preferably up to 10 consecutive arginine amino acids.
The nucleic acid according to the present invention comprises a replication element that is essential for the replication of the nucleic acid. The replication element can be a gene encoding for a product (particularly a protein) that is necessary in the reproduction of said nucleic acid in a target cell. The term "element" can also refer to a structural element of the nucleic acid indispensable for replication, particularly an origin of replication, a regulatorily active site, or a termination region.
According to the present invention, the replication element of the nucleic acid is under control of a regulatory factor, so that replication of the nucleic acid is allowed or promoted in the target cell and minimized or inhibited in a non-target cell. The regulatory factor can be encoded by the nucleic acid of the invention. The regulatory factor can also be encoded by the genomic DNA of the target or non-target cell; in this case, the regulatory factor can be a trans-acting factor. The regulatory factor can also be encoded by a helper nucleic acid; in this case, the regulatory factor can also be a trans-acting factor. Suitable regulatory factors are described below in detail. The regulatory factor is part of a regulatory switch which reacts to a factor most preferably provided by the target cell, and either directly or indirectly influences the functioning of the replication element. A direct influence can be exerted by directly promoting or enhancing the expression of the replication element, if said replication element is a gene, or, for example, by binding to the replication element in order to promote the replication of said nucleic acid, if said replication element is a structural element. A.n indirect influence is preferably exerted by enhancing the expression or function of another factor, preferably a protein, that itself or via further factors enhances the functioning of the regulatory element. An indirect influence can also preferably be exerted by suppressing a suppressor of said replication element, again either directly or through interceding further factors.
In particularly preferred nucleic acids according to the present invention, the replication element is a gene essential for replication of said nucleic acid in eucaryotic cells, most preferably a packaging or cell lysis gene. In case the nucleic acid is one of a virus, it is preferred that the replication element is one of the genes essential for viral replication. In the particularly preferred case that the nucleic acid is an adenovirus derived viral nucleic acid, it is particularly preferred when the replication element is one of the early genes of the adenoviral reproduction cycle.
In particularly 'preferred nucleic acids according to the present invention, the* replication element is under control of transcriptionally active and/or repressive elements which can be 1) constitutively active, such as the CMV-promoter, the
RSV-promoter, the HSV-tk promoter, the E1A promoter and the SV4O promoter as well as subtypes and artificial derivatives thereof, 2) promoters active in a tumor-specific environment, such as, but not limited to the hTERT promoter and the alpha fetoprotein promoter, 3) sites for DNA-binding factors attracting the regulatory protein encoded and controlled by the said regulatory switch and 4) sites binding the hypoxia inducible factor.
The adapter encoded by the nucleic acid of the present invention is designed to directly or indirectly couple to said nucleic acid and simultaneously display a protein transduction domain as described above. Through the action of the adapter, the cell tropism of the nucleic acid is easily broadened so that even when treating living target organisms like mammals and humans, in principle all cell types accessible by protein transduction domains are prone to transformation by the nucleic acid according to the present invention. Although a vast number of different cells are thus rendered susceptible to transformation even in a living organism, the nucleic acid replication element under the control of the regulatory factor prevents unacceptable high replication and titres of said nucleic acid in non-target cells of the target organism which in turn limits incidence of the adapter protein within said cells and in the environment thereof. The nucleic acid of the present invention therefore offers for the first time the possibility to infect permissive and hitherto non-permissive tumor cells in a receptor-independent manner and simultaneously balances the potentially harmful stress exerted on non-target cells of the target organism by restricting the replication to target cells.
Particularly preferred nucleic acids according to the present invention further comprise a gene coding for a restriction nuclease, said restriction nuclease being heterologous to the target cell and/or to the target organism, and a restriction site specifically recognized by the heterologous restriction nuclease, wherein said restriction nuclease gene is under control of a regulatory factor, to allow functioning of the restriction nuclease in a non-target cell and to minimise functioning of the restriction nuclease in a target cell. The restriction nuclease encoded by the restriction nuclease gene preferably is harmless to the integrity of the non-target cell genome, and is further preferably a restriction endonuclease, a rare cutting restriction endonuclease, such as but not limited to intron-encoded ('homing') endonucleases or designed zincfinger endonucleases. The restriction nuclease recognises a restriction site inserted in the nucleic acid of the present invention, said restriction site being alien or heterologous to the genomic DNA at least of a non-target cell of the target organism. The restriction nuclease encoded by the nucleic acid of the present invention is therefore capable of selectively digesting the nucleic acid of the present invention in non-target cells of the target organism, and preferably rendering it incapable of replication in the digested form, thus leading to irreversible destruction of said nucleic acid in non-target cells.
The restriction nuclease is under direct or indirect control of a regulatory factor to allow functioning of the restriction nuclease in a non-target cell and to minimize or inhibit functioning of the restriction nuclease in a target cell. The control of the regulatory factor, functioning as part of a regulatory switch, on the restriction nuclease of the present invention can be exerted correspondingly to the control of the regulatory factor exerted on the nucleic acid replication element as described above. In preferred embodiments of the nucleic acid according to the present invention, the regulating factor controlling the nucleic acid replication element is also the regulating factor controlling the functioning of the restriction nuclease. The restriction nuclease can be controlled either by up- or downregulation of the restriction nuclease gene or by enhancing or inhibiting the function of the restriction nuclease protein itself. The nucleic acid of the present invention therefore further minimizes potentially harmful influences of said nucleic acid to non-target cells by actively destroying the nucleic acid in such non-target cells.
In particularly preferred nucleic acids of the present invention, the regulating factor is a nucleic acid binding, trans-acting factor produced by non-target cells, and preferably is the p53 gene product. The regulating factor enhances expression of the restriction nuclease gene, and also enhances expression of a further regulator protein encoded by the nucleic acid of the present invention. The further regulator protein suppresses expression of a replication gene essential for the reproduction of the nucleic acid of the present invention. Thus, one regulating factor directly controls the expression of the restriction nuclease and indirectly J controls expression of the replication gene essential for reproduction of the nucleic acid of the invention.
Further preferred nucleic acids of the present invention comprise a regulator gene coding for a regulator protein for i) allowing or enhancing the functioning of the replication element in a target cell and/or for preventing or repressing the functioning of the replication element in a non-target cell, ii) allowing or enhancing the functioning of the adapter protein in a target cell and/or for preventing or repressing the functioning of the adapter protein in a non-target cell, iii) allowing or enhancing the functioning of the restriction nuclease in a non-target cell and/or for preventing or repressing the functioning of the restriction nuclease in a target cell, and/or for iv) allowing or enhancing the functioning of a target gene in a target cell and/or for preventing or repressing the functioning of the target gene in a non-target cell. Particularly preferred regulator proteins comprise a DNA binding moiety and a moiety for modification of transcription. Particularly preferred DNA binding moieties are those of Gal4, LexA, TetR and rTetR (NCBI accession numbers NC001148, J01643 bzw. X00694). Transcription modification domains can be activator (enhancer) domains, particularly preferred those with acetyltransferase activity or those attracting another factor with histone acetyltransferase activity. A particularly preferred activator domain is that of the Herpesvirus-VP16 protein (NCBI accession number: M57289). Transcription modification domains can also be repressor domains, particularly preferred those derived from Krϋppel-like zink- finger proteins, such as KIRAB (NCBI accession number: M67509) and can also be proteins that influence the methylation status of their target DNA, such as MeCP (NM004992), or they can be derived from factors which display Histone Deacetylase activity or attract other factors that have Histonedeacetylase activity, such as for example, but not exclusively, the SIN family (AF 038848) or NcoR (NM006311).
The regulator protein encoded by the regulated gene in case i) allows or enhances the functioning of the replication element in a target cell and/or prevents or represses the functioning of the replication element in a non-target cell. The regulator protein can be the regulating factor described above, under " whose control the replication element is. In this case, the regulator protein is responsive to an element, particularly preferred a metabolic product, of a target or non-target cell such as to allow and/or repress, respectively, the functioning of the replication element in a target and non-target cell as described just before. The element the regulator protein is responsive to preferably allows to distinguish between target and non-target cells, and preferably is the p53 gene product.
To support the regulatory function of the regulatory protein said nucleic acid can be provided with nucleic acid sequences coding for siRNAs when transcribed along with the messenger coding for the regulatory protein. This can be achieved by application of either intron-encoded siRNA or linking the shRNA coding nucleic acid downstream of the polyadenylation signal. SiRNA can either applied as shRNA or MicroRNA. Preferred shRNA sequences target and interfere with factors promoting replication of said nucleic acid such as, but not exclusively, target-cell factors like E2F, c-myc and/or HSP70, and/or viral factors like E1 A and several ORFs of the E4 locus.
In case ii) the regulator protein can allow or enhance the functioning of the adapter protein in a target cell and/or prevent or repress the functioning of the adapter protein in a non-target cell. In the same manner as for case i), the regulator protein can up- and downregulate the expression of the adapter protein gene, and/or it can also stimulate or repress the functioning of the adapter protein itself.
In case iii), the regulator protein is adapted to allow or enhance the functioning of the restriction nuclease in a non-target cell and/or prevent or repress the function of the restriction nuclease in a target cell. The influence of the regulator protein on the restriction nuclease can again be exerted by up- and downregulation of the corresponding gene and/or by stimulation or repression of the restriction nuclease itself, in a manner corresponding to that of cases i) to ii).
In a further preferred case iv), the regulator protein is adapted to allow or enhance the functioning of the target gene in a target cell and/or prevent or repress the functioning of the target gene in a non-target cell. The influence of the regulator protein on the restriction nuclease can again be exerted by up- and downregulation of the corresponding gene and/or by stimulation or repression of the restriction nuclease itself, in a manner corresponding to that of cases i) - iii). This is particularly useful in the case that the target gene is a therapeutically active gene, preferably one which leads to induction of apoptosis.
The cases i) to iv) are not meant to be mutually exclusive, but can be combined on the same nucleic acid of the present invention. Also, the regulator protein does not have to be identical for all of these cases.
A particularly preferred nucleic acid according to the present invention is one wherein the function of regulator gene, the nucleic acid replication element, the adapter protein gene and the restriction nuclease gene are directly or indirectly linked to the status of the human p53 gene product within the target and non- target cells, respectively. The p53 tumour suppressor gene is a gene upregulated following DNA damage, viral infection, hypoxia or other factors threatening the cellular and genomic integrity. Aberrant forms of the p53 gene product are frequently found in tumours, where the p53 gene product is e.g. functionally altered (lack of DNA-binding activity), bound in complexes together with oncogenic proteins or has decreased protein stability. As active ρ53 serves as e. g. as strong transcriptional activator and is able to sensitively and specifically control the said regulatory switch, the nucleic acid according to the present invention is therefore most suitable for the treatment of tumours, as its effects on non-tumour cells is kept in check by at least by one, preferably two, more preferably three and most preferably all four of the security measures implemented by the nucleic acid of the present invention (i. e. controlled regulator gene and subsequent nucleic acid replication, controlled functioning and expression of the adapter and/or the corresponding gene, controlled functioning and/or expression of the restriction nuclease and controlled functioning and/or expression of the regulator gene.
A particularly preferred nucleic acid according to the present invention is one wherein the function of regulator gene, the nucleic acid replication element, the adapter protein gene and the restriction nuclease gene are not linked to the status of neither p73 plus derived subforms nor p63 and derived subforms thereof, within the target and non-target cells, respectively. This is of particular importance as many ρ53 altered tumor cells harbour normal p63/p73, a subgroup of proteins which are able to replace p53 function under certain circumstances, such as genotoxic stress. The nucleic acid replication element, the adapter gene, the restriction nuclease gene and/or the regulator gene is therefore preferably under control of a normal human p53 gene product and independent of p73 and/or p63 and/or subtypes thereof. The independence from p73 and p63 and their respective subtypes is preferably achieved by using doxorubicine, an adenovirus or an adenoviral vector for transduction of the target and non-target cells.
Particularly preferred according to the present invention is a virus for transforming a target cell of a target organism, said virus comprising a nucleic acid according to the present invention. Viruses are particularly capable of infecting eucaryotic, mammalian and human cells and have in the past been extensively studied for gene therapy applications. A virus comprising a nucleic acid of the present invention is therefore particularly suited for gene therapeutic applications as attempted in the prior art and described above.
The virus of the present invention most suitably is one wherein the adapter protein encoded by the nucleic acid of the present invention is capable of coupling to an adenoviral fibreknob protein. It is most useful when the virus itself is derived from an adenovirus and comprises a fibreknob protein to allow cell infection. Adenoviruses and viruses derived therefrom are particularly well studied for gene therapeutic applications.
The virus of the present invention is preferably also suitable for coapplication with chemotherapy, e.g. Doxorubicin. DNA damaging agents lead to an upregulation of p53 und therefore support correct and sensitive function of the regulatory switch as described above according to the invention. In a most preferred case chemotherapy is applied prior to the application of the virus of the present invention as this leads to preactivated p53 in non-target cells and diminishes negative interference loops of the replication unit with the transcriptional activity of p53. It is therefore also preferred to manufacture a medicament for treatment of tumors, said medicament comprising a) a chemotherapeutic agent, and b) a virus according to the invention.
In another form of this virus not the entire gene for the adapter protein is included in the viral DNA. Instead, short nucleic acid sequences coding for protein transduction domains, corresponding to those used in the said adapter proteins, are directly inserted into the Hl-Loop region of the adenoviral fiberknob protein. In this way, the derived virus displaying the protein transduction, domain in the fiberknob protein can directly couple to target and/or non-target cells in a receptor-independent manner. The viral tropism alteration is therefore not limited to sites of high replication. This property limits safety of the vector but enhances efficacy of viral spreading.
In another form of this virus the protein transduction domains present in the Hl- Loop region are provided with stretches of several cystein residues. The presence of cystein residues allow the virus to multimerize with each other to build concatamers or aggregates via disulfide bonds. Viral aggregation, preferably occurring in the oxidative environment of a solid tumor as a result of elevated apoptosis rate and nutrient deprivation, should hinder the viral progeny being washed out of the tumor by the blood flow. Therefore viral replication should be more concentrated to the tumor area.
The nucleic acid according to the present invention and the corresponding virus are preferably constructed to destroy connective tissue within solid tumors, mainly consisting of fibroblasts, which serves as physical backbone for the tumor as well as supplier with nutrients and survival factors. Furthermore, as fibroblasts are refractory to adenoviral infection, tumor connective tissue serves as physical barriers for effective expansion of adenoviral infection, thus greatly limiting therapeutic efficacy when using adenoviral vectors with natural tropism. The nucleic acid according to the present invention and the corresponding virus therefore allow the vector to target and to destroy those preestablished barriers, subsequently allowing viral distribution throughout the entire tumor mass. Additionally, these vectors can also target tumor stem cells, as stem cells are known to be CAR-downregulated.
The nucleic acid according to the present invention and the corresponding virus preferably are a medicament, preferably for the treatment of a tumor, and are < used alone or in combination with a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier is one that does not or only to an acceptable extend damage the health of a target organism treated with the medicament of the present invention. Particularly preferred carriers are water and physiological saline.
The invention is now further described with reference to the following examples and figures. The examples and figures are not meant to limit the invention, but are given for illustration purposes.
Figure 1 is a schematic illustration of the reproductive cycle of a virus according to the present invention. Figure 2 is a set of three construction schemes for viruses according to the present invention.
Figure 3 is a construction scheme for a further virus according to the present invention.
A virus according to the present invention preferably is an adenoviral based virus. Suitable viruses and their construction schemes can be found in the publications of Kϋhnel et al., Journal of Virology 2004 and Cancer Gene Therapy 2004, as cited above.
Figure 1 is a schematic representation of the functioning of a virus according to the present invention. The virus comprises a regulator gene, namely a repressor gene GAL4-KRAB, wherein the GAL4-domain is a specifically DNA binding domain and the KRAB-dornain is a repressor domain that primarily affects the basal transcription machinery.
The regulator gene is under the control of an artificial p53 dependent promoter prMin-RGC. This promoter contains thirteen p53 binding sites derived from the ribosomal gene cluster, in combination with a minimal CMV-promoter providing a
TATA-box motive. The regulator protein GAL4-KRAB is therefore expressed in the presence of p53, as is indicated in the lower left portion of figure 1 representing a normal, mammalian cell. In the absence of normal p53 protein, e.g. when the p53 protein is completely absent or replaced by a mutant form, the regulator protein GAL4-KFLAB is not expressed, as is indicated in the lower right portion of figure 1 which represents a degenerate, mammalian tumor cell.
The adenovirus of figure 1 further comprises an E1A gene as nucleic acid replication element under the control of a CMV-gal promoter. In the presence of GAL4-KRAB repressor, expression of the adenoviral E1A gene is downregulated and the gene is effectively silenced. This is indicated in figure 1 by a crossed-out arrow indicating the absence of transcription starting from the CMV-gal promoter and by crossed-out icons of packaged adenoviruses. In the absence of the regulator protein GAL4-KRAB, transcription of the E1A early adenoviral replication gene starts from the CMV-gal promoter, so that the E1A gene is effectively transcribed and the corresponding gene product is expressed. This is indicated by a left-pointing arrow starting from the CMV-gal promoter, circular representations of the E1A gene product and by icons of packaged adenoviral vectors in the lower right half of Figure 1.
Essentially, the regulator protein GAL4-KRAB acts as a frans-acting factor to prevent replication of the virus in the presence of normal p53 protein, while the absence of GAL4-KRAB allows reproduction of the adenovirus of the preferred embodiment in the absence of normal p53 protein. For details relating to the construction of appropriate adenoviral vectors, the skilled person will refer to the publication of Kϋhnel et al., Cancer Gene Therapy 2004, as cited above.
Figure 2 shows three construction schemes for viral nucleic acids of the present invention. In the first scheme, a CMV-gal promoter as described above is operably fused to an expression cassette com prising adenoviral early replication genes E1A and E1B and also the reporter ge ne EGFP. Furthermore, under the control of a p53 dependent prMin-RGC promoter is an expression cassette comprising a gene for the restriction endonuclease I-SCE I and the regulator gene coding for the GAL4-KRAB fusion repressor protein. Between the CMV-gal promoter and the E1A early adenoviral replication gene, a I-SCE I restriction endonuclease site is inserted to allow cleavage of the nucleic acid between the CMV-gal promoter and the nucleic acid replication element (the E1 A gene). Thus, at the onset of expression of the restriction endonuclease gene as a consequence of normal p53 action, the nucleic acid will be cleaved to secure its non-replication.
In the second scheme of figure 2, a further construction scheme is depicted. Under the control of a normal adenoviral E1/V promoter (E1Apr), an expression cassette comprising the adenoviral early replication genes E1A and E1B is operably located. Between the E1Apr promoter and the E1A gene, a I-SCE I restriction endonuclease site is located to allow cleavage of the nucleic acid. The nucleic acid further comprises a I-SCE I restriction endonuclease gene under the control of a p53 dependent promoter prMin-RGC. A nucleic acid constructed according to this construction scheme functions like a nucleic acid constructed in accordance to the first construction scheme of figure 2 in that at the onset of I-SCE I expression, the nucleic acid is cleaved between the promoter responsible for expression of the nucleic acid replication element (here: the E1A gene), so that replication of the nucleic acid is prevented. In the absence of a normal p53 gene product, no I-SCE I restriction endonuclease enzyme is produced, so that normal reproduction of the nucleic acid via the adenoviral repl ϊcation pathway commences, which ultimately leads to cell death of the target cell.
The third construction scheme of figure 2 parallels the first construction scheme but lacks the EGFP reporter gene in the CMV-gal dependent expression cassette. Again, replication of the nucleic acid is prevented t>oth by I-SCE I cleavage between the CMV-gal promoter and the early adenoviral replication gene E1A in the presence of normal p53 protein. Furthermore, replication is also prevented by p53 dependent expression of the GAL4-KRAB fusion repressor protein which silences the CMV-gal dependent expression cassette comprising the nucleic acid replication element essential for replication of "the nucleic acid (here: the adenoviral E1A and E1B genes). In the absence of normal p53 protein, expression levels of both GAL4-KRAB repressor and I-SCE I restriction endonuclease is very low or does not occur at all, so that the nucleic acid is not cleaved at the I-SCE I restriction site and the CMV-gal promoter is not silenced. In this case, normal replication of the nucleic acid occurs, which ultimately leads to death of the host cell containing the nucleic acid (target cell). '*"
Figure 3 shows a construction scheme of a nucleic acid according to the present invention comprising an adapter gene coding for an adapter protein comprising a protein transduction domain and a coupling domain for coupling to an adenoviral fibreknob protein. The nucleic acid comprises, under the control of a cytomegalovirus promoter (CMV), a gene coding for a fusion protein as described in Kϋhnel et al., Journal of Virology 2004, as cited above. In one variant, the protein transduction domain is VP22 domain, in the other variant, the protein transduction domain is Tatjs-57 protein transduction domain. For the construction and function of the adapter gene, the skilled person will refer to the above publication of Kϋhnel et al. just cited.
The adapter protein comprising any of the protein transduction domains described above will be released from the target cell and will bind to adenoviruses, particularly to adenoviruses of the present invention comprising the nucleic acid of the present invention. When binding to the adenoviral fibreknob protein, the target cell range is greatly expanded as described above.

Claims

Claims
1. Nucleic acid for transforming a target cell of a target organism, comprising a) a nucleic acid replication element essential for replication of the nucleic acid, wherein said replication element is under control of a regulating factor, to allow replication of the nucleic acid in the target cell and to minimise replication of the nucleic acid in a non-target cell, b) an adapter gene coding for an adapter protein comprising a) a protein transduction domain and b) a coupling domain for coupling to said nucleic acid.
2. Nucleic acid according to claim 1 , further comprising c) a restriction nuclease gene coding for a restriction nuclease, said restriction nuclease being heterologous to the target cell and/or to the target organism, and d) a restriction site specifically recognized by the heterologous restriction nuclease, wherein said restriction nuclease gene is under control of a regulating factor, to allow functioning of the restriction nuclease in a non-target cell and to minimise functioning of the restriction nuclease in a target cell.
3. Nucleic acid according to any of claims 1 to 2, further comprising e) a regulator gene coding for a regulator protein for i) allowing or enhancing the functioning of the replication element in a target cell and/or for preventing or repressing the functioning of the replication element in a non-target cell, ii) allowing or enhancing the functioning of the adapter protein in a target cell and/or for preventing or repressing the functioning of the adapter protein in a non-target cell, iii) allowing or enhancing the functioning of the restriction nuclease in a non-target cell and/or for preventing or repressing the functioning of the restriction nuclease in a target cell, and/or for iv) allowing or enhancing the functioning of a target gene in a target cell and/or for preventing or repressing the functioning of the target gene in a non- target cell.
4. Nucleic acid according to any of claims 1 to 3, wherein the nucleic acid replication element, the adapter gene, the restriction nuclease gene and/or the regulator gene is under the control of a normal human p53 gene product.
5. Virus for transforming a target cell of a target organism, comprising a 5 nucleic acid according to any of claims 1 to 4.
6. Virus according to claim 5, wherein the adapter protein is capable of coupling to an adenoviral fibre knob protein.
7. Nucleic acid according to any of claims 1 to 4 as a medicament.
8. Virus according to any of claims 5 to 6 as a medicament.
io 9. Medicament comprising a) a nucleic acid according to any of claims 1 to 4, and/or b) a virus according to any of claims 5 to 6, and a pharmaceutically acceptable carrier.
10. Use of a nucleic acid according to any of claims 1 to 4 and/or a virus w 15 according to any of claims 5 to 6 for preparing a medicament for transforming target cells of a target organism.
11. Use of a nucleic acid according to any of claims 1 to 4 and/or a virus according to any of claims 5 to 6 for preparing a medicament for treatment of a tumor.
20
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