WO2007068772A1 - Novel conditionally-replicating recombinant adenoviruses (crad) - Google Patents
Novel conditionally-replicating recombinant adenoviruses (crad) Download PDFInfo
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- WO2007068772A1 WO2007068772A1 PCT/ES2006/000676 ES2006000676W WO2007068772A1 WO 2007068772 A1 WO2007068772 A1 WO 2007068772A1 ES 2006000676 W ES2006000676 W ES 2006000676W WO 2007068772 A1 WO2007068772 A1 WO 2007068772A1
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Classifications
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- 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
- A61K38/19—Cytokines; Lymphokines; Interferons
- A61K38/20—Interleukins [IL]
- A61K38/208—IL-12
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- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/45—Transferases (2)
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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
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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
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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
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- C12N15/8613—Chimaeric vector systems comprising heterologous sequences for production of another viral vector
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- C12N2710/00011—Details
- C12N2710/10011—Adenoviridae
- C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
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- C12N2710/00011—Details
- C12N2710/10011—Adenoviridae
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- C12N2710/10341—Use of virus, viral particle or viral elements as a vector
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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
- C12N2830/002—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
Definitions
- the present invention relates to new recombinant adenoviruses for the treatment of solid tumors, and in particular to new selectively replicative adenoviruses in tumor cells that have altered the pathway of retinoblastoma protein (pRB) and that develop in hypoxia conditions
- SUBSTITUTE SHEET (RULE 26) advances Probably due to an inefficient transduction of the entire mass of solid tumors.
- adenoviruses possess to penetrate cells and, using cellular machinery, replicate and package their own viral genome, ultimately causing cell lysis and the propagation of their progeny.
- adenoviruses are genetically modified so that this replicative and cytopathic activity is attenuated or nullified in normal cells, but not diminished in the target tumor cells.
- These adenoviruses are commonly known as conditioned replication adenoviruses.
- the first CRAds incorporate genomic mutations that cause a functional loss only compensated by some specific alterations of the target tumor cells.
- the incorporation of deletions in the ElA or ElB genes results in the production of the mutated proteins, unable to bind with cellular proteins and block control elements that possess normal cells, but that are already intrinsically blocked in tumor cells.
- the E1B-55 Kda protein which normally binds and inactivates p53 by inducing entry into S phase (synthesis phase of the cell cycle), is prevented from binding to p53 and consequently will not activate the phase. S and replication, except in tumor cells with an altered p53 pathway.
- Delta24 adenovirus which incorporates a 24 base pair deletion in the constant region 2 (CR2) of the ElA viral gene.
- CR2 constant region 2
- the mutated ElA protein is unable to bind to the pRB retinoblastoma protein, a binding that in normal cells is necessary for induction of the S phase and viral replication.
- CRAds would only replicate in cells in which the ElA / pRB interaction is not necessary, for example in tumor cells with an altered RB-pl ⁇ pathway.
- a second type of CRAds is those in which one or more tumor-specific promoters are introduced instead of the endogenous promoters of some of their viral genes (eg ElA, ElB, E4).
- the endogenous promoters of some of their viral genes eg ElA, ElB, E4.
- viral replication would be restricted to those tumors in which the specific tumor promoter is re-activated or over-activated. Since many of these promoters are not activated in all tumors, the most recent developments seek to design promoters whose activation is universal in all types of tumors.
- CRAds have been developed where the transcription of the ElA gene is controlled by the promoter of the E2F-1 gene, by a promoter derived from the gene
- SUBSTITUTE SHEET (RULE 26) of telomerase reverse transcriptase (TERT), or also by a hypoxia inducible promoter.
- US6900049, US2005 / 0074430 and WO2004 / 031357 have oncolytic adenoviruses whose replication is dependent on the hypoxia-inducible factor (hypoxia-inducible factor, HIF).
- the replicative and cytolytic activity of these viruses would be limited to cells whose HIF activity is increased, and in particular to cells that grow under hypoxic conditions.
- viruses carry one or more genes (ElA, ElB, E4) whose transcription is controlled by a promoter, usually artificial, that incorporates one or more hypoxia-responsive hypoxia-responsive elements.
- Ad9xHRElA hypoxia-inducible oncolytic adenovirus
- E2F for example tumor.
- SUBSTITUTE SHEET (RULE 26) of new generations of oncolytic adenoviruses to maximize their replicative and oncolytic efficiency, maintaining their selectivity for tumor cells, and ultimately, presenting maximum anti-tumor activity and safety. This may be possible by developing new CRAds that "exploit" new regulatory mechanisms, and by selecting CRAds that, by combining different control mechanisms, have better performance. In view of the pre-clinical and clinical trials conducted to date, some specialists have ventured that oncolytic virotherapy alone will hardly be effective in the complete eradication of tumors. However, the clinical data obtained incline them to think that the use of oncolytic adenovirus in combination with radiotherapy, chemotherapy or gene therapy will allow greater anti-tumor efficacy.
- a first object of the invention relates to a recombinant conditioned replication adenovirus, which for simplicity we will refer to as adenovirus
- SUBSTITUTE SHEET (RULE 26) recombinant of the invention characterized in that it comprises: a) a promoter operatively linked to the ElA adenoviral gene comprising at least one HRE hypoxia response element; b) a promoter operatively linked to the adenoviral region E4 comprising at least one element of response to the E2F factor. c) an adenoviral ElA gene deleted in the constant region CR2; and d) an exogenous gene of interest.
- any human adenoviral variety or serotype (which has been isolated in humans) can be used, for example serotypes 2 (Ad2), 5 (Ad5), 11 (AdIl) or 3 (Ad3).
- Ad5 serotype whose complete sequence is available in GeneBank (Human adenovirus type 5, complete genome; Accession number: AC_000008; 35938 bp linear DNA VRL 26 -JAN-2005).
- the viral promoter that regulates and controls the transcription of the gene encoding the ElA protein has been replaced by a promoter comprising at least one hypoxia response element (HRE) inducible by the HIF-I factor, preferably more than 3.
- said promoter operatively linked to the ElA adenoviral gene comprises 9 tandem copies of a hypoxia response element (HRE).
- HIF-I-inducible hypoxia responses can be obtained from promoters of other genes that include such elements.
- HREs can be obtained, for example, from the promoter of the VEGF vascular endothelium growth factor gene, the erythropoietin gene, or some glycolytic enzyme genes, for example enolasa-1.
- a promoter operably linked to a gene means a DNA fragment functionally associated with the coding sequence of said gene, such that said fragment is sufficient to regulate and control the transcription of said sequence.
- gene coding is the minimum promoter region that includes regulatory (minimum) sequences that allow efficient control of gene transcription.
- the promoter that regulates and controls transcription of the ElA gene is an artificial promoter that includes several tandem copies of the HRE derived from the human VEGF gene promoter.
- said promoter comprises the sequence SEQ. ID. NO: 1, which includes 9 tandem copies of the hypoxia response element of the VEGF-A gene, linked in a 3 'position to a TATA sequence from the rat prolactin gene promoter.
- This minimal artificial promoter (9xHRE promoter) has already been described by Cuevas y cois. (Cancer Research 2003; cited above).
- the natural promoter of the genes encoding the E4 proteins has been replaced by a promoter that
- SUBSTITUTE SHEET (RULE 26) it comprises at least one element of response to the E2F factor, preferably more than one.
- said promoter is a fragment of the human E2F-1 factor promoter comprising the regulatory sequences of the E2F-1 gene, for example a minimal promoter.
- said promoter comprises the sequence SEQ. ID. NO: 2. This minimal artificial promoter has already been described by Hernández-Alcoceba and cois ("New oncolytic adenoviruses with hypoxia- and estrogen receptor-regulated replication"; Human Gene Therapy, 2002; 13: 1737-1750).
- the use of the E2F-1 promoter to direct the expression of the ElA, ElB and E4 viral genes has been described in WO01 / 36650.
- the gene encoding the ElA protein of the recombinant virus of the invention has a deletion in the constant region CR2. More specifically, said deletion affects, totally or partially, the region of CR2 necessary for protein binding of pRB retinoblastoma, so that the mutated ElA protein is unable to bind to pRB.
- the deletion comprises 8 "LTCHEAGF” amino acids (amino acids 122-129 of the ElA protein, corresponding to nucleotides 923-946 of the aforementioned sequence AC_000008). This deletion corresponds to the Delta24 deletion (Fueyo J. et al. Oncogene; 2000/19: 2-12).
- the recombinant adenovirus of the invention comprises an ElA viral gene deleted with the Delta24 deletion and, operatively
- SUBSTITUTE SHEET (RULE 26) linked to said gene, a 9xHRE promoter with SEQ sequence. ID. NO: 1; and an E4 viral gene operably linked to the human E2F-1 promoter of sequence SEQ ID NO: 2.
- the recombinant virus of the invention comprises an exogenous gene or transgene.
- said exogenous gene is introduced in the place occupied by the viral genes encoding the gpl9k and 6.1k proteins, which are deleted
- the exogenous gene introduced into the recombinant adenovirus of the invention may be a reporter gene.
- luciferase eg luciferase
- a therapeutic gene such as a tumor suppressor gene, an apoptosis inducing gene, an anti-angiogenic gene, a suicide gene that encodes a pro-drug activating enzyme, or an immunostimulatory gene.
- the exogenous gene is the luciferase reporter gene.
- said exogenous gene is the thymidine kinase (TK) suicide gene.
- the exogenous gene is the interleukin 12 gene (IL-12).
- WO2004 / 031357 describes a hypoxic-regulated oncolytic virus with the IL-12 gene as a transgene.
- SUBSTITUTE SHEET (RULE 26)
- the design of the new CRAd has advantages for its use as an oncolytic virus compared to previous versions, since several elements that determine its efficacy and specificity have been optimized: 1.-
- the use of a promoter that responds to hypoxia to control the ElA gene allows that the replication and cytopathic effect of the virus be stimulated under conditions of low oxygen tension. These conditions are present in solid tumors, and it has been described that they can decrease the activity of different oncolytic adenoviruses regulated by other mechanisms.
- an advantage of the recombinant adenovirus of the invention over ONYX-411 is the control of . the ElA and E4 regions by different promoters, and specifically the use of the E2F-1 promoter in the E4 region. This allows a double replication control mechanism, while ONYX-411 depends exclusively on the pRB path in the cells. On the other hand, avoiding the repetition of sequences reduces the
- SUBSTITUTE SHEET (RULE 26) possibility of recombination, increasing the stability of the viral genome.
- Interleukin 12 This cytokine has several mechanisms of action that contribute to its antitumor effect. On the one hand, it stimulates the reaction of the immune system against tumors through the production of other cytokines and the activation of effector cells. On the other hand, it has an antiangiogenic effect, which hinders the vascularization of tumors and prevents their growth. These actions make the expression of IL-12 in the context of the CRAd described above present important advantages.
- SUBSTITUTE SHEET (RULE 26) causes hypoxia, which stimulates the replication of CRAd.
- HSV-TK Thymidine kinase from Herpes virus type I
- This enzyme has the function of converting a relatively harmless drug (ganciclovir) into a potent cytotoxic agent.
- ganciclovir a relatively harmless drug
- the cells that express HSV-TK in the presence of exogenously administered ganciclovir, die and cause the death of the cells that are in their vicinity. Since the administration of ganciclovir can be done days after administering the CRAd, this can achieve an increase in the destruction of tumor cells once the virus has been amplified, and thus increase its oncolytic capacity. At the same time, this stops viral replication, due to the death of infected cells, and can be considered a safety mechanism in the case of replication in healthy tissues.
- HSV-TK can be used as a reporter gene in humans using the technique of
- PET Positron Emission Tomography
- the CRAd genome has been modified so that exogenous genes can be included in the E3 region. These genes take the place of the viral genes that code for the gpl9k and ⁇ .7k proteins. (Hawkins et al., Gene Therapy (2001) 8, 1123-1131). The deletion of these genes
- SUBSTITUTE SHEET (RULE 26) adenovirus) does not affect the replication of the virus in tumor cells, since the gpl9k and ⁇ .7k proteins have the function of inhibiting the immune response against infected cells. Since tumor cells have their own mechanisms to inhibit their recognition by the immune system, these deletions can favor the elimination of the virus in normal cells, but not in tumor cells, and therefore contribute to the specificity of viral replication.
- the inclusion of exogenous genes in this location has additional advantages. On the one hand, the endogenous promoter and polyphenylation sequences of the virus are used, so it is not necessary to include these sequences exogenously. This saves space in the viral genome and larger genes can be included.
- DNA constructs for the preparation of the recombinant adenovirus of the invention can be obtained by conventional methods of molecular biology, many of them collected in general laboratory manuals (for example, "Molecular Cloning: a
- SUBSTITUTE SHEET (RULE 26) specifically oriented to the preparation of adenovirus, for example:
- any cell line that is permissive for the replication and formation of selectively replicative recombinant virions of the recombinant adenovirus of the invention can be used for the propagation of the recombinant adenovirus of the invention.
- Almost any conventional human tumor cell line could be used.
- the packaging cells transfected with the adenovirus genome have increased HIF expression and activity, except for HEK293 or PER.C6 cells, which constitutively contribute the El viral genes.
- SUBSTITUTE SHEET (RULE 26) cells can be subjected to an inducing treatment of HIF expression, such as culture under hypoxia conditions (eg in a hypoxic chamber adjusted to a gaseous composition 93% N 2 / ⁇ % CÜ 2 /1% O 2 ) , culture in the presence of cobalt chloride (eg 10OOM), or any other mimetic treatment of hypoxia conditions that is an inducer of HIF.
- hypoxia conditions eg in a hypoxic chamber adjusted to a gaseous composition 93% N 2 / ⁇ % CÜ 2 /1% O 2
- cobalt chloride eg 10OOM
- the present invention relates to a host cell comprising a recombinant adenovirus previously described and object of the present invention.
- the invention also relates to a process for the in vitro propagation of said adenovirus object of the present invention which comprises culturing a host cell containing a recombinant adenovirus of the invention, under conditions that allow the expression of said adenovirus.
- the conditions for optimizing the culture of the host cell will depend on the type of host cell used.
- the method of producing the recombinant adenovirus of the invention will include isolation and purification thereof.
- a further object of the invention relates to a pharmaceutical composition
- a pharmaceutical composition comprising a recombinant adenovirus of the invention and a pharmaceutically acceptable excipient.
- the pharmaceutical composition of the invention can be formulated with a variety of conventional excipients that improve adenovirus stability during the manufacturing, handling, storage and distribution processes of the product, or that are appropriate for therapeutic administration.
- cryoprotectant sugars, polyols, surfactants, amino acids, polymers, buffers and salts can be used to adjust the pH of the composition, antioxidants and other chelating agents, as well as bacteriostatics and bactericides (Parkins et al. "The formulation of biopharmaceutical products "; Pharmaceutical Science and Technology Today, 2000; 3: 129-137).
- a sterile aqueous or non-aqueous suspension could also be used, which may contain suspending agents or thickening agents. Concrete excipients and formulations useful for the preparation of the pharmaceutical composition of the invention can be found for example in the Journal of Pharmaceutical Sciences (Evans RK et al.
- the pharmaceutical composition will contain from 10 3 to 10 15 or more adenoviral particles in aqueous solution.
- Figure 1 Schematic representation of the generic plasmid for the production of CRAds. The sequence of
- Type 5 adenovirus is in the form of a plasmid with
- SUBSTITUTE SHEET (RULE 26) Kanamycin resistance, and can be released by digestion with the restriction enzyme Pac ⁇ .
- ElAp ElA promoter, which is flanked by recognition sequences for BstBI.
- E4p promoter of the E4 region, flanked by sequences for SwaI and
- E3 for the insertion of exogenous genes.
- LP late promoter of the virus.
- Figure 2. Schematic representation of the AdHLuc, AdDHLuc, AdDHTK, AdDHIL-12 and AdWTLuc viruses.
- ITR Inverted Terminal Repeat; HRE, ' Hypoxia Response Element; E2F-lp, promoter of the transcription factor E2F-1.
- D24 deletion of the CR2 domain of ElA.
- Figure 3. Cytopathic effect of AdHLuc, AdDHLuc and AdWT in Huh-7 human hepatocarcinoma cells ( Figure A) and in normal IMR-90 fibroblasts ( Figure B).
- Relative survival is represented with respect to uninfected cells cultured under the same conditions, comparing survival when the cells were maintained in hypoxia conditions compared to that observed in normoxia conditions.
- Huh-7 cells were infected with 5 viruses / cell, and IMR-90 fibroblasts with 135 viruses / cell to compensate for their lower adenovirus infectivity. The cytopathic effect was evaluated at 5 days after infection for Huh-7 cells and at 10 days for IMR-90 fibroblasts. The asterisk indicates significant differences (p ⁇ 0.05)
- SUBSTITUTE SHEET (RULE 26) The cells were grown under normoxia or hypoxia. The cultures were photographed (20Ox) 5 days after infection. Cells that undergo cytopathic effect by viric replication are distinguished by loss of adhesion, with increased refringence and rounded morphology.
- FIG. 1 Cytopathic effect of the AdHLuc, AdDHLuc and AdWT viruses on BJ cells (infected with 500 viruses / cell) and IMR-90 (125 viruses / cell), for cells maintained under normoxia or hypoxia conditions. Photographed (20Ox) also at 5 days. Cells that undergo cytopathic effect by viric replication are distinguished by loss of adhesion, with increased refringence and rounded morphology. Figure 6. Cell viability assay in Huh-7 and IMR-90 cells infected with AdDHLuc virus
- AdDHLuc or AdWT when they remained in hypoxia or normoxia conditions for 4 days. units
- FIG. 8 Graphical comparison of in vitro expression of luciferase (represented as RLU / ⁇ g of total protein; RLU, relative luciferase units) in Huh-7 cells infected by the AdDHLuc, AdHLuc viruses,
- AdWTLuc or AdCMVLuc when they were kept in hypoxia or normoxia conditions. Infection: MOI of 10 viruses / cell.
- FIG. 9 Cytotoxicity of the AdDHTK virus in response to hypoxia and GCV treatment. Graphic comparison of the viability of A549 cells infected with an MOI of 0.12 virus / cell under normoxia and hypoxia conditions, after 5 days of incubation with a normal culture medium or supplemented with 100 ⁇ M GCV. The asterisk indicates significant differences (p ⁇ 0.05).
- FIG. 10 Control of the expression of IL12 in the AdDHIL12 virus. Graphical comparison of the increase in IL12 production measured in the supernatant of HeLa cells infected with the AdDHIL12 virus under conditions of normoxia or hypoxia. The asterisk indicates significant differences (p ⁇ 0.05).
- FIG. 11 In vivo expression of luciferase in athymic mice with human tumor xenografts (Huh-7 cells), after infection (by intratumoral injection) with the AdDHLuc, AdDHWT and Ad-CMV-Luc viruses.
- A Luciferase activity in subcutaneous tumors at different times after infection with 2xlO 8 iu of the virus to be tested.
- B Increase in luciferase activity compared to day 1 in the same mice.
- C Tumors induced by intrahepatic injection of Huh-7 cells;
- Example 1 Construction and propagation of recombinant adenoviruses
- AdDHTK which incorporates the Herpes simplex virus thymidine kinase gene (HSV-TK, suicide gene); and - AdDHIL-12, to which the interleukin-12 gene (IL-12, therapeutic gene) has been incorporated.
- HSV-TK Herpes simplex virus thymidine kinase gene
- IL-12 interleukin-12 gene
- the ElA gene promoter has been replaced by a synthetic promoter (SEQ. ID. NO: 1) that responds to hypoxia; ii) the E4 region promoter has been replaced by the E2F-1 transcription factor promoter (SEQ. ID. NO: 2); iii) a deletion (Delta24 deletion) has been made in the CR2 domain of the ElA gene; and iv) the genes of interest have been introduced to replace the gpl9k / ⁇ .7k genes of the E3 region.
- SEQ. ID. NO: 1 that responds to hypoxia
- the E4 region promoter has been replaced by the E2F-1 transcription factor promoter (SEQ. ID. NO: 2);
- iii) a deletion (Delta24 deletion) has been made in the CR2 domain of the ElA gene; and iv) the genes of interest have been introduced to replace the gpl9k / ⁇ .7k genes of the E3 region.
- AdWT wild type 5 adenovirus
- AdWTLuc which differs from AdWT in the inclusion of the luciferase gene in place of the gpl9k / ⁇ .7k genes of the E3 region
- Ad-CMV-Luc a defective adenovirus that expresses the luciferase gene under the control of the CMV promoter (Vector Biolabs, Philadelphia, Ref. 1000); Y
- AdHLuc which differs from AdDHLuc because it has not been deleted (Delta24 deletion) in the CR2 domain of the ElA gene.
- the plasmid pSEHE2F
- This plasmid is based on the adenovirus type 5 genome, modified to facilitate the replacement of the ElA and E4 promoters with tumor-specific promoters (Hernández Alcoceba R. et al. Human Gene Therapy (2002) 13: 1737-1750).
- recognition sequences have been introduced for restriction enzymes in specific regions and exogenous promoters that can be easily substituted:
- ElAp ElA promoter region flanked by recognition sequences for the BstBI enzyme (pSEHE2F contains in this region the artificial promoter 5XEH3, which will be replaced by the 9XHRE promoter in the viruses object of the invention);
- SUBSTITUTE SHEET (RULE 26) - E4 promoter region (E4p) flanked by recognition sequences for the I-Ceul and SwaI enzymes (pSEHE2F contains in this region the promoter for factor E2F-1, which will be maintained in the viruses object of the invention).
- This last promoter was obtained by Polymerase Chain Reaction (PCR) from human genomic DNA using the following oligonucleotides: SEQ ID NO 3: 5 'TACTGTAACTATAACGGTCCTAAGGTAGCGTGGTACCATCCGGACAAAGCC-S' and, SEQ ID NO 4: 5 'TAAGTATTTAGG .
- An insulating sequence (referred to herein as "Ins") is present in pSEHE2F between the adenovirus packaging sequence and the ElA promoter region.
- This sequence transcription stop sequence of the bovine growth hormone gene
- Plasmid pSHE2F was modified in successive stages to obtain the plasmids necessary to produce the viruses of the invention and their controls: .- Introduction of the 9XHRE promoter in the ElA region.
- Plasmid PSEHE2F was digested with BstBI, and the 5XEH3 promoter was replaced by the 9XHRE promoter.
- an adapter consisting of the following pair of oligonucleotides was used:
- SUBSTITUTE SHEET (RULE 26) The correct orientation of the promoter was verified by digestion with the restriction enzyme BamHI and sequencing using the following oligonucleotides: SEQ ID NO 7: 5Ad5St: 5 'TAGTGTGGCGGAAGTGTGATGTTG 3' and SEQ ID NO 8: 3Ad5St: 5 'TCTTCGGTAATAACACCTCCG.
- This new plasmid was called pSHIFE2F. .- Partial deletion of the E3 region.
- Plasmid pSHIFE2F was modified to delete the genes encoding gpl9k / 6.7k (Dgpl9k / ⁇ .7K) and flanking this area with recognition sequences for the Pl-Scel enzyme, which will allow the incorporation of exogenous genes. To obtain the deletion, successive stages of subcloning were carried out. First, a 4.7 kb fragment was obtained by digestion of plasmid pSHIFE2F with the AgeI enzyme, and subcloned into the Agel site of commercial plasmid pMIB / V5-HisC
- pMIB-AgeC This new plasmid (called pMIB-AgeC) was digested simultaneously with the Spel and Xbal enzymes and a fragment obtained by PCR was introduced in this position into which a restriction site for the Xmnl enzyme has been introduced at position 28555 relative to the genome of adenovirus type 5.
- This PCR fragment was obtained with the following pair of oligonucleotides:
- 3XmnI 5 'CCGATTCTAGAGAAACCTGAATTAGAATAGCCCGTAGAGTTGCTTGA AATTGTTCTAAACCCCAC 3', using plasmid pSEHE2F as a template.
- the new plasmid was called pMIB-E3Xmn.
- 5PI-Sce 5 'ACGTAATCTATGTCGGGTGCGGAGAAAGAGGTAATGAAATGGCA 3' and, SEQ ID NO 12:
- 3PI-Sce 5 'TGCCATTTCATTACCTCTTTCTCCGCACCCGACATAGATTACGT 3'.
- plasmid pMIB-PIScel The resulting plasmid is called pMIB-PIScel, and it has the Pl-Scel site where the adenoviral genes that code for To incorporate this deletion into the genome of adenoviral vectors, plasmid pMIB-PIScel was digested with Agel and Seal enzymes simultaneously, and the 4.1 kb fragment obtained was introduced into plasmid pSHIFE2F digested with the same enzymes. In this way, a plasmid is generated that contains the desired deletion, but is incomplete because it has lost the initial regions of the adenoviral genome. However, from this plasmid the 7.7 kb fragment comprised between the SwaI and Spel enzymes was obtained, which was introduced into the plasmid pSHIFE2F instead of the homonymous fragment. In this way, the genes that
- SUBSTITUTE SHEET (RULE 26) coding for gpl9K / 6.7k viral proteins were deleted and instead the Pl-Scel restriction site was introduced that can be used for the incorporation of exogenous genes in this area, since it is not present in any other region of the virus.
- the first exogenous gene that was introduced in the E3 region was the luciferase reporter gene, obtained from plasmid pGL3-Basic (Promega).
- the 1.6 kb fragment between the HindIII and Xbal sites encoding luciferase was treated with the enzyme T4 polymerase to achieve blunt ends, and was introduced into the Pl-Scel site that had been treated in the same way in the modified adenoviral plasmid previously.
- the resulting plasmid is called pSHE2F-Luc.
- Pl-Scel recognition sites for the CIaI enzyme instead of Pl-Scel, because the latter is very large and its symmetrical repetition can cause plasmid recombinations.
- sequence that is intended to be introduced can be flanked by CIaI sites by ligation of these adapters or by PCR reaction using oligonucleotides that include the recognition sequence for CIaI. If the exogenous gene of interest has internal CIaI sites, as with the
- SUBSTITUTE SHEET (RULE 26) interleukin 12, NarI sites, which are compatible with CIaI, can be used.
- SEQ ID NO 16 BElA: 5 'ATCGATCACCTCCGGTACAAGGTTTGG 3'.
- the AB and BC fragments have a homology region, so that if they are mixed they can partially hybridize. This region has been designed to exclude the bases between position 922 and 947. If the hybridization of these two fragments is used as a template for a PCR reaction with oligonucleotides AElA and
- SUBSTITUTE SHEET (RULE 26) incorporated the PCR fragment.
- This intermediate plasmid was named pSdlElA, which has incorporated the modified ElA gene but has lost the 9XHRE promoter and the sequence between bases 9197 and 33756.
- the 9XHRE promoter was reintroduced using the BstBI site.
- the 14.8 kb fragment comprised between the Sdal sites was initially subcloned. A triple ligation was then carried out to fuse 3 fragments between RsrII sites that restore the modified viral genome.
- the 14 kb fragment comes from the plasmid that contains the deletion in the CR2 domain of ElA and the 9XHRE promoter, while the 17 kb fragment (from plasmid pSHE2F-Luc) contains the luciferase gene in the E3 region. Finally, the genome is completed with the 7.7 kb fragment also from pSHE2F-Luc. The plasmid resulting from this fusion was called pSDHE2F-Luc. .- Construction of pAdLuc. For this, the 12.5 kb fragment between the Ndel sites of plasmid pSHE2F-Luc was obtained and introduced into the homonymous sites of plasmid pTG3602.
- SUBSTITUTE SHEET (RULE 26) CIaI recognition.
- the oligonucleotides used were: SEQ ID NO 20:
- 5CIaTK 5 'GTACTATCGATGCTAGCCACCATGGCTTCGTACC 3' and SEQ ID NO 21:
- 3CIaTK 5 'GTACTATCGATAAGCTTAAGTCAGTTAGCCTCC 3' This fragment was digested with CIaI and subcloned into a plasmid based on pSDHE2F-Luc, in which the E3 deletion is flanked by CIaI sites, as described in the previous section. In this way the luciferase gene is replaced by the TK gene. .- Construction of pSDHE2F-IL12.
- 5NARIL12 5 'GTACTGGCGCCACCATGGGTCCTCAGAAGCTAACC 3'
- 3NarIL12 5 'GTACTGGCGCCTAATCCGGATCAATTCTCAGG 3' This fragment was digested with NarI and subcloned into the CIaI site of the pro-viral plasmid described above. In this way the luciferase gene is replaced by the interleukin 12 gene.
- SUBSTITUTE SHEET (RULE 26) The general structure of the modified plasmids is depicted in Figure 1, and a genome skeleton of the recombinant viruses is shown in Figure 2.
- the plasmids described above were digested with the enzyme Pac ⁇ , which releases the viral genome from the rest of the bacterial sequences, and transfected into 293 cells (ATCC CRL-1573) by the calcium phosphate precipitation method. Once the cytopathic effect was observed (typically 7-10 days after transfection), the cells were used by 3 consecutive cycles of freezing and thawing. Serial dilutions of the lysate were made and with them A549 cells (ATCC CCL-185; from human lung cancer) were infected under hypoxic conditions (1% oxygen). In this way several clones of the viruses were obtained, which were verified by PCR.
- the new viruses obtained from plasmids pSHE2F-Luc, pSDHHE2F-Luc, pSDHE2F-TK, pSDHE2F-IL12 and pAdLuc were named AdHLuc, AdDHLuc, AdDHTK, AdDHIL12 and AdWTLuc, respectively.
- viruses were amplified in A549 cells maintained under hypoxia in the case of AdHLuc, AdDHLuc,
- AdDHTK AdDHTK, AdDHIL12, and in 293 cells in the case of AdWTLuc. Purification was carried out by standard techniques using a Cesium gradient and subsequent passage through an exclusion column. The virus obtained was stored in a medium buffered with 10% glycerol at -80 ° C
- AdDHLuc virus The activity of the AdDHLuc virus against control viruses in different cell types was analyzed, both in normal conditions and in hypoxia.
- the main feature sought in CRAds is their ability to selectively induce the death of tumor cells.
- SUBSTITUTE SHEET (RULE 26) 2. At 24 hours, infection with the virus to be tested (one type of virus per well, eg AdWT, AdHLuc or AdDHLuc); The viral concentration was adjusted to each cell line based on the infectivity for that line. 3. Incubation at 37 ° C under hypoxic conditions (chamber adjusted to a gaseous composition of 93% N2 / 6% CO2 / 1% O 2 ) or normoxia (environmental gaseous composition).
- Huh-7 tumor cells (Nakabayashi et al. 1982 were infected. Cancer Res., 42: 3858-3863; from human hepatocarcinoma) and A549 (ATTC CCL-185; from human lung cancer), and normal IMR cells -90 (ATCC CCL-186; from primary human lung fibroblasts) with the various viruses.
- AdHLuc has a cytotoxic potency equal to
- AdHLuc and close to AdWT on tumor cells are advantageously it is strongly attenuated in normal cells.
- SUBSTITUTE SHEET (RULE 26) (ATCC CCL-75.1), which have been maligned by the expression of SV40 T antigen, which blocks the pRB path. WI38-VA13 cells have impaired cell cycle control, and therefore it is expected that the AdDHLuc virus is not attenuated with respect to AdHLuc. Likewise, tests were carried out with normal BJ cells (ATCC CRL-2522; from primary human skin fibroblasts).
- FIG 4 shows photomicrographs of normal WI-38 and malignant WI38-VA13 fibroblasts infected with the different viruses under hypoxia or normoxia conditions.
- the cells that are suffering from the cytopathic effect of the virus are distinguished because they lose adhesion to the culture plate and acquire a rounded morphology.
- AdDHLuc is the virus that is most attenuated in normal cells, while retaining its ability to kill malignant cells.
- AdHLuc which confirms greater attenuation and the operation of the double control system for its replication. It is important to note that in this type of experiments the decrease in cell viability at low doses is dependent on viral amplification, so this test also indirectly reflects the control of virus replication.
- AdDHLuc replication In order to directly analyze the control of AdDHLuc replication, A549 cells were infected with the virus under normoxia or hypoxia conditions, and the production of new infective particles was quantified after days.
- the cells were seeded, infected and grown in the same manner as in the tests to evaluate the cytopathic effect. In this case, 4 days after infection, the cells were used by 3 freeze-thaw cycles and the infective particles were quantified by limit dilution in 293 cells or by immunohistochemistry with anti-adenovirus antibodies (Adeno-X Rapad Titer Kit , BD Biosciences Clontech Cat. No. K1653-1) following the instructions
- hypoxia activated the amplification of the AdDHLuc virus, while the same does not happen with AdWT, whose replication did not undergo changes in these experimental conditions.
- luciferase reporter gene in the E3 region of the AdDHLuc virus allows monitoring of the expression of exogenous genes introduced into the virus.
- luciferase expression is increased in response to viral replication, due to the activation of late promoters and the increase in the number of copies of the viral genome in the cell. Therefore, measuring luciferase activity is an indicator of viral replication.
- Huh-7 cells were seeded and infected with the AdDHLuc, AdHLuc, AdWTLuc replicative viruses, and with the Ad-CMV-Luc defective virus (Vector Biolabs Cat. No. 1000).
- the dose of virus used (MOI) was 10 viruses / cell, and the cells were cultured in the same manner as in the tests for cytopathic effect evaluation.
- Two days after infection, the cells were used to measure Luciferase activity using a commercial kit (Luciferase Assay System, Promega Cat. No. E4030), following the manufacturer's instructions. The activity was expressed as RLü / ⁇ g of total protein (RLU: Relative Luciferase Units).
- Example 3 Characterization of the AdDHTK virus In v ⁇ tro
- the cytopathic effect obtained after infection of the A549 cells, in the presence or absence of the Ganciclovir pro-drug (GCV) was analyzed.
- the test was performed as described in example 2, with the following modifications.
- the cells were infected with different AdDHTK MOIs under conditions of normoxia or hypoxia, and 24 hours later the infectious medium was removed and two types of wells were differentiated. In one of them standard culture medium with 2% fetal bovine serum was added, and in the other GCV (100 ⁇ M) was included. Cell survival was quantified 5 days later.
- Figure 9 shows the data obtained when A549 cells were infected with an MOI of 0.12 virus / cell. As can be seen, this small amount of virus did not significantly decrease the survival of tumor cells under conditions
- HeLa cells (ATTC CCL-2) were seeded in 24-well plates (2xlO 4 cells / well), and infected with the AdDHIL12 virus under normoxia or hypoxia conditions. Since IL12 is a secretable protein, its production in the culture medium was quantified at different times after infection. The detection of murine IL12 was carried out by a commercial ELISA kit (BD
- IL12 practically does not increase under normoxia conditions, but there is a significant increase at 3 days if the cells are maintained in hypoxia conditions.
- human tumor xenotransplants were performed in atomic mice (immunosuppressed) by subcutaneous injection (IxIO 7 cells in 150 ⁇ l saline serum) or intrahepatic (1.5 x 6 cells in 50 ⁇ l serum) of Huh-7 cells.
- the virus to be tested AdDHLuc, Ad-CMV-Luc and AdWTLuc
- the measurement of luciferase activity was started, performed daily for 10 days.
- the luciferin substrate was administered intraperitoneally.
- the expression kinetics and biodistribution of viruses can be monitored in live animals by measuring the emission of light using a high-sensitivity luminometric camera (In vivo imaging system, Xenogen). Under these conditions, the light emission
- a defective adenovirus such as Ad-CMV-Luc maintains the initial expression levels during the first week and then initiates a slow decrease.
- AdDHLuc showed a significant increase in luciferase activity during the first 4 days.
- Figure HB illustrates the luciferase activity relative to the first day post-infection and shows how only in the case of AdDHLuc there is a significant increase in successive days. This is compatible with the achievement of several replication cycles in the
- Huh-7 cells were injected into the liver of the mice and subsequently the virus was administered (10 9 i.).
- the virus was administered (10 9 i.).
- figure HC it can be observed how very high levels of expression are achieved and the increase in the first days is confirmed and then stabilized and a slow descent begins.
- AdDHLuc is a CRAd capable of serving as an expression vector for exogenous genes with efficacy superior to non-replicative adenoviruses.
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Abstract
The invention relates to a conditionally-replicating recombinant adenovirus which is characterised in that it comprises a promoter which is linked to gene E1A comprising hypoxia response elements, a promoter which is linked to region E4 comprising at least one factor E2F response element, a deletion in domain CR2 of gene E1A, and an exogenous gene of interest. The invention also relates to a method for obtaining said adenovirus and to a composition containing same.
Description
NUEVOS ADENOVIRUS RECOMBINANTES DE REPLICACION CONDICIONADA (CRAD) NEW ADENOVIRUS RECOMBINANTS OF CONDITIONED REPLICATION (CRAD)
CAMPO TÉCNICO DE LA INVENCIÓN La presente invención se refiere a nuevos adenovirus recombinantes para el tratamiento de tumores sólidos, y en particular a nuevos adenovirus selectivamente replicativos en células tumorales que presentan alterada la via de la proteina del retinoblastoma (pRB) y que se desarrollan en condiciones de hipoxia.TECHNICAL FIELD OF THE INVENTION The present invention relates to new recombinant adenoviruses for the treatment of solid tumors, and in particular to new selectively replicative adenoviruses in tumor cells that have altered the pathway of retinoblastoma protein (pRB) and that develop in hypoxia conditions
ESTADO DE LA TÉCNICA ANTERIOR A LA INVENCIÓNSTATE OF THE PRIOR ART OF THE INVENTION
A pesar de los últimos avances en las técnicas quirúrgicas y de imagen, asi como de las mejoras realizadas en los procedimientos combinados de radioterapia y quimioterapia, la supervivencia de pacientes con metástasis y ciertos tipos de tumoresDespite the latest advances in surgical and imaging techniques, as well as improvements in the combined radiotherapy and chemotherapy procedures, the survival of patients with metastases and certain types of tumors
(cerebrales, pancreáticos, hepáticos, etc.) no ha mej-orado significativamente. En su esfuerzo por buscar terapias alternativas, los investigadores han visto en la viroterapia, y más particularmente e'n los adenovirus, una herramienta terapéutica atractiva y prometedora.(cerebral, pancreatic, hepatic, etc.) has not improved significantly. In its effort to seek alternative therapies, researchers have seen in Virotherapy, and more particularly e 'n adenoviruses, attractive and promising therapeutic tool.
En un primer momento se abordó la utilización de los adenovirus como vectores adecuados para la transferencia y terapia génica. Sin embargo, aunque los datos preclinicos obtenidos con diversas estrategias de terapia génica adenoviral son alentadores (buena capacidad de transducción y eficacia anti-tumoral) , los ensayos clínicos realizados no han proporcionado todavía grandesInitially, the use of adenoviruses as suitable vectors for gene transfer and therapy was discussed. However, although the preclinical data obtained with various adenoviral gene therapy strategies are encouraging (good transduction capacity and anti-tumor efficacy), the clinical trials performed have not yet provided large
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avances. Probablemente debido a una ineficiente transducción de toda la masa de los tumores sólidos.SUBSTITUTE SHEET (RULE 26) advances Probably due to an inefficient transduction of the entire mass of solid tumors.
Una estrategia alternativa busca aprovechar la capacidad natural que los adenovirus poseen para penetrar en las células y, utilizando la maquinaria celular, replicar y empaquetar su propio genoma viral, provocando finalmente la lisis celular y la propagación de su progenie. Asi, los adenovirus son modificados genéticamente para que esta actividad replicativa y citopática quede atenuada o anulada en las células normales, pero no se vea disminuida en las células tumorales diana. Estos adenovirus se conocen comúnmente como adenovirus de replicación condicionadaAn alternative strategy seeks to take advantage of the natural ability that adenoviruses possess to penetrate cells and, using cellular machinery, replicate and package their own viral genome, ultimately causing cell lysis and the propagation of their progeny. Thus, adenoviruses are genetically modified so that this replicative and cytopathic activity is attenuated or nullified in normal cells, but not diminished in the target tumor cells. These adenoviruses are commonly known as conditioned replication adenoviruses.
("conditionally replicatíve adenoviruses" o CRAds) , adenovirus oncoliticos o también como adenovirus de replicación selectiva. Una revisión reciente de las diferentes construcciones virales desarrolladas puede encontrarse en Chu R. L. y cois: Use of replicating oncolytic adenoviruses in combination therapy for cáncer; Clinical Cáncer Research 2004; 10:5299-5312. •("conditionally replicative adenoviruses" or CRAds), oncolytic adenoviruses or also as selective replication adenoviruses. A recent review of the different viral constructs developed can be found in Chu R. L. and cois: Use of replicating oncolytic adenoviruses in combination therapy for cancer; Clinical Cancer Research 2004; 10: 5299-5312. •
Los primeros CRAds incorporan mutaciones genómicas que provocan una pérdida funcional solamente compensada por algunas alteraciones especificas de las células tumorales diana. Asi, la incorporación de deleciones en los genes ElA o ElB (genes de transcripción temprana necesarios para la replicación "immediate early genes") , resulta en la producción de proteínas El mutadas, incapaces de unirse con proteínas celulares y bloquear elementos de control que poseen las células normales, pero que se encuentran ya intrínsecamente bloqueados en las células tumorales.The first CRAds incorporate genomic mutations that cause a functional loss only compensated by some specific alterations of the target tumor cells. Thus, the incorporation of deletions in the ElA or ElB genes (early transcription genes necessary for "immediate early genes" replication), results in the production of the mutated proteins, unable to bind with cellular proteins and block control elements that possess normal cells, but that are already intrinsically blocked in tumor cells.
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Asi por ejemplo, Onyx-015 - uno de los CRAds mejor estudiados - porta 2 mutaciones en el gen que codifica para la proteína E1B-55 Kda. De esta manera, la proteina E1B-55 Kda, que en condiciones normales se une e inactiva a p53 induciendo la entrada en fase S (fase de síntesis del ciclo celular) , queda impedida para la unión a p53 y en consecuencia no activará la fase S y la replicación, salvo en células tumorales con una vía p53 alterada.SUBSTITUTE SHEET (RULE 26) Thus, for example, Onyx-015 - one of the best studied CRAds - carries 2 mutations in the gene that codes for the E1B-55 Kda protein. In this way, the E1B-55 Kda protein, which normally binds and inactivates p53 by inducing entry into S phase (synthesis phase of the cell cycle), is prevented from binding to p53 and consequently will not activate the phase. S and replication, except in tumor cells with an altered p53 pathway.
Otra modalidad también ampliamente estudiada es el adenovirus Delta24, que incorpora una deleción de 24 pares de bases en la región constante 2 (CR2) del gen viral ElA. De esta manera, la proteína ElA mutada es incapaz de unirse a la proteína del retinoblastoma pRB, unión que en células normales es necesaria para la inducción de la fase S y la replicación viral. EstosAnother modality also widely studied is the Delta24 adenovirus, which incorporates a 24 base pair deletion in the constant region 2 (CR2) of the ElA viral gene. In this way, the mutated ElA protein is unable to bind to the pRB retinoblastoma protein, a binding that in normal cells is necessary for induction of the S phase and viral replication. These
CRAds solamente se replicarían en células en las que no es necesaria la interacción ElA / pRB, por ejemplo en células tumorales con una vía RB-plβ alterada.CRAds would only replicate in cells in which the ElA / pRB interaction is not necessary, for example in tumor cells with an altered RB-plβ pathway.
Un segundo tipo de CRAds lo constituyen aquellos en los que se introducen uno o varios promotores específicos de tumores en sustitución de los promotores endógenos de algunos de sus genes virales (p.ej. ElA, ElB, E4) . De esta manera la replicación viral quedaría restringida a aquellos tumores en los que el promotor tumoral específico está re-activado o sobre-activado. Dado que muchos de estos promotores no están activados en todos los tumores, los desarrollos más recientes buscan diseñar promotores cuya activación sea universal en todo tipo de tumores. Así por ejemplo se han desarrollado CRAds donde la transcripción del gen ElA está controlada por el promotor del gen E2F-1, por un promotor derivado del genA second type of CRAds is those in which one or more tumor-specific promoters are introduced instead of the endogenous promoters of some of their viral genes (eg ElA, ElB, E4). In this way viral replication would be restricted to those tumors in which the specific tumor promoter is re-activated or over-activated. Since many of these promoters are not activated in all tumors, the most recent developments seek to design promoters whose activation is universal in all types of tumors. Thus, for example, CRAds have been developed where the transcription of the ElA gene is controlled by the promoter of the E2F-1 gene, by a promoter derived from the gene
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de la transcriptasa inversa de la telomerasa (TERT) , o también por un promotor inducible por hipoxia.SUBSTITUTE SHEET (RULE 26) of telomerase reverse transcriptase (TERT), or also by a hypoxia inducible promoter.
US6900049, US2005/0074430 y WO2004/031357 presentan adenovirus oncoliticos cuya replicación es dependiente del factor inducible por hipoxia (hypoxia-inducible factor, HIF) . La actividad replicativa y citolitica de estos virus estarla limitada a células cuya actividad HIF está aumentada, y en particular a células que crecen en condiciones de hipoxia. A tal efecto, los virus portan uno o varios genes (ElA, ElB, E4) cuya transcripción está controlada por un promotor, generalmente artificial, que incorpora uno o varios elementos de respuesta a hipoxia inducibles por HIF.US6900049, US2005 / 0074430 and WO2004 / 031357 have oncolytic adenoviruses whose replication is dependent on the hypoxia-inducible factor (hypoxia-inducible factor, HIF). The replicative and cytolytic activity of these viruses would be limited to cells whose HIF activity is increased, and in particular to cells that grow under hypoxic conditions. For this purpose, viruses carry one or more genes (ElA, ElB, E4) whose transcription is controlled by a promoter, usually artificial, that incorporates one or more hypoxia-responsive hypoxia-responsive elements.
En un trabajo reciente, Cuevas Y. y cois. (Specific oncolytic effect of a new hypoxia-inducible factor- dependent replicative adenovirus on von Hippel-Lindau- defective renal cell carcinomas; Cáncer Research 2003;In a recent work, Cuevas Y. and cois. (Specific oncolytic effect of a new hypoxia-inducible factor-dependent replicative adenovirus on von Hippel-Lindau- defective renal cell carcinomas; Cancer Research 2003;
63:6877-6884) presentan un adenovirus oncolitico inducible por hipoxia (Ad9xHRElA) en el que la expresión del gen ElA está controlada por un promotor artificial con 9 copias en tándem del elemento de respuesta a hipoxia procedente del promotor del gen VEGF (factor de crecimiento del endotelio vascular) . Ad9xHRElA, porta además un gen E4 controlado por un promotor E2F-1, de manera que la expresión de E4 quedarla restringida a células en proliferación con una importante actividad63: 6877-6884) have a hypoxia-inducible oncolytic adenovirus (Ad9xHRElA) in which the expression of the ElA gene is controlled by an artificial promoter with 9 tandem copies of the hypoxia response element from the VEGF gene promoter ( vascular endothelial growth). Ad9xHRElA, also carries an E4 gene controlled by an E2F-1 promoter, so that E4 expression will be restricted to proliferating cells with significant activity
E2F, por ejemplo tumorales.E2F, for example tumor.
Se ha podido constatar sin embargo, la existencia de un compromiso entre la selectividad replicativa de los virus atenuados y su eficiencia replicativa-oncolitica.It has been possible to verify, however, the existence of a compromise between the replicative selectivity of the attenuated viruses and their replicative-oncolytic efficiency.
Sigue siendo necesario y deseable el diseño y selecciónDesign and selection is still necessary and desirable
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de nuevas generaciones de adenovirus oncolíticos para maximizar su eficiencia replicativa y oncolitica, manteniendo su selectividad por las células tumorales, y en último término, que presenten la máxima actividad anti-tumoral y seguridad. Esto puede ser posible mediante el desarrollo de nuevos CRAds que "exploten" nuevos mecanismos reguladores, y mediante la selección de CRAds que, combinando distintos mecanismos de control, presenten mejores prestaciones. A la vista de los ensayos pre-clínicos y clinicos realizados hasta la fecha, algunos especialistas han aventurado que la viroterapia oncolitica por si sola difícilmente será efectiva en la completa erradicación de los tumores. Sin embargo, los datos clinicos obtenidos les inclinan a pensar que la utilización de adenovirus oncoliticos en combinación con radioterapia, quimioterapia o terapia génica permitirán una mayor eficacia anti-tumoral.SUBSTITUTE SHEET (RULE 26) of new generations of oncolytic adenoviruses to maximize their replicative and oncolytic efficiency, maintaining their selectivity for tumor cells, and ultimately, presenting maximum anti-tumor activity and safety. This may be possible by developing new CRAds that "exploit" new regulatory mechanisms, and by selecting CRAds that, by combining different control mechanisms, have better performance. In view of the pre-clinical and clinical trials conducted to date, some specialists have ventured that oncolytic virotherapy alone will hardly be effective in the complete eradication of tumors. However, the clinical data obtained incline them to think that the use of oncolytic adenovirus in combination with radiotherapy, chemotherapy or gene therapy will allow greater anti-tumor efficacy.
Es por tanto interesante y deseable, y constituye el objeto de esta invención, el diseño de nuevos adenovirus de replicación condicionada para el tratamiento de tumores sólidos y metástasis en general que, junto con un control optimizado de la replicación (selectividad/oncolisis) , sean portadores de genes exógenos que contribuyan a mejorar la eficacia anti- tumoral .It is therefore interesting and desirable, and is the object of this invention, the design of new adenovirus conditioned replication for the treatment of solid tumors and metastases in general that, together with an optimized control of replication (selectivity / oncolysis), are carriers of exogenous genes that contribute to improve antitumor efficacy.
DESCRIPCIÓN DETALLADA DE LA INVENCIÓNDETAILED DESCRIPTION OF THE INVENTION
Un primer objeto de la invención se refiere a un adenovirus recombinante de replicación condicionada, al que por simplificar nos referiremos como adenovirusA first object of the invention relates to a recombinant conditioned replication adenovirus, which for simplicity we will refer to as adenovirus
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recombinante de la invención, caracterizado porque comprende: a) un promotor operativamente unido al gen adenoviral ElA que comprende al menos un elemento de respuesta a hipoxia HRE; b) un promotor operativamente unido a la región adenoviral E4 que comprende al menos un elemento de respuesta al factor E2F. c) un gen adenoviral ElA delecionado en la región constante CR2; y d) un gen exógeno de interés .SUBSTITUTE SHEET (RULE 26) recombinant of the invention, characterized in that it comprises: a) a promoter operatively linked to the ElA adenoviral gene comprising at least one HRE hypoxia response element; b) a promoter operatively linked to the adenoviral region E4 comprising at least one element of response to the E2F factor. c) an adenoviral ElA gene deleted in the constant region CR2; and d) an exogenous gene of interest.
Para la construcción del adenovirus recombinante de la invención puede utilizarse cualquier variedad o serotipo adenoviral humano (que haya sido aislado en humanos), por ejemplo los serotipos 2 (Ad2), 5 (Ad5), 11 (AdIl) ó 3 (Ad3) . Para facilitar la comprensión, el adenovirus recombinante de la invención se describe e ilustra con ejemplos realizados utilizando el serotipo Ad5, cuya secuencia completa está disponible en GeneBank (Human adenovirus type 5, complete genome; Accesión number: AC_000008; 35938 bp DNA linear VRL 26-JAN-2005) .For the construction of the recombinant adenovirus of the invention any human adenoviral variety or serotype (which has been isolated in humans) can be used, for example serotypes 2 (Ad2), 5 (Ad5), 11 (AdIl) or 3 (Ad3). For ease of understanding, the recombinant adenovirus of the invention is described and illustrated with examples made using the Ad5 serotype, whose complete sequence is available in GeneBank (Human adenovirus type 5, complete genome; Accession number: AC_000008; 35938 bp linear DNA VRL 26 -JAN-2005).
En el virus recombinante de la invención el promotor viral que regula y controla la transcripción del gen que codifica la proteina ElA ha sido sustituido por un promotor que comprende al menos un elemento de respuesta a hipoxia (HRE) inducible por el factor HIF-I, preferentemente más de 3. En una realización más preferente, dicho promotor operativamente unido al gen adenoviral ElA comprende 9 copias en tándem de un elemento de respuesta a hipoxia (HRE) . Estos elementos deIn the recombinant virus of the invention the viral promoter that regulates and controls the transcription of the gene encoding the ElA protein has been replaced by a promoter comprising at least one hypoxia response element (HRE) inducible by the HIF-I factor, preferably more than 3. In a more preferred embodiment, said promoter operatively linked to the ElA adenoviral gene comprises 9 tandem copies of a hypoxia response element (HRE). These elements of
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respuesta a hipoxia inducibles por HIF-I pueden obtenerse de promotores de otros genes que incluyen dichos elementos. Dichos HRE pueden obtenerse por ejemplo del promotor del gen del factor de crecimiento del endotelio vascular VEGF, del gen de la eritropoyetina, o de algunos genes de enzimas glicoliticas, por ejemplo enolasa-1.SUBSTITUTE SHEET (RULE 26) HIF-I-inducible hypoxia responses can be obtained from promoters of other genes that include such elements. Such HREs can be obtained, for example, from the promoter of the VEGF vascular endothelium growth factor gene, the erythropoietin gene, or some glycolytic enzyme genes, for example enolasa-1.
Tal y como se utiliza en la presente invención, "un promotor operativamente unido a un gen" significa un fragmento de DNA funcionalmente asociado a la secuencia codificante de dicho gen, de manera que dicho fragmento es suficiente para regular y controlar la transcripción de dicha secuencia codificante del gen. Asi "un promotor minimo" es la región mínima del promotor que incluye las secuencias reguladoras (mínimas) que permiten un control eficiente de la transcripción del gen.As used in the present invention, "a promoter operably linked to a gene" means a DNA fragment functionally associated with the coding sequence of said gene, such that said fragment is sufficient to regulate and control the transcription of said sequence. gene coding Thus "a minimum promoter" is the minimum promoter region that includes regulatory (minimum) sequences that allow efficient control of gene transcription.
En una realización particular del adenovirus recombinante de la invención, el promotor que regula y controla la transcripción del gen ElA es un promotor artificial que incluye varias copias en tándem del HRE derivado del promotor del gen VEGF humano. En una realización más particular, dicho promotor comprende la secuencia SEQ. ID. NO: 1, que incluye 9 copias en tándem del elemento de respuesta a hipoxia del gen VEGF-A, unidas en posición 3' a una secuencia TATA procedente del promotor del gen de la prolactina de rata. Este promotor artificial minimo (promotor 9xHRE) ha sido ya descrito por Cuevas y cois. (Cáncer Research 2003; citado anteriormente) .In a particular embodiment of the recombinant adenovirus of the invention, the promoter that regulates and controls transcription of the ElA gene is an artificial promoter that includes several tandem copies of the HRE derived from the human VEGF gene promoter. In a more particular embodiment, said promoter comprises the sequence SEQ. ID. NO: 1, which includes 9 tandem copies of the hypoxia response element of the VEGF-A gene, linked in a 3 'position to a TATA sequence from the rat prolactin gene promoter. This minimal artificial promoter (9xHRE promoter) has already been described by Cuevas y cois. (Cancer Research 2003; cited above).
Por otra parte, en el adenovirus recombinante de la invención, el promotor natural de los genes que codifican las proteínas E4 ha sido sustituido por un promotor queOn the other hand, in the recombinant adenovirus of the invention, the natural promoter of the genes encoding the E4 proteins has been replaced by a promoter that
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comprende al menos un elemento de respuesta al factor E2F, preferiblemente más de uno. En una realización particular dicho promotor es un fragmento del promotor del factor E2F-1 humano que comprende las secuencias reguladoras del gen E2F-1, por ejemplo un promotor minimo. En una realización más particular, dicho promotor comprende la secuencia SEQ. ID. NO: 2. Este promotor artificial minimo ha sido ya descrito por Hernández- Alcoceba y cois ("New oncolytic adenoviruses with hypoxia- and estrogen receptor-regulated replication"; Human Gene Therapy, 2002; 13:1737-1750). Por otra parte, la utilización del promotor E2F-1 para dirigir la expresión de los genes virales ElA, ElB y E4 ha sido descrita en WO01/36650. Como mecanismo adicional para el control de la replicación, el gen que codifica la proteina ElA del virus recombinante de la invención presenta una deleción en la región constante CR2. Más concretamente, dicha deleción afecta, total o parcialmente, a la región de CR2 necesaria para la unión a la proteina del retinoblastoma pRB, de manera que la proteina ElA mutada es incapaz de unirse a pRB.SUBSTITUTE SHEET (RULE 26) it comprises at least one element of response to the E2F factor, preferably more than one. In a particular embodiment said promoter is a fragment of the human E2F-1 factor promoter comprising the regulatory sequences of the E2F-1 gene, for example a minimal promoter. In a more particular embodiment, said promoter comprises the sequence SEQ. ID. NO: 2. This minimal artificial promoter has already been described by Hernández-Alcoceba and cois ("New oncolytic adenoviruses with hypoxia- and estrogen receptor-regulated replication"; Human Gene Therapy, 2002; 13: 1737-1750). On the other hand, the use of the E2F-1 promoter to direct the expression of the ElA, ElB and E4 viral genes has been described in WO01 / 36650. As an additional mechanism for replication control, the gene encoding the ElA protein of the recombinant virus of the invention has a deletion in the constant region CR2. More specifically, said deletion affects, totally or partially, the region of CR2 necessary for protein binding of pRB retinoblastoma, so that the mutated ElA protein is unable to bind to pRB.
En una realización particular la deleción comprende 8 aminoácidos "LTCHEAGF" (aminoácidos 122-129 de la proteina ElA, correspondientes a los nucleótidos 923-946 de la secuencia AC_000008 ya citada) . Esta deleción se corresponde con la deleción Delta24 (Fueyo J. y cois. Oncogene; 2000/ 19:2-12) .In a particular embodiment, the deletion comprises 8 "LTCHEAGF" amino acids (amino acids 122-129 of the ElA protein, corresponding to nucleotides 923-946 of the aforementioned sequence AC_000008). This deletion corresponds to the Delta24 deletion (Fueyo J. et al. Oncogene; 2000/19: 2-12).
En una realización preferente, el adenovirus recombinante de la invención comprende un gen viral ElA delecionado con la deleción Delta24 y, operativamenteIn a preferred embodiment, the recombinant adenovirus of the invention comprises an ElA viral gene deleted with the Delta24 deletion and, operatively
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unido a dicho gen, un promotor 9xHRE con secuencia SEQ. ID. NO: 1; y un gen viral E4 operativamente unido al promotor E2F-1 humano de secuencia SEQ ID NO: 2.SUBSTITUTE SHEET (RULE 26) linked to said gene, a 9xHRE promoter with SEQ sequence. ID. NO: 1; and an E4 viral gene operably linked to the human E2F-1 promoter of sequence SEQ ID NO: 2.
Adicionalmente, el virus recombinante de la invención comprende un gen exógeno o transgén. En una realización preferida dicho gen exógeno se introduce en el lugar que ocupan los genes virales que codifican para las proteínas gpl9k y 6.1k, que son delecionadosAdditionally, the recombinant virus of the invention comprises an exogenous gene or transgene. In a preferred embodiment said exogenous gene is introduced in the place occupied by the viral genes encoding the gpl9k and 6.1k proteins, which are deleted
(nucleótidos 28.555 a 29.355 de la secuencia del adenovirus, Gene Bank AC_000008) . La introducción de esta sustitución ha sido ya descrita por Hawkins y cois.(nucleotides 28,555 to 29,355 of the adenovirus sequence, Gene Bank AC_000008). The introduction of this substitution has already been described by Hawkins and cois.
("Gene delivery from the E3 región of replication human adenovirus: evaluation of the 6.7 K/gpl9K región"; Gene("Gene delivery from the E3 region of replication human adenovirus: evaluation of the 6.7 K / gpl9K region"; Gene
Therapy, 2001; 8: 1123-1131). El gen exógeno introducido en el adenovirus recombinante de la invención puede ser un gen reporteroTherapy, 2001; 8: 1123-1131). The exogenous gene introduced into the recombinant adenovirus of the invention may be a reporter gene.
(por ejemplo luciferasa) o un gen terapéutico, como un gen supresor de tumor, un gen inductor de apoptosis, un gen anti-angiogénico, un gen suicida que codifica una enzima activadora de pro-fármacos, o un gen inmunoestimulador.(eg luciferase) or a therapeutic gene, such as a tumor suppressor gene, an apoptosis inducing gene, an anti-angiogenic gene, a suicide gene that encodes a pro-drug activating enzyme, or an immunostimulatory gene.
En una realización particular de la invención el gen exógeno es el gen reportero de la luciferasa. En otra realización particular dicho gen exógeno es el gen suicida de la timidina quinasa (TK) . En otra realización particular de la invención el gen exógeno es el gen de la interleuquina 12 (IL-12) . WO2004/031357 describe un virus oncolitico regulado por hipoxia con el gen de la IL-12 como transgén.In a particular embodiment of the invention the exogenous gene is the luciferase reporter gene. In another particular embodiment said exogenous gene is the thymidine kinase (TK) suicide gene. In another particular embodiment of the invention the exogenous gene is the interleukin 12 gene (IL-12). WO2004 / 031357 describes a hypoxic-regulated oncolytic virus with the IL-12 gene as a transgene.
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El diseño del nuevo CRAd presenta ventajas para su uso como virus oncolitico frente a versiones anteriores, ya que se han optimizado diversos elementos que determinan su eficacia y especificidad: 1.- El uso de un promotor que responde a hipoxia para controlar el gen ElA permite que la replicación y el efecto citopático del virus se vea estimulado en condiciones de baja tensión de oxigeno. Estas condiciones están presentes en los tumores sólidos, y se ha descrito que pueden disminuir la actividad de diferentes adenovirus oncoliticos regulados por otros mecanismosSUBSTITUTE SHEET (RULE 26) The design of the new CRAd has advantages for its use as an oncolytic virus compared to previous versions, since several elements that determine its efficacy and specificity have been optimized: 1.- The use of a promoter that responds to hypoxia to control the ElA gene allows that the replication and cytopathic effect of the virus be stimulated under conditions of low oxygen tension. These conditions are present in solid tumors, and it has been described that they can decrease the activity of different oncolytic adenoviruses regulated by other mechanisms.
(Pipiya T. et al., Gene Therapy (2005) 12:911-917; Shen(Pipiya T. et al., Gene Therapy (2005) 12: 911-917; Shen
BH and Hermiston T.W., Gene Therapy (2005) 12:902-910).BH and Hermiston T.W., Gene Therapy (2005) 12: 902-910).
2.- El uso combinado de la deleción Delta24 en el gen ElA con el control transcripcional del gen ElA y la región E4 consigue una mayor atenuación del virus en células normales, sin afectar negativamente a su capacidad de matar las células tumorales. Este concepto ha sido demostrado para el virus ONYX-411, en el que la deleción del dominio CR2 de ElA se combinó con el control transcripcional de las regiones ElA y E4 por el promotor2.- The combined use of the Delta24 deletion in the ElA gene with the transcriptional control of the ElA gene and the E4 region achieves greater attenuation of the virus in normal cells, without negatively affecting its ability to kill tumor cells. This concept has been demonstrated for the ONYX-411 virus, in which the deletion of the El2 CR2 domain was combined with the transcriptional control of the ElA and E4 regions by the promoter
E2F-1 (Johnson et al., Cáncer CeIl (2002), 1: 325-337).E2F-1 (Johnson et al., CeIl Cancer (2002), 1: 325-337).
Es importante señalar que una ventaja del adenovirus recombinante de la invención frente a ONYX-411 es el control de . las regiones ElA y E4 por promotores diferentes, y concretamente el uso del promotor E2F-1 en la región E4. Esto permite un doble mecanismo de control de la replicación, mientras que ONYX-411 depende exclusivamente de la ruta de pRB en las células . Por otra parte, evitando la repetición de secuencias se reduce laIt is important to note that an advantage of the recombinant adenovirus of the invention over ONYX-411 is the control of . the ElA and E4 regions by different promoters, and specifically the use of the E2F-1 promoter in the E4 region. This allows a double replication control mechanism, while ONYX-411 depends exclusively on the pRB path in the cells. On the other hand, avoiding the repetition of sequences reduces the
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posibilidad de recombinación, aumentando la estabilidad del genoma viral.SUBSTITUTE SHEET (RULE 26) possibility of recombination, increasing the stability of the viral genome.
3.- La posibilidad de introducir genes terapéuticos en la estructura del virus incrementa su eficacia porque al efecto del gen exógeno se une la capacidad oncolitica propia del adenovirus. En el caso de los genes reporteros, se hace posible la visualización de las células infectadas a lo largo del tiempo.3.- The possibility of introducing therapeutic genes into the structure of the virus increases its effectiveness because the oncolytic ability of adenovirus is linked to the exogenous gene effect. In the case of reporter genes, the visualization of infected cells over time is made possible.
Ventajas de los genes terapéuticos.Advantages of therapeutic genes.
1. Interleuquina 12 (IL-12) . Esta citoquina tiene diversos mecanismos de acción que contribuyen a su efecto antitumoral. Por una parte, estimula la reacción del sistema inmunológico frente a los tumores mediante la producción de otras citoquinas y la activación de células efectoras . Por otra parte, tiene un efecto antiangiogénico, que dificulta la vascularización de los tumores e impide su crecimiento . Estas acciones hacen que la expresión de IL-12 en el contexto del CRAd anteriormente descrito, presente importantes ventajas.1. Interleukin 12 (IL-12). This cytokine has several mechanisms of action that contribute to its antitumor effect. On the one hand, it stimulates the reaction of the immune system against tumors through the production of other cytokines and the activation of effector cells. On the other hand, it has an antiangiogenic effect, which hinders the vascularization of tumors and prevents their growth. These actions make the expression of IL-12 in the context of the CRAd described above present important advantages.
En primer lugar, puede haber una sinergia en la estimulación del sistema inmune frente al tumor, ya que las células tumorales serán infectadas por un agente infeccioso replicativo. Este hecho es de por si un evento inmunoestimulador, que se refuerza por la expresión local de IL-12.First, there may be a synergy in the stimulation of the immune system against the tumor, since the tumor cells will be infected by a replicative infectious agent. This fact is in itself an immunostimulatory event, which is reinforced by the local expression of IL-12.
En segundo lugar, el efecto antiangiogénico de la IL-12 dificulta la oxigenación del tumor ySecond, the anti-angiogenic effect of IL-12 hinders tumor oxygenation and
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provoca hipoxia, lo cual estimula la replicación del CRAd. 2. Timidina kinasa del Herpes virus tipo I (HSV-TK) .SUBSTITUTE SHEET (RULE 26) causes hypoxia, which stimulates the replication of CRAd. 2. Thymidine kinase from Herpes virus type I (HSV-TK).
Esta enzima tiene como función convertir un pro- fármaco relativamente inocuo (ganciclovir) en un potente agente citotóxico. Las células que expresan HSV-TK, en presencia de ganciclovir administrado exógenamente, mueren y provocan la muerte de las células que se encuentran en su proximidad. Dado que la administración de ganciclovir puede realizarse dias después de administrar el CRAd, esto puede conseguir un aumento de la destrucción de células tumorales una vez el virus se ha amplificado, y asi aumentar su capacidad oncolitica. Al mismo tiempo, esto detiene la replicación viral, debido a la muerte de las células infectadas, y puede considerarse un mecanismo de seguridad en el caso de observarse replicación en tejidos sanos.This enzyme has the function of converting a relatively harmless drug (ganciclovir) into a potent cytotoxic agent. The cells that express HSV-TK, in the presence of exogenously administered ganciclovir, die and cause the death of the cells that are in their vicinity. Since the administration of ganciclovir can be done days after administering the CRAd, this can achieve an increase in the destruction of tumor cells once the virus has been amplified, and thus increase its oncolytic capacity. At the same time, this stops viral replication, due to the death of infected cells, and can be considered a safety mechanism in the case of replication in healthy tissues.
Además, HSV-TK puede utilizarse como gen reportero en humanos mediante la técnica deIn addition, HSV-TK can be used as a reporter gene in humans using the technique of
Tomografia de Emisión de Positrones (PET) . De este modo, permite visualizar las células infectadas y determinar la diseminación del virus.Positron Emission Tomography (PET). In this way, it allows visualizing infected cells and determining the spread of the virus.
Localización de los genes exógenos .Location of exogenous genes.
El genoma del CRAd ha sido modificado de modo que pueden incluirse genes exógenos en la región E3- Estos genes ocupan el lugar de los genes virales que codifican para las proteínas gpl9k y β.7k. (Hawkins y cois., Gene Therapy (2001) 8, 1123-1131) . La deleción de estos genesThe CRAd genome has been modified so that exogenous genes can be included in the E3 region. These genes take the place of the viral genes that code for the gpl9k and β.7k proteins. (Hawkins et al., Gene Therapy (2001) 8, 1123-1131). The deletion of these genes
(nucleótidos 28.555 a 29.355 en la secuencia del(nucleotides 28,555 to 29,355 in the sequence of
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adenovirus) no afecta a la replicación del virus en células tumorales, ya que las proteínas gpl9k y β.7k tienen como función inhibir la respuesta inmune frente a las células infectadas. Dado que las células tumorales tienen mecanismos propios para inhibir su reconocimiento por el sistema inmune, estas deleciones pueden favorecer la eliminación del virus en las células normales, pero no en las tumorales, y por tanto contribuyen a la especificidad de la replicación viral. La inclusión de los genes exógenos en esta localización tiene ventajas adicionales. Por un lado, se utiliza el promotor endógeno y las secuencias de poli- adenilación del virus, con lo cual no se necesita incluir estas secuencias exógenamente . Con ello se ahorra espacio en el genoma viral y se pueden incluir genes más grandes. Por otro lado, se ha demostrado que el promotor endógeno que regula gpl9k/6.7k se activa en la fase tardia del ciclo viral, con lo cual la replicación del CRAd y la expresión del gen exógeno se regulan de la misma manera. Por último, se ha demostrado que los niveles de expresión superan los obtenidos con los promotores habituales que se utilizan en terapia génica.SUBSTITUTE SHEET (RULE 26) adenovirus) does not affect the replication of the virus in tumor cells, since the gpl9k and β.7k proteins have the function of inhibiting the immune response against infected cells. Since tumor cells have their own mechanisms to inhibit their recognition by the immune system, these deletions can favor the elimination of the virus in normal cells, but not in tumor cells, and therefore contribute to the specificity of viral replication. The inclusion of exogenous genes in this location has additional advantages. On the one hand, the endogenous promoter and polyphenylation sequences of the virus are used, so it is not necessary to include these sequences exogenously. This saves space in the viral genome and larger genes can be included. On the other hand, it has been shown that the endogenous promoter that regulates gpl9k / 6.7k is activated in the late phase of the viral cycle, whereby replication of the CRAd and expression of the exogenous gene are regulated in the same way. Finally, it has been shown that expression levels exceed those obtained with the usual promoters used in gene therapy.
Las construcciones de DNA para la preparación del adenovirus recombinante de la invención pueden obtenerse mediante métodos convencionales de biologia molecular, muchos de ellos recogidos en manuales generales de laboratorio (por ejemplo, "Molecular Cloning: aDNA constructs for the preparation of the recombinant adenovirus of the invention can be obtained by conventional methods of molecular biology, many of them collected in general laboratory manuals (for example, "Molecular Cloning: a
Laboratory manual". Joseph Sambrook, David W. Russel Eds . 2001, 3a ed. CoId Spring Harbor, Nueva York) . Pueden consultarse también en otros manuales o referenciasLaboratory manual ". Joseph Sambrook, David W. Russel Eds. 2001, 3rd ed. Cold Spring Harbor, New York). Also users can check in other manuals or references
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orientados específicamente a la preparación de adenovirus , por ejemplo:SUBSTITUTE SHEET (RULE 26) specifically oriented to the preparation of adenovirus, for example:
"Adenovirus Methods and Protocols. Methods in Molecular Medicine". WoId, William S. M. Ed. 1998, Humana Press;"Adenovirus Methods and Protocols. Methods in Molecular Medicine". WoId, William S. M. Ed. 1998, Humana Press;
Mizuguchi H. y Kay M. A. "Efficient construction of a recombinant adenovirus vector by an improved in vitro ligation method"; Hum Gene Ther., 1998; 9: 2577-2583; yMizuguchi H. and Kay M. A. "Efficient construction of a recombinant adenovirus vector by an improved in vitro ligation method"; Hum Gene Ther., 1998; 9: 2577-2583; Y
He T. C y cois. "A simplified system for generating recombinant adenoviruses"; Proc . Nati. Acad. Sci . USA, 1998; 95:2509-2514.I have T. C and cois. "A simplified system for generating recombinant adenoviruses"; Proc. Nati Acad. Sci. USA, 1998; 95: 2509-2514.
Para la propagación del adenovirus recombinante de la invención puede utilizarse cualquier linea celular que sea permisiva para la replicación y formación de viriones recombinantes selectivamente replicativos del adenovirus recombinante de la invención (linea celular empaquetadora) . Podria utilizarse casi cualquier linea celular humana tumoral convencional. Pueden utilizarse entre otras las lineas HEK293 (derivada de células embrionarias), PER.Cβ (derivada de retinoblastos embrionarios), A549 (derivada de cáncer de pulmón humano), HeLa (p.ej. HeLaS3; ATCC Cat.No. CCL-2.2; Yuk et al. "Perfusión cultures of human tumor cells: a scalable production platform for oncolytic adenoviral vectors". Biotechnology and Bioengineering, 2004; 86:637-642).Any cell line that is permissive for the replication and formation of selectively replicative recombinant virions of the recombinant adenovirus of the invention (packaging cell line) can be used for the propagation of the recombinant adenovirus of the invention. Almost any conventional human tumor cell line could be used. Among others, lines HEK293 (derived from embryonic cells), PER.Cβ (derived from embryonic retinoblasts), A549 (derived from human lung cancer), HeLa (eg HeLaS3; ATCC Cat.No. CCL-2.2) ; Yuk et al. "Perfusion cultures of human tumor cells: a scalable production platform for oncolytic adenoviral vectors". Biotechnology and Bioengineering, 2004; 86: 637-642).
Para la propagación eficiente del adenovirus recombinante de la invención es necesario también que las células empaquetadoras transfectadas con el genoma del adenovirus tengan aumentada la expresión y actividad HIF, excepto las células HEK293 o PER.C6, que aportan constitutivamente los genes virales El. A tal efecto, lasFor efficient propagation of the recombinant adenovirus of the invention it is also necessary that the packaging cells transfected with the adenovirus genome have increased HIF expression and activity, except for HEK293 or PER.C6 cells, which constitutively contribute the El viral genes. effect the
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células pueden ser sometidas a un tratamiento inductor de la expresión de HIF, como el cultivo en condiciones de hipoxia (p. ej . en una cámara hipóxica ajustada a una composición gaseosa 93% N2/β% CÜ2/1% O2) , cultivo en presencia de cloruro de cobalto (p.ej. lOOμM) , o cualquier otro tratamiento mimético de condiciones de hipoxia que sea inductor de HIF.SUBSTITUTE SHEET (RULE 26) cells can be subjected to an inducing treatment of HIF expression, such as culture under hypoxia conditions (eg in a hypoxic chamber adjusted to a gaseous composition 93% N 2 / β% CÜ 2 /1% O 2 ) , culture in the presence of cobalt chloride (eg 10OOM), or any other mimetic treatment of hypoxia conditions that is an inducer of HIF.
Asi, en un aspecto concreto, la presente invención se refiere a una célula hospedadora que comprende un adenovirus recombinante previamente descrito y objeto de la presente invención. Por otro lado, la invención también se refiere a un procedimiento para la propagación in vitro de dicho adenovirus objeto de la presente invención que comprende cultivar una célula hospedadora que contenga un adenovirus recombinante de la invención, en condiciones que permitan la expresión de dicho adenovirus. Las condiciones para optimizar el cultivo de la célula hospedadora dependerán del tipo de célula hospedadora empleado. Si se desea, el método para producir el adenovirus recombinante de la invención incluirá el aislamiento y purificación del mismo.Thus, in a particular aspect, the present invention relates to a host cell comprising a recombinant adenovirus previously described and object of the present invention. On the other hand, the invention also relates to a process for the in vitro propagation of said adenovirus object of the present invention which comprises culturing a host cell containing a recombinant adenovirus of the invention, under conditions that allow the expression of said adenovirus. The conditions for optimizing the culture of the host cell will depend on the type of host cell used. If desired, the method of producing the recombinant adenovirus of the invention will include isolation and purification thereof.
Un objeto adicional de la invención se refiere a una composición farmacéutica que comprende un adenovirus recombinante de la invención y un excipiente farmacéuticamente aceptable. La composición farmacéutica de la invención puede formularse con una variedad de excipientes convencionales que mejoran la estabilidad del adenovirus durante los procesos de fabricación, manipulación, almacenaje y distribución del producto, o que sean apropiadas para su administración terapéutica.A further object of the invention relates to a pharmaceutical composition comprising a recombinant adenovirus of the invention and a pharmaceutically acceptable excipient. The pharmaceutical composition of the invention can be formulated with a variety of conventional excipients that improve adenovirus stability during the manufacturing, handling, storage and distribution processes of the product, or that are appropriate for therapeutic administration.
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Pueden utilizarse por ejemplo, azúcares crio- o lio- protectores, polioles, surfactantes, aminoácidos, polímeros, tampones y sales para ajustar el pH de la composición, antioxidantes y otros agentes quelantes, asi como bacteriostáticos y bactericidas (Parkins et al. "The formulation of biopharmaceutical products"; Pharmaceutical Science and Technology Today, 2000; 3:129-137). Podria utilizarse también una suspensión estéril acuosa o no acuosa, que puede contener agentes suspensores o agentes espesantes. Excipientes y formulaciones concretas útiles para la preparación de la composición farmacéutica de la invención pueden encontrarse por ejemplo en Journal of Pharmaceutical Sciences (Evans R. K. et al. "Development of stable liquid formulations for adenovirus-based vaccines"; 2004, 93:2458-2475), Gene Therapy (Croyle M.A. et al. "Development of formulations that enhance physical stability of viral vectors for gene therapy"; 2001, 8:1281-1290), J. Virol . Methods (Hosokawa M. et al. "Preparation of purified, sterilized, and stable adenovirus vectors using albumin"; 2002; 103:191-199), WO98/02522, WO00/34444, WO00/61726, WO03/049763, US6544769, US20020031527 , entre otros.SUBSTITUTE SHEET (RULE 26) For example, cryoprotectant sugars, polyols, surfactants, amino acids, polymers, buffers and salts can be used to adjust the pH of the composition, antioxidants and other chelating agents, as well as bacteriostatics and bactericides (Parkins et al. "The formulation of biopharmaceutical products "; Pharmaceutical Science and Technology Today, 2000; 3: 129-137). A sterile aqueous or non-aqueous suspension could also be used, which may contain suspending agents or thickening agents. Concrete excipients and formulations useful for the preparation of the pharmaceutical composition of the invention can be found for example in the Journal of Pharmaceutical Sciences (Evans RK et al. "Development of stable liquid formulations for adenovirus-based vaccines"; 2004, 93: 2458-2475 ), Gene Therapy (Croyle MA et al. "Development of formulations that enhance physical stability of viral vectors for gene therapy"; 2001, 8: 1281-1290), J. Virol. Methods (Hosokawa M. et al. "Preparation of purified, sterilized, and stable adenovirus vectors using albumin";2002; 103: 191-199), WO98 / 02522, WO00 / 34444, WO00 / 61726, WO03 / 049763, US6544769, US20020031527, among others.
En una realización particular la composición farmacéutica contendrá de 103 a 1015 o más partículas adenovirales en solución acuosa.In a particular embodiment the pharmaceutical composition will contain from 10 3 to 10 15 or more adenoviral particles in aqueous solution.
BREVE DESCRIPCIÓN DE LAS FIGURASBRIEF DESCRIPTION OF THE FIGURES
Figura 1. Representación esquemática del plásmido genérico para la producción de CRAds . La secuencia deFigure 1. Schematic representation of the generic plasmid for the production of CRAds. The sequence of
Adenovirus tipo 5 se encuentra en forma de plásmido conType 5 adenovirus is in the form of a plasmid with
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resistencia a kanamicina, y puede liberarse mediante digestión con el enzima de restricción Pací. ElAp, promotor de ElA, que se encuentra flanqueado por secuencias de reconocimiento para BstBI . E4p, promotor de la región E4, flanqueado por secuencias para SwaI ySUBSTITUTE SHEET (RULE 26) Kanamycin resistance, and can be released by digestion with the restriction enzyme Pací. ElAp, ElA promoter, which is flanked by recognition sequences for BstBI. E4p, promoter of the E4 region, flanked by sequences for SwaI and
I-Ceul. ElA D24, deleción del dominio CR2 de ElAI-Ceul. ElA D24, deletion of the CR2 domain of ElA
(opcional). E3Dgpl9k/β .7k, deleción parcial de la región(optional). E3Dgpl9k / β .7k, partial deletion of the region
E3 para la inserción de genes exógenos. LP, promotor tardio del virus. Figura 2. Representación esquemática de los virus AdHLuc, AdDHLuc, AdDHTK, AdDHIL-12 y AdWTLuc . ITR, Inverted Terminal Repeat; HRE,' Hypoxia Response Element; E2F-lp, promotor del factor de transcripción E2F-1. D24, deleción del dominio CR2 de ElA. Figura 3. Efecto citopático de AdHLuc, AdDHLuc y AdWT en células de hepatocarcinoma humano Huh-7 (figura A) y en fibroblastos normales IMR-90 (figura B) . Se representa la supervivencia relativa respecto a células no infectadas cultivadas en las mismas condiciones, comparando la supervivencia cuando las células se mantuvieron en condiciones de hipoxia frente a la observada en condiciones de normoxia. Las células Huh-7 se infectaron con 5 virus/célula, y los fibroblastos IMR- 90 con 135 virus/célula para compensar su menor infectividad por adenovirus. El efecto citopático se evaluó a los 5 días después de la infección para células Huh-7 y a los 10 días para los fibroblastos IMR-90. El asterisco indica diferencias significativas (p<0.05)E3 for the insertion of exogenous genes. LP, late promoter of the virus. Figure 2. Schematic representation of the AdHLuc, AdDHLuc, AdDHTK, AdDHIL-12 and AdWTLuc viruses. ITR, Inverted Terminal Repeat; HRE, ' Hypoxia Response Element; E2F-lp, promoter of the transcription factor E2F-1. D24, deletion of the CR2 domain of ElA. Figure 3. Cytopathic effect of AdHLuc, AdDHLuc and AdWT in Huh-7 human hepatocarcinoma cells (Figure A) and in normal IMR-90 fibroblasts (Figure B). Relative survival is represented with respect to uninfected cells cultured under the same conditions, comparing survival when the cells were maintained in hypoxia conditions compared to that observed in normoxia conditions. Huh-7 cells were infected with 5 viruses / cell, and IMR-90 fibroblasts with 135 viruses / cell to compensate for their lower adenovirus infectivity. The cytopathic effect was evaluated at 5 days after infection for Huh-7 cells and at 10 days for IMR-90 fibroblasts. The asterisk indicates significant differences (p <0.05)
Figura 4. Efecto citopático de los virus AdHLuc, AdDHLuc y AdWT sobre células WI-38 (infectadas conFigure 4. Cytopathic effect of AdHLuc, AdDHLuc and AdWT viruses on WI-38 cells (infected with
250 virus/célula) y WI38-VA13 (125 virus/célula) , cuando250 virus / cell) and WI38-VA13 (125 virus / cell), when
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las células se cultivaron en condiciones de normoxia o hipoxia. Los cultivos fueron fotografiados (20Ox) a los 5 dias desde la infección. Las células que sufren efecto citopático por replicación virica se distinguen por pérdida de adherencia, con aumento de la refringencia y morfologia redondeada.SUBSTITUTE SHEET (RULE 26) The cells were grown under normoxia or hypoxia. The cultures were photographed (20Ox) 5 days after infection. Cells that undergo cytopathic effect by viric replication are distinguished by loss of adhesion, with increased refringence and rounded morphology.
Figura 5. Efecto citopático de los virus AdHLuc, AdDHLuc y AdWT sobre células BJ (infectadas con 500 virus/célula) e IMR-90 (125 virus/célula) , para células mantenidas en condiciones de normoxia o hipoxia. Fotografiados (20Ox) también a los 5 dias. Las células que sufren efecto citopático por replicación virica se distinguen por pérdida de adherencia, con aumento de la refringencia y morfologia redondeada. Figura 6. Ensayo de viabilidad celular en células Huh-7 e IMR-90 infectadas con virus AdDHLucFigure 5. Cytopathic effect of the AdHLuc, AdDHLuc and AdWT viruses on BJ cells (infected with 500 viruses / cell) and IMR-90 (125 viruses / cell), for cells maintained under normoxia or hypoxia conditions. Photographed (20Ox) also at 5 days. Cells that undergo cytopathic effect by viric replication are distinguished by loss of adhesion, with increased refringence and rounded morphology. Figure 6. Cell viability assay in Huh-7 and IMR-90 cells infected with AdDHLuc virus
(triángulos) o AdHLuc (cuadrados), a diferentes dosis virales (MOI, "multiplicidad de infección", número de virus por cada célula) . Los cultivos se mantuvieron en condiciones de hipoxia (linea discontinua) o normoxia(triangles) or AdHLuc (squares), at different viral doses (MOI, "multiplicity of infection", number of viruses per cell). The cultures were maintained in hypoxic conditions (dashed line) or normoxia.
(linea continua) . La supervivencia se cuantificó por tinción con cristal violeta a los 6 dias (Huh-7) o los 8 dias (IMR-90) desde la infección con el virus a ensayar.(continuous line) . Survival was quantified by staining with violet crystal at 6 days (Huh-7) or 8 days (IMR-90) from infection with the virus to be tested.
El asterisco indica diferencias significativas (p<0.05) Figura 7. Control de la replicación por hipoxia.The asterisk indicates significant differences (p <0.05) Figure 7. Control of replication by hypoxia.
Comparación gráfica de la cantidad de partículas virales infectivas (i.u./ml) producidas en células A549 infectadas con una MOI de 1 virus/célula de los virusGraphical comparison of the amount of infective viral particles (i.u./ml) produced in A549 cells infected with an MOI of 1 virus / virus cell
AdDHLuc o AdWT, cuando se mantuvieron en condiciones de hipoxia o normoxia durante 4 dias. i.u., unidadesAdDHLuc or AdWT, when they remained in hypoxia or normoxia conditions for 4 days. units
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infectivas. El asterisco indica diferencias significativas (p<0.05).SUBSTITUTE SHEET (RULE 26) infective The asterisk indicates significant differences (p <0.05).
Figura 8. Comparación gráfica de la expresión in vitro de luciferasa (representada como RLU/μg de proteina total; RLU, relative luciferasa units) en células Huh-7 infectadas por los virus AdDHLuc, AdHLuc,Figure 8. Graphical comparison of in vitro expression of luciferase (represented as RLU / μg of total protein; RLU, relative luciferase units) in Huh-7 cells infected by the AdDHLuc, AdHLuc viruses,
AdWTLuc o AdCMVLuc cuando se mantuvieron en condiciones de hipoxia o normoxia. Infección: MOI de 10 virus/célula.AdWTLuc or AdCMVLuc when they were kept in hypoxia or normoxia conditions. Infection: MOI of 10 viruses / cell.
El asterisco indica diferencias significativas (p<0.05). Figura 9. Citotoxicidad del virus AdDHTK en respuesta a hipoxia y tratamiento con GCV. Comparación gráfica de la viabilidad de células A549 infectadas con una MOI de 0.12 virus/célula en condiciones de normoxia e hipoxia, tras 5 dias de incubación con un medio de cultivo normal o suplementado con 100 μM GCV. El asterisco indica diferencias significativas (p<0.05).The asterisk indicates significant differences (p <0.05). Figure 9. Cytotoxicity of the AdDHTK virus in response to hypoxia and GCV treatment. Graphic comparison of the viability of A549 cells infected with an MOI of 0.12 virus / cell under normoxia and hypoxia conditions, after 5 days of incubation with a normal culture medium or supplemented with 100 μM GCV. The asterisk indicates significant differences (p <0.05).
Figura 10. Control de la expresión de IL12 en el virus AdDHIL12. Comparación gráfica del incremento de producción de IL12 medida en el sobrenadante de células HeLa infectadas con el virus AdDHIL12 en condiciones de normoxia o hipoxia. El asterisco indica diferencias significativas (p<0.05).Figure 10. Control of the expression of IL12 in the AdDHIL12 virus. Graphical comparison of the increase in IL12 production measured in the supernatant of HeLa cells infected with the AdDHIL12 virus under conditions of normoxia or hypoxia. The asterisk indicates significant differences (p <0.05).
Figura 11. Expresión in vivo de luciferasa en ratones atimicos con xenoinjertos de tumores humanos (células Huh-7), tras infección (mediante inyección intratumoral) con los virus AdDHLuc, AdDHWT y Ad-CMV-Luc.Figure 11. In vivo expression of luciferase in athymic mice with human tumor xenografts (Huh-7 cells), after infection (by intratumoral injection) with the AdDHLuc, AdDHWT and Ad-CMV-Luc viruses.
A: Actividad luciferasa en tumores subcutáneos a distintos tiempos tras la infección con 2xlO8 i.u. del virus a ensayar. B: Incremento de la actividad luciferasa respecto al dia 1 en los mismos ratones. C: Tumores inducidos por inyección intrahepática de células Huh-7;A: Luciferase activity in subcutaneous tumors at different times after infection with 2xlO 8 iu of the virus to be tested. B: Increase in luciferase activity compared to day 1 in the same mice. C: Tumors induced by intrahepatic injection of Huh-7 cells;
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Infección con 109 i.u. del virus AdDHLuc. La emisión de luz (expresada en fotones/segundo) se cuantificó a lo largo del tiempo mediante una cámara luminométrica . El asterisco indica diferencias significativas (p<0.05).SUBSTITUTE SHEET (RULE 26) Infection with 10 9 iu of the AdDHLuc virus. The light emission (expressed in photons / second) was quantified over time by means of a luminometric chamber. The asterisk indicates significant differences (p <0.05).
MODO DE REALIZACIÓN DE LA INVENCIÓNEMBODIMENT OF THE INVENTION
Los siguientes ejemplos pretenden ilustrar, en modo alguno limitativo, la realización de la invención objeto de la presente solicitud de patente.The following examples are intended to illustrate, in no way limitative, the embodiment of the invention object of the present patent application.
Ejemplo 1. Construcción y propagación de los adenovirus recombinantesExample 1. Construction and propagation of recombinant adenoviruses
Conforme a las especificaciones de la invención, se construyeron 3 adenovirus recombinantes, cada uno de ellos con un transgen diferente:In accordance with the specifications of the invention, 3 recombinant adenoviruses were constructed, each with a different transgene:
- AdDHLuc, que lleva incorporado el gen de la luciferasa (reportero) ;- AdDHLuc, which incorporates the luciferase gene (reporter);
- AdDHTK, que incorpora el gen de la timidin quinasa del virus Herpes simplex (HSV-TK, gen suicida) ; y - AdDHIL-12, al que se le ha incorporado el gen de la interleucina-12 (IL-12, gen terapéutico).- AdDHTK, which incorporates the Herpes simplex virus thymidine kinase gene (HSV-TK, suicide gene); and - AdDHIL-12, to which the interleukin-12 gene (IL-12, therapeutic gene) has been incorporated.
En los 3 adenovirus: i) se ha sustituido el promotor del gen ElA por un promotor sintético (SEQ. ID. NO: 1) que responde a hipoxia; ii) se ha sustituido el promotor de la región E4 por el promotor del factor de transcripción E2F-1 (SEQ. ID. NO: 2); iii) se ha realizado una deleción (deleción Delta24) en el dominio CR2 del gen ElA; y iv) se han introducido los genes de interés en sustitución de los genes gpl9k/β.7k de la región E3.In the 3 adenoviruses: i) the ElA gene promoter has been replaced by a synthetic promoter (SEQ. ID. NO: 1) that responds to hypoxia; ii) the E4 region promoter has been replaced by the E2F-1 transcription factor promoter (SEQ. ID. NO: 2); iii) a deletion (Delta24 deletion) has been made in the CR2 domain of the ElA gene; and iv) the genes of interest have been introduced to replace the gpl9k / β.7k genes of the E3 region.
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Además, se prepararon y utilizaron otros adenovirus recombinantes que han sido utilizados como controles en los experimentos:SUBSTITUTE SHEET (RULE 26) In addition, other recombinant adenoviruses that have been used as controls in the experiments were prepared and used:
- AdWT, adenovirus silvestre tipo 5 (ATCC VR-5); - AdWTLuc, que difiere de AdWT en la inclusión del gen de luciferasa en sustitución de los genes gpl9k/β.7k de la región E3;- AdWT, wild type 5 adenovirus (ATCC VR-5); - AdWTLuc, which differs from AdWT in the inclusion of the luciferase gene in place of the gpl9k / β.7k genes of the E3 region;
- Ad-CMV-Luc, un adenovirus defectivo que expresa el gen de la luciferasa bajo el control del promotor CMV (Vector Biolabs, Philadelphia, Ref. 1000); y- Ad-CMV-Luc, a defective adenovirus that expresses the luciferase gene under the control of the CMV promoter (Vector Biolabs, Philadelphia, Ref. 1000); Y
- AdHLuc, que difiere de AdDHLuc porque no ha sido delecionado (deleción Delta24) en el dominio CR2 del gen ElA.- AdHLuc, which differs from AdDHLuc because it has not been deleted (Delta24 deletion) in the CR2 domain of the ElA gene.
Construcción de los plásmidosPlasmid Construction
Para la construcción de los adenovirus recombinantes utilizados en los ejemplos se utilizó como reactivo inicial el plásmido, pSEHE2F. Este plásmido está basado en el genoma del adenovirus tipo 5, modificado para facilitar la sustitución de los promotores ElA y E4 por promotores específicos de tumores (Hernández Alcoceba R. et al. Human Gene Therapy (2002) 13: 1737-1750) . Sobre el genoma adenoviral se han introducido secuencias de reconocimiento para enzimas de restricción en regiones concretas y promotores exógenos que pueden ser sustituidos con facilidad:For the construction of the recombinant adenoviruses used in the examples, the plasmid, pSEHE2F, was used as the initial reagent. This plasmid is based on the adenovirus type 5 genome, modified to facilitate the replacement of the ElA and E4 promoters with tumor-specific promoters (Hernández Alcoceba R. et al. Human Gene Therapy (2002) 13: 1737-1750). On the adenoviral genome, recognition sequences have been introduced for restriction enzymes in specific regions and exogenous promoters that can be easily substituted:
- región del promotor de ElA (ElAp) flanqueada por secuencias de reconocimiento para el enzima BstBI (pSEHE2F contiene en esta región el promotor artificial 5XEH3, que será sustituido por el promotor 9XHRE en los virus objeto de la invención);- ElA promoter region (ElAp) flanked by recognition sequences for the BstBI enzyme (pSEHE2F contains in this region the artificial promoter 5XEH3, which will be replaced by the 9XHRE promoter in the viruses object of the invention);
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- Región del promotor de E4 (E4p) flanqueada por secuencias de reconocimiento para los enzimas I-Ceul y SwaI (pSEHE2F contiene en esta región el promotor para el factor E2F-1, que se mantendrá en los virus objeto de la invención) . Este último promotor se obtuvo mediante Reacción en Cadena de la Polimerasa (PCR) a partir de ADN genómico humano utilizando los siguientes oligonucleótidos : SEQ ID NO 3: 5' TACTGTAACTATAACGGTCCTAAGGTAGCGTGGTACCATCCGGACAAAGCC-S' y, SEQ ID NO 4: 5' TAAGTATTTAAATGGCGAGGGCTCGATCCCGC-3' .SUBSTITUTE SHEET (RULE 26) - E4 promoter region (E4p) flanked by recognition sequences for the I-Ceul and SwaI enzymes (pSEHE2F contains in this region the promoter for factor E2F-1, which will be maintained in the viruses object of the invention). This last promoter was obtained by Polymerase Chain Reaction (PCR) from human genomic DNA using the following oligonucleotides: SEQ ID NO 3: 5 'TACTGTAACTATAACGGTCCTAAGGTAGCGTGGTACCATCCGGACAAAGCC-S' and, SEQ ID NO 4: 5 'TAAGTATTTAGG .
Una secuencia aislante (denominada aqui "Ins") está presente en pSEHE2F entre la secuencia de empaquetamiento del adenovirus y la región del promotor ElA. Esta secuencia (secuencia de parada de la transcripción del gen de la hormona de crecimiento bovino) se obtuvo a partir del plásmido pCDNA3 (Invitrogen) mediante digestión con los enzimas PvuII y Notl. El plásmido pSHE2F se modificó en sucesivas etapas para obtener los plásmidos necesarios para producir los virus de la invención y sus controles : .- Introducción del promotor 9XHRE en la región ElA.An insulating sequence (referred to herein as "Ins") is present in pSEHE2F between the adenovirus packaging sequence and the ElA promoter region. This sequence (transcription stop sequence of the bovine growth hormone gene) was obtained from plasmid pCDNA3 (Invitrogen) by digestion with the enzymes PvuII and Notl. Plasmid pSHE2F was modified in successive stages to obtain the plasmids necessary to produce the viruses of the invention and their controls: .- Introduction of the 9XHRE promoter in the ElA region.
El plásmido PSEHE2F fue digerido con BstBI, y se sustituyó el promotor 5XEH3 por el promotor 9XHRE . Para introducir los sitios de restricción BstBI a ambos lados del promotor, se utilizó un adaptador consistente en el siguiente par de oligonucleótidos:Plasmid PSEHE2F was digested with BstBI, and the 5XEH3 promoter was replaced by the 9XHRE promoter. To introduce the BstBI restriction sites on both sides of the promoter, an adapter consisting of the following pair of oligonucleotides was used:
SEQ ID NO 5: 5Bstbam: 5' CGAAGATCTGACTCCAAG 3' y SEQ ID NO 6: 3Bstbam: 5' GATCCTTGGAGTCAGATCTT 3' .SEQ ID NO 5: 5Bstbam: 5 'CGAAGATCTGACTCCAAG 3' and SEQ ID NO 6: 3Bstbam: 5 'GATCCTTGGAGTCAGATCTT 3'.
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La orientación correcta del promotor se verificó mediante digestión con el enzima de restricción BamHI y secuenciación utilizando los siguientes oligonucleótidos : SEQ ID NO 7: 5Ad5St: 5' TAGTGTGGCGGAAGTGTGATGTTG 3' y SEQ ID NO 8: 3Ad5St: 5' TCTTCGGTAATAACACCTCCGTGG 3'.SUBSTITUTE SHEET (RULE 26) The correct orientation of the promoter was verified by digestion with the restriction enzyme BamHI and sequencing using the following oligonucleotides: SEQ ID NO 7: 5Ad5St: 5 'TAGTGTGGCGGAAGTGTGATGTTG 3' and SEQ ID NO 8: 3Ad5St: 5 'TCTTCGGTAATAACACCTCCG.
Este nuevo plásmido se llamó pSHIFE2F. .- Deleción parcial de la región E3.This new plasmid was called pSHIFE2F. .- Partial deletion of the E3 region.
El plásmido pSHIFE2F se modificó para delecionar los genes que codifican para gpl9k/6.7k (Dgpl9k/β .7K) y flanquear esta zona con secuencias de reconocimiento para el enzima Pl-Scel, que permitirán la incorporación de genes exógenos . Para obtener la deleción se llevaron a cabo sucesivas etapas de subclonaje. En primer lugar se obtuvo un fragmento de 4.7 kb mediante digestión del plásmido pSHIFE2F con el enzima AgeI, y se subclonó en el sitio Agel del plásmido comercial pMIB/V5-HisCPlasmid pSHIFE2F was modified to delete the genes encoding gpl9k / 6.7k (Dgpl9k / β .7K) and flanking this area with recognition sequences for the Pl-Scel enzyme, which will allow the incorporation of exogenous genes. To obtain the deletion, successive stages of subcloning were carried out. First, a 4.7 kb fragment was obtained by digestion of plasmid pSHIFE2F with the AgeI enzyme, and subcloned into the Agel site of commercial plasmid pMIB / V5-HisC
(Invitrogen) . Este nuevo plásmido (llamado pMIB-AgeC) se digirió simultáneamente con los enzimas Spel y Xbal y se introdujo en esta posición un fragmento obtenido mediante PCR en el que se ha introducido un sitio de restricción para el enzima Xmnl en la posición 28555 relativa al genoma del adenovirus tipo 5. Este fragmento de PCR se obtuvo con el siguiente par de oligonucleótidos:(Invitrogen). This new plasmid (called pMIB-AgeC) was digested simultaneously with the Spel and Xbal enzymes and a fragment obtained by PCR was introduced in this position into which a restriction site for the Xmnl enzyme has been introduced at position 28555 relative to the genome of adenovirus type 5. This PCR fragment was obtained with the following pair of oligonucleotides:
SEQ ID NO 9: 5Spel: 5' AAGGACTAGTTTCGCGCCCTTTCTCAAATTTAAGC 3', y SEQ ID NO 10:SEQ ID NO 9: 5Spel: 5 'AAGGACTAGTTTCGCGCCCTTTCTCAAATTTAAGC 3', and SEQ ID NO 10:
3XmnI: 5' CCGATTCTAGAGAAACCTGAATTAGAATAGCCCGTAGAGTTGCTTGA AATTGTTCTAAACCCCAC 3', utilizando como molde el plásmido pSEHE2F. El nuevo plásmido se llamó pMIB-E3Xmn.3XmnI: 5 'CCGATTCTAGAGAAACCTGAATTAGAATAGCCCGTAGAGTTGCTTGA AATTGTTCTAAACCCCAC 3', using plasmid pSEHE2F as a template. The new plasmid was called pMIB-E3Xmn.
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A continuación se efectuó una digestión de pMIB- E3Xmn con el enzima Muñí (que corta en la posición 29355 relativa al adenovirus tipo 5) , y una digestión parcial con Xmnl de modo que solo se corta en la posición 28555. Una vez abierto el plásmido por estos dos sitios y eliminado el fragmento comprendido entre ellos, se llevó a cabo una reacción de ligación para insertar una secuencia reconocible por el enzima Pl-Scel (adaptador Pl-Scel) . Este adaptador se obtuvo mezclando el siguiente par de oligonucleótidos que hibridan entre si: SEQ ID NO 11:SUBSTITUTE SHEET (RULE 26) Subsequently, a digestion of pMIB-E3Xmn was carried out with the enzyme Muñí (which cuts at position 29355 relative to adenovirus type 5), and a partial digestion with Xmnl so that it is only cut at position 28555. Once the plasmid is opened through these two sites and removing the fragment between them, a ligation reaction was carried out to insert a sequence recognizable by the enzyme Pl-Scel (adapter Pl-Scel). This adapter was obtained by mixing the following pair of oligonucleotides that hybridize with each other: SEQ ID NO 11:
5PI-Sce: 5' ACGTAATCTATGTCGGGTGCGGAGAAAGAGGTAATGAAATGGCA 3' y, SEQ ID NO 12:5PI-Sce: 5 'ACGTAATCTATGTCGGGTGCGGAGAAAGAGGTAATGAAATGGCA 3' and, SEQ ID NO 12:
3PI-Sce: 5' TGCCATTTCATTACCTCTTTCTCCGCACCCGACATAGATTACGT 3' .3PI-Sce: 5 'TGCCATTTCATTACCTCTTTCTCCGCACCCGACATAGATTACGT 3'.
El plásmido resultante se denomina pMIB-PIScel, y presenta el sitio Pl-Scel en el lugar donde han sido delecionados los genes adenovirales que codifican para
Para incorporar esta deleción en el genoma de los vectores adenovirales, el plásmido pMIB-PIScel se digirió con los enzimas Agel y Seal simultáneamente, y el fragmento de 4.1 kb obtenido se introdujo en el plásmido pSHIFE2F digerido con los mismos enzimas. De esta manera se genera un plásmido que contiene la deleción deseada, pero que es incompleto por haber perdido las regiones iniciales del genoma adenoviral. Sin embargo, a partir de este plásmido se obtuvo el fragmento de 7.7 kb comprendido entre los enzimas SwaI y Spel, que fue introducido en el plásmido pSHIFE2F en sustitución del fragmento homónimo. De esta manera, los genes queThe resulting plasmid is called pMIB-PIScel, and it has the Pl-Scel site where the adenoviral genes that code for To incorporate this deletion into the genome of adenoviral vectors, plasmid pMIB-PIScel was digested with Agel and Seal enzymes simultaneously, and the 4.1 kb fragment obtained was introduced into plasmid pSHIFE2F digested with the same enzymes. In this way, a plasmid is generated that contains the desired deletion, but is incomplete because it has lost the initial regions of the adenoviral genome. However, from this plasmid the 7.7 kb fragment comprised between the SwaI and Spel enzymes was obtained, which was introduced into the plasmid pSHIFE2F instead of the homonymous fragment. In this way, the genes that
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codifican para las proteinas víricas gpl9K/6.7k fueron delecionados y en su lugar se introdujo el sitio de restricción Pl-Scel que puede utilizarse para la incorporación de genes exógenos en esta zona, ya que no está presente en ninguna otra región del virus. El primer gen exógeno que se introdujo en la región E3 fue el gen reportero de la luciferasa, obtenido a partir del plásmido pGL3-Basic (Promega) . El fragmento de 1.6 kb comprendido entre los sitios HindIII y Xbal que codifica para luciferasa fue tratado con el enzima T4 polimerasa para conseguir extremos romos, y se introdujo en el sitio Pl-Scel que había sido tratado de la misma manera en el plásmido adenoviral modificado anteriormente. El plásmido resultante se denomina pSHE2F-Luc. Para facilitar el subclonaje de otros genes en la región E3, es más conveniente utilizar sitios de reconocimiento para el enzima CIaI en lugar de Pl-Scel, porque este último es muy grande y su repetición simétrica puede provocar recombinaciones en el plásmido. Para ello se puede digerir con Pl-Scel, hacer romos los extremos con T4 DNA Polimerasa, e introducir un adaptador de CIaI consistente en el par de oligonucleótidos : SEQ ID NO 13: 5CIa: 5' CCTATATCGATAGCCT 3' y SEQ ID NO 14: 3CIa: 5' AGGCTATCGATATAGG 3'. La secuencia que se pretende introducir puede flanquearse por sitios CIaI mediante ligación de estos adaptadores o por reacción de PCR utilizando oligonucleótidos que incluyan la secuencia de reconocimiento para CIaI. Si el gen exógeno de interés posee sitios CIaI internos, como sucede con laSUBSTITUTE SHEET (RULE 26) coding for gpl9K / 6.7k viral proteins were deleted and instead the Pl-Scel restriction site was introduced that can be used for the incorporation of exogenous genes in this area, since it is not present in any other region of the virus. The first exogenous gene that was introduced in the E3 region was the luciferase reporter gene, obtained from plasmid pGL3-Basic (Promega). The 1.6 kb fragment between the HindIII and Xbal sites encoding luciferase was treated with the enzyme T4 polymerase to achieve blunt ends, and was introduced into the Pl-Scel site that had been treated in the same way in the modified adenoviral plasmid previously. The resulting plasmid is called pSHE2F-Luc. To facilitate the subcloning of other genes in the E3 region, it is more convenient to use recognition sites for the CIaI enzyme instead of Pl-Scel, because the latter is very large and its symmetrical repetition can cause plasmid recombinations. To do this, it is possible to digest with Pl-Scel, blunt the ends with T4 DNA Polymerase, and introduce a CIaI adapter consisting of the pair of oligonucleotides: SEQ ID NO 13: 5CIa: 5 'CCTATATCGATAGCCT 3' and SEQ ID NO 14: 3CIa: 5 'AGGCTATCGATATAGG 3'. The sequence that is intended to be introduced can be flanked by CIaI sites by ligation of these adapters or by PCR reaction using oligonucleotides that include the recognition sequence for CIaI. If the exogenous gene of interest has internal CIaI sites, as with the
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interleuquina 12, pueden utilizarse sitios NarI, que son compatibles con CIaI .SUBSTITUTE SHEET (RULE 26) interleukin 12, NarI sites, which are compatible with CIaI, can be used.
.- Deleción del dominio CR2 del gen ElA..- Deletion of the CR2 domain of the ElA gene.
La deleción de las bases 922 a 947 del genoma adenoviral se realizó mediante PCR, utilizando como molde el genoma de adenovirus tipo 5. Inicialmente se generaron dos fragmentos. Uno de ellos, llamado AB se amplificó utilizando el par de oligonucleótidos : SEQ ID NO 15: AElA: 5' TCAGTATTCGAATCGCGACTCTTGAGTGCCAGCGAG 3' yDeletion of bases 922 to 947 of the adenoviral genome was performed by PCR, using the adenovirus genome type 5 as a template. Initially two fragments were generated. One of them, called AB, was amplified using the oligonucleotide pair: SEQ ID NO 15: AElA: 5 'TCAGTATTCGAATCGCGACTCTTGAGTGCCAGCGAG 3' and
SEQ ID NO 16: BElA: 5 ' ATCGATCACCTCCGGTACAAGGTTTGG 3'.SEQ ID NO 16: BElA: 5 'ATCGATCACCTCCGGTACAAGGTTTGG 3'.
El segundo, llamado BC se amplificó con los oligonucleótidos : SEQ ID NO 17: CElA: 5' AACCTTGTACCGGAGGTGATCGATCCACCCAGTGACGACGAGGATGAA GAGG 3' y, SEQ ID NO 18:The second, called BC, was amplified with the oligonucleotides: SEQ ID NO 17: CElA: 5 'AACCTTGTACCGGAGGTGATCGATCCACCCAGTGACGACGAGGATGAA GAGG 3' and, SEQ ID NO 18:
DElA: 5' AAGGCGTTAACCACACACGCAATCACAGGTTTACACCTTATGGCC 3'DElA: 5 'AAGGCGTTAACCACACACGCAATCACAGGTTTACACCTTATGGCC 3'
Los fragmentos AB y BC poseen una región de homología, de modo que si se mezclan pueden hibridar parcialmente. Esta región ha sido diseñada para excluir las bases comprendidas entre la posición 922 y 947. Si la hibridación de estos dos fragmentos se utiliza como molde para una reacción de PCR con los oligonucleótidos AElA yThe AB and BC fragments have a homology region, so that if they are mixed they can partially hybridize. This region has been designed to exclude the bases between position 922 and 947. If the hybridization of these two fragments is used as a template for a PCR reaction with oligonucleotides AElA and
DElA, se obtiene un fragmento (AD) flanqueado por sitios BstBI y Hpal en el que el dominio CR2 del gen ElA ha sido delecionado. A continuación este fragmento debe ser introducido en el plásmido pSHE2F-Luc. Para hacerlo posible, pSHE2F-Luc fue digerido con el enzima EcoRV y re-ligado. De esta manera se obtiene un plásmido mucho menor, que contiene sólo un sitio Hpal. A continuación se digirió este nuevo plásmido con BstBI y Hpal y seDElA, a fragment (AD) flanked by BstBI and Hpal sites is obtained in which the CR2 domain of the ElA gene has been deleted. This fragment must then be introduced into plasmid pSHE2F-Luc. To make this possible, pSHE2F-Luc was digested with the EcoRV enzyme and re-ligated. In this way a much smaller plasmid is obtained, which contains only one Hpal site. This new plasmid was then digested with BstBI and Hpal and
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incorporó el fragmento de PCR. Este plásmido intermedio se denomi-nó pSdlElA, que ha incorporado el gen ElA modificado pero ha perdido el promotor 9XHRE y la secuencia comprendida entre las bases 9197 y 33756. El promotor 9XHRE se volvió a introducir utlizando el sitio BstBI . Para restituir el resto del genoma adenoviral, inicialmente se subclonó el fragmento de 14.8 kb comprendido entre los sitios Sdal . A continuación se llevó a cabo una ligación triple para fusionar 3 fragmentos comprendidos entre sitios RsrII que restituyen el genoma viral modificado. El fragmento de 14 kb procede del plásmido que contiene la deleción en el dominio CR2 de ElA y el promotor 9XHRE, mientras que el fragmento de 17 kb (procedente del plásmido pSHE2F-Luc) contiene el gen de la luciferasa en la región E3. Por último, el genoma se completa con el fragmento de 7.7 kb procedente también de pSHE2F-Luc. El plásmido resultante de esta fusión se llamó pSDHE2F-Luc. .- Construcción de pAdLuc. Para ello se obtuvo el fragmento de 12.5 kb comprendido entre los sitios Ndel del plásmido pSHE2F- Luc, y se introdujo en los sitios homónimos del plásmido pTG3602. De esta manera se obtiene un plásmido con el gen de la luciferasa sustituyendo a los genes gpl9k/6.7k en el contexto del genoma de Adenovirus tipo 5, sin otras modificaciones . .- Construcción de pSDHE2F-TK.SUBSTITUTE SHEET (RULE 26) incorporated the PCR fragment. This intermediate plasmid was named pSdlElA, which has incorporated the modified ElA gene but has lost the 9XHRE promoter and the sequence between bases 9197 and 33756. The 9XHRE promoter was reintroduced using the BstBI site. To restore the rest of the adenoviral genome, the 14.8 kb fragment comprised between the Sdal sites was initially subcloned. A triple ligation was then carried out to fuse 3 fragments between RsrII sites that restore the modified viral genome. The 14 kb fragment comes from the plasmid that contains the deletion in the CR2 domain of ElA and the 9XHRE promoter, while the 17 kb fragment (from plasmid pSHE2F-Luc) contains the luciferase gene in the E3 region. Finally, the genome is completed with the 7.7 kb fragment also from pSHE2F-Luc. The plasmid resulting from this fusion was called pSDHE2F-Luc. .- Construction of pAdLuc. For this, the 12.5 kb fragment between the Ndel sites of plasmid pSHE2F-Luc was obtained and introduced into the homonymous sites of plasmid pTG3602. In this way a plasmid with the luciferase gene is obtained by replacing the gpl9k / 6.7k genes in the context of the adenovirus type 5 genome, without other modifications. .- Construction of pSDHE2F-TK.
Para ello se obtuvo mediante PCR un fragmento que consiste en la secuencia codificante para la proteina - TK optimizada (SEQ ID NO 19, flanqueada por sitios deFor this purpose, a fragment consisting of the coding sequence for the optimized TK-protein (SEQ ID NO 19, flanked by sites) was obtained by PCR.
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reconocimiento CIaI. Los oligonucleótidos utilizados fueron : SEQ ID NO 20:SUBSTITUTE SHEET (RULE 26) CIaI recognition. The oligonucleotides used were: SEQ ID NO 20:
5CIaTK: 5' GTACTATCGATGCTAGCCACCATGGCTTCGTACC 3' y SEQ ID NO 21:5CIaTK: 5 'GTACTATCGATGCTAGCCACCATGGCTTCGTACC 3' and SEQ ID NO 21:
3CIaTK: 5' GTACTATCGATAAGCTTAAGTCAGTTAGCCTCC 3' Este fragmento fue digerido con CIaI y subclonado en un plásmido basado en pSDHE2F-Luc, en el que la deleción de E3 está flanqueada por sitios CIaI, tal y como se describe en el apartado anterior. De este modo el gen de la luciferasa queda sustituido por el gen de la TK. .- Construcción de pSDHE2F-IL12.3CIaTK: 5 'GTACTATCGATAAGCTTAAGTCAGTTAGCCTCC 3' This fragment was digested with CIaI and subcloned into a plasmid based on pSDHE2F-Luc, in which the E3 deletion is flanked by CIaI sites, as described in the previous section. In this way the luciferase gene is replaced by the TK gene. .- Construction of pSDHE2F-IL12.
Para ello se obtuvo mediante PCR un fragmento que consiste en la secuencia codificante para una versión optimizada de la interleuquina 12 murina (proteina de fusión de las subunidades p35 y p40, SEQ ID NO 22, Lieschke y cois., Nat Biotechnol (1997)15: 35-40), flanqueada por sitios de reconocimiento NarI . Los oligonucleótidos utilizados fueron: SEQ ID NO 23:For this purpose, a fragment consisting of the coding sequence for an optimized version of murine interleukin 12 (fusion protein of subunits p35 and p40, SEQ ID NO 22, Lieschke et al., Nat Biotechnol (1997) 15 was obtained by PCR. : 35-40), flanked by NarI recognition sites. The oligonucleotides used were: SEQ ID NO 23:
5NarIL12: 5' GTACTGGCGCCACCATGGGTCCTCAGAAGCTAACC 3' y5NARIL12: 5 'GTACTGGCGCCACCATGGGTCCTCAGAAGCTAACC 3' and
SEQ ID NO 24:SEQ ID NO 24:
3NarIL12: 5' GTACTGGCGCCTAATCCGGATCAATTCTCAGG 3' Este fragmento fue digerido con NarI y subclonado en el sitio CIaI del plásmido pro-viral descrito anteriormente. De este modo el gen de la luciferasa queda sustituido por el gen de la interleuquina 12.3NarIL12: 5 'GTACTGGCGCCTAATCCGGATCAATTCTCAGG 3' This fragment was digested with NarI and subcloned into the CIaI site of the pro-viral plasmid described above. In this way the luciferase gene is replaced by the interleukin 12 gene.
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En la figura 1 se representa la estructura general de los plásmidos modificados, y en la figura 2 se muestra un esguema del genoma de los virus recombinantes .SUBSTITUTE SHEET (RULE 26) The general structure of the modified plasmids is depicted in Figure 1, and a genome skeleton of the recombinant viruses is shown in Figure 2.
Obtención de las partículas víricasObtaining viral particles
Los plásmidos descritos anteriormente se digirieron con el enzima Pací, que libera el genoma vírico del resto de secuencias bacterianas, y se transfectaron en células 293 (ATCC CRL-1573) mediante el método de precipitación con fosfato calcico. Una vez observado el efecto citopático (típicamente 7-10 dias después de la transfección) , se Usaron las células mediante 3 ciclos consecutivos de congelación y descongelación. Se efectuaron diluciones seriadas del lisado y con ellas se infectaron células A549 (ATCC CCL-185; procedente de cáncer de pulmón humano) en condiciones de hipoxia (1% oxigeno) . De esta manera se obtuvieron varios clones de los virus, que fueron verificados mediante PCR. Los nuevos virus obtenidos a partir de los plásmidos pSHE2F- Luc, pSDHHE2F-Luc ,pSDHE2F-TK, pSDHE2F-IL12 y pAdLuc se denominaron AdHLuc, AdDHLuc, AdDHTK, AdDHIL12 y AdWTLuc, respectivamente .The plasmids described above were digested with the enzyme Pací, which releases the viral genome from the rest of the bacterial sequences, and transfected into 293 cells (ATCC CRL-1573) by the calcium phosphate precipitation method. Once the cytopathic effect was observed (typically 7-10 days after transfection), the cells were used by 3 consecutive cycles of freezing and thawing. Serial dilutions of the lysate were made and with them A549 cells (ATCC CCL-185; from human lung cancer) were infected under hypoxic conditions (1% oxygen). In this way several clones of the viruses were obtained, which were verified by PCR. The new viruses obtained from plasmids pSHE2F-Luc, pSDHHE2F-Luc, pSDHE2F-TK, pSDHE2F-IL12 and pAdLuc were named AdHLuc, AdDHLuc, AdDHTK, AdDHIL12 and AdWTLuc, respectively.
Amplificación y purificación de los virus Los virus se amplificaron en células A549 mantenidas en condiciones de hipoxia en el caso de AdHLuc, AdDHLuc,Amplification and purification of viruses Viruses were amplified in A549 cells maintained under hypoxia in the case of AdHLuc, AdDHLuc,
AdDHTK, AdDHIL12, y en células 293 en el caso de AdWTLuc. La purificación se llevó a cabo mediante técnicas estándar utilizando un gradiente de Cesio y posterior paso por una columna de exclusión. El virus obtenido se conservó en un medio tamponado con 10% glicerol a -80 °CAdDHTK, AdDHIL12, and in 293 cells in the case of AdWTLuc. Purification was carried out by standard techniques using a Cesium gradient and subsequent passage through an exclusion column. The virus obtained was stored in a medium buffered with 10% glycerol at -80 ° C
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hasta el momento de su utilización. La titulación se llevó a cabo mediante observación del efecto citopático tras dilución limite en células 293, verificado por ensayos de inmunohistoquimica con un anticuerpo anti- adenovirus (Adeno-X Rapad Titer Kit, BD Biosciences Clontech Cat . No. K1653-1) .SUBSTITUTE SHEET (RULE 26) until the moment of its use. Titration was carried out by observing the cytopathic effect after limited dilution in 293 cells, verified by immunohistochemical assays with an anti-adenovirus antibody (Adeno-X Rapad Titer Kit, BD Biosciences Clontech Cat. No. K1653-1).
Ejemplo 2. Caracterización del virus AdDHLuc In vitroExample 2. Characterization of the AdDHLuc virus in vitro
Se analizó la actividad del virus AdDHLuc frente a virus control en distintos tipos celulares, tanto en condiciones normales como en hipoxia.The activity of the AdDHLuc virus against control viruses in different cell types was analyzed, both in normal conditions and in hypoxia.
Se analizaron los siguientes parámetros: - el efecto citopático, mediante tinción con el colorante cristal violeta; - la replicación y producción de nuevas partículas virales mediante dilución límite en células 293 o inmunohistoquimica con anticuerpos anti-adenovirus (Adeno-X Rapad Titer Kit, BD Biosciences Clontech Cat. No. K1653-1); y - la expresión de genes exógenos (luciferasa) .The following parameters were analyzed: - the cytopathic effect, by staining with the violet crystal dye; - replication and production of new viral particles by limiting dilution in 293 cells or immunohistochemistry with anti-adenovirus antibodies (Adeno-X Rapad Titer Kit, BD Biosciences Clontech Cat. No. K1653-1); and - the expression of exogenous genes (luciferase).
Efecto citopáticoCytopathic effect
La principal característica que se busca en los CRAds es su capacidad de inducir de modo selectivo la muerte de las células tumorales.The main feature sought in CRAds is their ability to selectively induce the death of tumor cells.
Los ensayos para evaluar este efecto citopático se realizaron de la siguiente manera:The tests to evaluate this cytopathic effect were performed as follows:
1. Siembra de las células en distintos pocilios en sendas placas (para crecimiento en normoxia e hipoxia respectivamente) : 5xlO3 células/pocilio en placas de 96 pocilios en medio apropiado para la línea celular.1. Sowing the cells in different wells in each plate (for growth in normoxia and hypoxia respectively): 5xlO 3 cells / well in 96-well plates in appropriate medium for the cell line.
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2. A las 24 horas, infección con los virus a ensayar (un tipo de virus por pocilio, p.ej. AdWT, AdHLuc o AdDHLuc) ; la concentración viral se ajustó a cada linea celular en función de la infectividad para dicha linea. 3. Incubación a 37 °C en condiciones de hipoxia (cámara ajustada a una composición gaseosa de 93% N2 / 6% CO2 / 1% O2) o normoxia (composición gaseosa ambiental) .SUBSTITUTE SHEET (RULE 26) 2. At 24 hours, infection with the virus to be tested (one type of virus per well, eg AdWT, AdHLuc or AdDHLuc); The viral concentration was adjusted to each cell line based on the infectivity for that line. 3. Incubation at 37 ° C under hypoxic conditions (chamber adjusted to a gaseous composition of 93% N2 / 6% CO2 / 1% O 2 ) or normoxia (environmental gaseous composition).
4. Cuando AdWT causó efecto citopático evidente en los cultivos, tinción con cristal violeta y cuantificación de las células supervivientes.4. When AdWT caused obvious cytopathic effect on the cultures, staining with violet crystal and quantification of the surviving cells.
5. Cálculo de la supervivencia (%) relativa respecto a células en pocilios control, cultivadas en las mismas condiciones pero que no han sido infectadas por el virus. Para el cultivo de las células Huh-7, A549 se utilizó medio DMEM (Gibco) y para las células IMR-90, WI-38, WI38-VA13 y BJ se utilizó medio MEM (Gibco) . El medio de mantenimiento estaba suplementado con suero bovino fetal al 10%, cuya proporción se redujo durante la infección al 2%. En todos los casos se añadió a los medios de cultivo una mezcla de antibióticos (penicilina 50 UI/L - estreptomicina 50 mg/mL; Biowhittaker) y antimicóticos (anfotericina-B 2,5 mg/mL; Gibco).5. Calculation of relative survival (%) with respect to cells in control wells, grown under the same conditions but not infected by the virus. For the culture of the Huh-7 cells, A549 DMEM medium (Gibco) was used and for the IMR-90, WI-38, WI38-VA13 and BJ cells MEM medium (Gibco) was used. The maintenance medium was supplemented with 10% fetal bovine serum, the proportion of which was reduced during 2% infection. In all cases a mixture of antibiotics (penicillin 50 IU / L - streptomycin 50 mg / mL; Biowhittaker) and antifungal agents (amphotericin-B 2.5 mg / mL; Gibco) was added to the culture media.
Inicialmente se infectaron células tumorales Huh-7 (Nakabayashi y cois. 1982. Cáncer Res., 42: 3858-3863; procedente de hepatocarcinoma humano) y A549 (ATTC CCL- 185; procedente de cáncer de pulmón humano), y células normales IMR-90 (ATCC CCL-186; procedentes de fibroblastos primarios humanos de pulmón) con los distintos virus.Initially Huh-7 tumor cells (Nakabayashi et al. 1982 were infected. Cancer Res., 42: 3858-3863; from human hepatocarcinoma) and A549 (ATTC CCL-185; from human lung cancer), and normal IMR cells -90 (ATCC CCL-186; from primary human lung fibroblasts) with the various viruses.
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Como puede apreciarse en la figura 3, tanto AdDHLuc como AdHLuc fueron capaces de eliminar las células tumorales cuando éstas crecen en condiciones de baja tensión de oxigeno (hipoxia) . El efecto citotóxico en hipoxia fue similar al observado con el adenovirus silvestre AdWT (figura 3A) . Estas condiciones reproducen la situación que se encuentra en los tumores sólidos. En cambio, los virus están atenuados en presencia de oxigenoSUBSTITUTE SHEET (RULE 26) As can be seen in Figure 3, both AdDHLuc and AdHLuc were able to eliminate tumor cells when they grow under conditions of low oxygen tension (hypoxia). The cytotoxic effect on hypoxia was similar to that observed with the AdWT wild adenovirus (Figure 3A). These conditions reproduce the situation found in solid tumors. Instead, viruses are attenuated in the presence of oxygen
(normoxia) . Para comprobar el grado de selectividad de los virus, se infectaron células normales (IMR-90) , figura 3B. Las condiciones más parecidas a los tejidos sanos son las células normales en normoxia, y en estas condiciones tanto AdDHLuc como AdHLuc están atenuados(normoxia). To check the degree of virus selectivity, normal cells (IMR-90) were infected, Figure 3B. The most similar conditions to healthy tissues are normal cells in normoxia, and in these conditions both AdDHLuc and AdHLuc are attenuated
(menos de un 30% de muerte celular) . Sin embargo, cuando las células normales se exponen a una baja tensión de oxigeno, AdHLuc es más citopático que AdDHLuc. Esto concuerda con el diseño de estos virus, ya que en el caso de AdHLuc la hipoxia es capaz de desencadenar la replicación viral por activación del promotor ElA, tanto en células tumorales como normales. En cambio, el mecanismo adicional de seguridad de AdDHLuc, consistente en la deleción del dominio CR2 del gen ElA, atenúa al virus en células normales incluso en presencia de hipoxia. En definitiva, los datos de la figura 3 sugieren que AdDHLuc tiene una potencia citotóxica igual que(less than 30% cell death). However, when normal cells are exposed to low oxygen tension, AdHLuc is more cytopathic than AdDHLuc. This is consistent with the design of these viruses, since in the case of AdHLuc hypoxia is capable of triggering viral replication by activation of the ElA promoter, both in tumor and normal cells. In contrast, the additional security mechanism of AdDHLuc, consisting of the deletion of the CR2 domain of the ElA gene, attenuates the virus in normal cells even in the presence of hypoxia. Ultimately, the data in Figure 3 suggest that AdDHLuc has a cytotoxic potency equal to
AdHLuc y cercana a AdWT sobre células tumorales, pero ventajosamente está fuertemente atenuado en células normales .AdHLuc and close to AdWT on tumor cells, but advantageously it is strongly attenuated in normal cells.
Para confirmar este punto se estudió el comportamiento de los virus en fibroblastos humanos normales WI-38 (ATCC CCL-75) y fibroblastos WI38-VA13To confirm this point, the behavior of the viruses in normal human fibroblasts WI-38 (ATCC CCL-75) and fibroblasts WI38-VA13 was studied
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(ATCC CCL-75.1), que han sido malignizadas por la expresión del antigeno T de SV40, que bloquea la ruta de pRB. Las células WI38-VA13 tienen alterado el control del ciclo celular, y por tanto se espera que el virus AdDHLuc no esté atenuado respecto a AdHLuc. Asimismo se realizaron ensayos con células normales BJ (ATCC CRL-2522; procedentes de fibroblastos primarios humanos de piel) .SUBSTITUTE SHEET (RULE 26) (ATCC CCL-75.1), which have been maligned by the expression of SV40 T antigen, which blocks the pRB path. WI38-VA13 cells have impaired cell cycle control, and therefore it is expected that the AdDHLuc virus is not attenuated with respect to AdHLuc. Likewise, tests were carried out with normal BJ cells (ATCC CRL-2522; from primary human skin fibroblasts).
En la figura 4 se muestran microfotografias de fibroblastos normales WI-38 y malignizados WI38-VA13 infectados con los distintos virus en condiciones de hipoxia o normoxia. Las células que están sufriendo el efecto citopático del virus se distinguen porque pierden adherencia a la placa de cultivo y adquieren una morfologia redondeada. Como puede observarse, AdDHLuc es el virus que está más atenuado en células normales, mientras que conserva su capacidad de matar células malignas .Figure 4 shows photomicrographs of normal WI-38 and malignant WI38-VA13 fibroblasts infected with the different viruses under hypoxia or normoxia conditions. The cells that are suffering from the cytopathic effect of the virus are distinguished because they lose adhesion to the culture plate and acquire a rounded morphology. As can be seen, AdDHLuc is the virus that is most attenuated in normal cells, while retaining its ability to kill malignant cells.
Estas observaciones se repitieron sobre otros tipos de células normales, como células IMR-90 y BJ, como se muestra en la figura 5.These observations were repeated on other types of normal cells, such as IMR-90 and BJ cells, as shown in Figure 5.
Citotoxicidad a diferentes concentraciones viralesCytotoxicity at different viral concentrations
Con el objetivo de estudiar con más detalle el efecto citopático de los virus AdDHLuc y AdHLuc, se determinó la viabilidad de células tumorales (Huh-7) y normales (IMR-90) infectadas con distintas concentraciones virales, tanto en condiciones de normoxia como de hipoxia (como se ha descrito anteriormente) . Como puede observarse en la figura 6, la hipoxia activó sensiblemente la citotoxicidad tanto de AdDHLucIn order to study in more detail the cytopathic effect of the AdDHLuc and AdHLuc viruses, the viability of tumor (Huh-7) and normal (IMR-90) infected cells with different viral concentrations was determined, both under normoxia and hypoxia (as described above). As can be seen in Figure 6, hypoxia significantly activated the cytotoxicity of both AdDHLuc
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como de AdHLuc en células tumorales, llegando a eliminar en torno al 90% de las células con una MOI de 5 virus/célula. El efecto fue similar al observado con AdWTSUBSTITUTE SHEET (RULE 26) as of AdHLuc in tumor cells, eliminating around 90% of the cells with an MOI of 5 viruses / cell. The effect was similar to that observed with AdWT
(no mostrado en la gráfica) . En cambio, la activación ejercida por hipoxia en células normales fue signifcativamente menor en el caso de AdDHLuc respecto a(not shown in the graph). On the other hand, the activation exerted by hypoxia in normal cells was significantly lower in the case of AdDHLuc compared to
AdHLuc, lo cual confirma una mayor atenuación y el funcionamiento del doble sistema de control para su replicación . Es importante señalar que en este tipo de experimentos la disminución de viabilidad celular a dosis bajas es dependiente de la amplificación viral, por lo que de una manera indirecta este ensayo también refleja el control de la replicación del virus .AdHLuc, which confirms greater attenuation and the operation of the double control system for its replication. It is important to note that in this type of experiments the decrease in cell viability at low doses is dependent on viral amplification, so this test also indirectly reflects the control of virus replication.
Replicación viralViral replication
Con el objetivo de analizar de modo directo el control de la replicación de AdDHLuc, se infectaron células A549 con el virus en condiciones de normoxia o hipoxia, y se cuantificó la producción de nuevas partículas infectivas al cabo de los dias .In order to directly analyze the control of AdDHLuc replication, A549 cells were infected with the virus under normoxia or hypoxia conditions, and the production of new infective particles was quantified after days.
Las células se sembraron, se infectaron y crecieron de la misma manera que en los ensayos para evaluar el efecto citopático. En este caso, a los 4 dias desde la infección, las células se usaron mediante 3 ciclos de congelación-descongelación y se cuantificaron las partículas infectivas mediante dilución límite en células 293 o mediante inmunohistoquímica con anticuerpos anti-adenovirus (Adeno-X Rapad Titer Kit, BD Biosciences Clontech Cat. No. K1653-1) siguiendo las instruccionesThe cells were seeded, infected and grown in the same manner as in the tests to evaluate the cytopathic effect. In this case, 4 days after infection, the cells were used by 3 freeze-thaw cycles and the infective particles were quantified by limit dilution in 293 cells or by immunohistochemistry with anti-adenovirus antibodies (Adeno-X Rapad Titer Kit , BD Biosciences Clontech Cat. No. K1653-1) following the instructions
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del fabricante. El resultado se expresó como unidades infectivas por mL (i.u./ml) .SUBSTITUTE SHEET (RULE 26) manufacturer. The result was expressed as infective units per mL (iu / ml).
Como puede observarse en la figura 7, la hipoxia activó la amplificación del virus AdDHLuc, mientras que no ocurre lo mismo con AdWT, cuya replicación no sufrió cambios en estas condiciones experimentales.As can be seen in Figure 7, hypoxia activated the amplification of the AdDHLuc virus, while the same does not happen with AdWT, whose replication did not undergo changes in these experimental conditions.
Expresión de genes exógenos (luciferasa)Exogenous gene expression (luciferase)
La localización del gen reportero luciferasa en la región E3 del virus AdDHLuc permite un seguimiento de la expresión de los genes exógenos introducidos en el virus. Al mismo tiempo, la expresión de luciferasa se incrementa en respuesta a la replicación viral, debido a la activación de los promotores tardíos y al aumento del número de copias del genoma viral en la célula. Por lo tanto, medir la actividad luciferasa es un indicador de la replicación viral.The location of the luciferase reporter gene in the E3 region of the AdDHLuc virus allows monitoring of the expression of exogenous genes introduced into the virus. At the same time, luciferase expression is increased in response to viral replication, due to the activation of late promoters and the increase in the number of copies of the viral genome in the cell. Therefore, measuring luciferase activity is an indicator of viral replication.
Con este fin, se sembraron células Huh-7 y se infectaron con los virus replicativos AdDHLuc, AdHLuc, AdWTLuc, y con el virus defectivo Ad-CMV-Luc (Vector Biolabs Cat. No. 1000) . La dosis de virus utilizada (MOI) fue de 10 virus/célula, y las células se cultivaron de la misma manera que en los ensayos para evaluación del efecto citopático. A los 2 dias desde la infección, las células se usaron para medir la actividad de la Luciferasa mediante un kit comercial (Luciferase Assay System, Promega Cat. No. E4030) , siguiendo las instrucciones del fabricante. La actividad se expresó como RLü/μg de proteina total (RLU: Relative Luciferase Units) .To this end, Huh-7 cells were seeded and infected with the AdDHLuc, AdHLuc, AdWTLuc replicative viruses, and with the Ad-CMV-Luc defective virus (Vector Biolabs Cat. No. 1000). The dose of virus used (MOI) was 10 viruses / cell, and the cells were cultured in the same manner as in the tests for cytopathic effect evaluation. Two days after infection, the cells were used to measure Luciferase activity using a commercial kit (Luciferase Assay System, Promega Cat. No. E4030), following the manufacturer's instructions. The activity was expressed as RLü / μg of total protein (RLU: Relative Luciferase Units).
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En la figura 8 se muestra como esta actividad aumenta con hipoxia en células infectadas con AdDHLuc, y AdHLuc, mientras que no ocurre lo mismo con AdWT y Ad- CMV-Luc, los dos virus no regulados por hipoxia. También puede observarse que la capacidad de expresar genes es mucho mayor en el caso de los virus replicativos frente al virus defectivo AdCMVLuc.SUBSTITUTE SHEET (RULE 26) Figure 8 shows how this activity increases with hypoxia in cells infected with AdDHLuc, and AdHLuc, while the same does not occur with AdWT and Ad-CMV-Luc, the two viruses not regulated by hypoxia. It can also be seen that the ability to express genes is much greater in the case of replicative viruses against the defective AdCMVLuc virus.
En definitiva, los experimentos descritos en este ejemplo 2 demuestran que todos los parámetros de actividad estudiados para AdDHLuc se incrementan en condiciones de hipoxia, de modo preferente en células tumorales .Ultimately, the experiments described in this example 2 demonstrate that all activity parameters studied for AdDHLuc are increased in hypoxic conditions, preferably in tumor cells.
Ejemplo 3. Caracterización del virus AdDHTK In v±tro Para comprobar la función del virus AdDHTK, se analizó el efecto citopático obtenido tras infección de las células A549, en presencia o ausencia del pro-fármaco Ganciclovir (GCV) . El ensayo se realizó tal y como se describe en el ejemplo 2, con las siguientes modificaciones. Las células se infectaron con distintas MOIs de AdDHTK en condiciones de normoxia o hipoxia, y 24 horas después el medio infectivo se retiró y se diferenciaron dos tipos de pocilios. En uno de ellos se añadió medio de cultivo estándar con 2% suero bovino fetal, y en el otro se incluyó GCV (100 μM) . La supervivencia de las células se cuantificó 5 dias después. En la figura 9 se representan los datos obtenidos cuando las células A549 se infectaron con una MOI de 0,12 virus/célula. Como puede comprobarse, esta pequeña cantidad de virus no disminuyó significativamente la supervivencia de las células tumorales en condicionesExample 3. Characterization of the AdDHTK virus In v ± tro To check the function of the AdDHTK virus, the cytopathic effect obtained after infection of the A549 cells, in the presence or absence of the Ganciclovir pro-drug (GCV), was analyzed. The test was performed as described in example 2, with the following modifications. The cells were infected with different AdDHTK MOIs under conditions of normoxia or hypoxia, and 24 hours later the infectious medium was removed and two types of wells were differentiated. In one of them standard culture medium with 2% fetal bovine serum was added, and in the other GCV (100 μM) was included. Cell survival was quantified 5 days later. Figure 9 shows the data obtained when A549 cells were infected with an MOI of 0.12 virus / cell. As can be seen, this small amount of virus did not significantly decrease the survival of tumor cells under conditions
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de normoxia, pero si se observó activación del efecto citopático cuando las células se mantuvieron en hipoxia. Tal y como cabe esperar si el virus expresa un enzima TK funcional, la viabilidad de las células tratadas con GCV fue sensiblemente menor. Esto indica que la regulación de la replicación viral en estas células se mantiene, y que el gen terapéutico TK puede incrementar el efecto antitumoral del virus. Esto es especialmente importante en el caso de que las células tumorales estén expuestas a una baja cantidad de virus, tal y como suele suceder en los tumores in vivo. Ejemplo 4. Caracterización del virus AdDHILl2 in vitroSUBSTITUTE SHEET (RULE 26) of normoxia, but activation of the cytopathic effect was observed when the cells remained in hypoxia. As expected if the virus expresses a functional TK enzyme, the viability of GCV treated cells was significantly lower. This indicates that the regulation of viral replication in these cells is maintained, and that the therapeutic TK gene can increase the antitumor effect of the virus. This is especially important in the case that tumor cells are exposed to a low amount of virus, as is usually the case in tumors in vivo. Example 4. Characterization of the AdDHILl2 virus in vitro
Para comprobar la función del virus AdDHIL12, se sembraron células HeLa (ATTC CCL-2) en placas de 24 pocilios (2xlO4 células/pocilio) , y se infectaron con el virus AdDHIL12 en condiciones de normoxia o hipoxia. Dado que la IL12 es una proteina secretable, se cuantificó su producción en el medio de cultivo a distintos tiempos después de la infección. La detección de IL12 murina se llevó a cabo mediante un kit de ELISA comercial (BDTo check the function of the AdDHIL12 virus, HeLa cells (ATTC CCL-2) were seeded in 24-well plates (2xlO 4 cells / well), and infected with the AdDHIL12 virus under normoxia or hypoxia conditions. Since IL12 is a secretable protein, its production in the culture medium was quantified at different times after infection. The detection of murine IL12 was carried out by a commercial ELISA kit (BD
Bioscience) . En la gráfica 10 se representa el incremento de la producción de IL12 en respuesta a hipoxia a lo largo del tiempo. Como puede comprobarse, la cantidad deBioscience) Graph 10 shows the increase in IL12 production in response to hypoxia over time. As can be seen, the amount of
IL12 prácticamente no se incrementa en condiciones de normoxia, pero si hay un aumento significativo a los 3 dias si las células se mantienen en condiciones de hipoxia. Estos datos confirman que el virus AdDHIL12 se amplifica en el tiempo en respuesta a hipoxia, y como resultado hay un incremento de IL12 en un microambiente que imita las condiciones esperables en los tumores sólidos .IL12 practically does not increase under normoxia conditions, but there is a significant increase at 3 days if the cells are maintained in hypoxia conditions. These data confirm that the AdDHIL12 virus is amplified over time in response to hypoxia, and as a result there is an increase in IL12 in a microenvironment that mimics the expected conditions in solid tumors.
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Ejemplo 5. Ensayos de actividad ±n vivoSUBSTITUTE SHEET (RULE 26) Example 5. Activity tests ± n live
Para evaluar la actividad in vivo de los adenovirus recombinantes se realizaron xenotransplantes de tumores humanos en ratones atimicos (inmunodeprimidos) mediante inyección subcutánea (IxIO7 células en 150 μl de suero salino) o intrahepática (l,5xlθ6 células en 50 μl de suero) de células Huh-7. A los 20 dias, se inyectó el virus a ensayar (AdDHLuc, Ad-CMV-Luc y AdWTLuc) mediante inyección intratumoral . A las 24 horas de la inyección del virus se inició la medición de actividad luciferasa, realizada diariamente durante 10 dias. Para ello se administró el sustrato luciferina por via intraperitonealTo evaluate the in vivo activity of the recombinant adenoviruses, human tumor xenotransplants were performed in atomic mice (immunosuppressed) by subcutaneous injection (IxIO 7 cells in 150 μl saline serum) or intrahepatic (1.5 x 6 cells in 50 μl serum) of Huh-7 cells. At 20 days, the virus to be tested (AdDHLuc, Ad-CMV-Luc and AdWTLuc) was injected by intratumoral injection. 24 hours after the injection of the virus, the measurement of luciferase activity was started, performed daily for 10 days. For this, the luciferin substrate was administered intraperitoneally.
(150 μg/ml disuelto en PBS) . Gracias al gen reportero luciferasa, la cinética de expresión y la biodistribución de los virus pueden monitorizarse en los animales vivos midiendo la emisión de luz mediante una cámara luminométrica de alta sensibilidad (In vivo imaging system, Xenogen) . En estas condiciones, la emisión de luz(150 μg / ml dissolved in PBS). Thanks to the luciferase reporter gene, the expression kinetics and biodistribution of viruses can be monitored in live animals by measuring the emission of light using a high-sensitivity luminometric camera (In vivo imaging system, Xenogen). Under these conditions, the light emission
(fotones/segundo) responde a un equilibrio entre la replicación viral y la muerte de las células infectadas.(photons / second) responds to a balance between viral replication and the death of infected cells.
Como puede observarse en la figura HA, un adenovirus defectivo (incapaz de replicarse) como Ad-CMV- Luc mantiene los niveles de expresión iniciales durante la primera semana y luego inicia una lenta disminución. En cambio, AdDHLuc mostró un incremento importante en la actividad luciferasa durante los 4 primeros dias. En la figura HB se ilustra la actividad luciferasa relativa al primer dia post-infección y se pone de manifiesto como solo en el caso de AdDHLuc existe un incremento significativo en dias sucesivos. Esto es compatible con la consecución de varios ciclos de replicación en elAs can be seen in Figure HA, a defective adenovirus (unable to replicate) such as Ad-CMV-Luc maintains the initial expression levels during the first week and then initiates a slow decrease. In contrast, AdDHLuc showed a significant increase in luciferase activity during the first 4 days. Figure HB illustrates the luciferase activity relative to the first day post-infection and shows how only in the case of AdDHLuc there is a significant increase in successive days. This is compatible with the achievement of several replication cycles in the
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tumor. En el caso de AdWTLuc, se observó una expresión inicial muy intensa y un incremento al segundo día, seguido de una disminución muy pronunciada. Esto puede deberse a la inducción de un efecto citopático más rápido en las células infectadas.SUBSTITUTE SHEET (RULE 26) tumor. In the case of AdWTLuc, a very intense initial expression and an increase on the second day were observed, followed by a very pronounced decrease. This may be due to the induction of a faster cytopathic effect on infected cells.
Para comprobar el comportamiento de AdDHLuc en tumores hepáticos, se inyectaron las células Huh-7 en el hígado de los ratones y posteriormente se administró el virus (109 i .u. ) . En la figura HC puede observarse como se consiguen niveles de expresión muy elevados y se confirma el incremento en los primeros dias para después estabilizarse e iniciar un descenso lento.To check the behavior of AdDHLuc in liver tumors, Huh-7 cells were injected into the liver of the mice and subsequently the virus was administered (10 9 i.). In figure HC it can be observed how very high levels of expression are achieved and the increase in the first days is confirmed and then stabilized and a slow descent begins.
En resumen, estos datos indican que AdDHLuc es un CRAd capaz de servir como vector de expresión de genes exógenos con eficacia superior a los adenovirus no replicativos .In summary, these data indicate that AdDHLuc is a CRAd capable of serving as an expression vector for exogenous genes with efficacy superior to non-replicative adenoviruses.
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SUBSTITUTE SHEET (RULE 26)
Claims
1. - Un adenovirus recombinante de replicación condicionada, caracterizado porque comprende: a) un promotor operativamente unido al gen adenoviral ElA que comprende al menos un elemento de respuesta a hipoxia HRE; b) un promotor operativamente unido al gen adenoviral E4 que comprende al menos un elemento de respuesta al factor E2F; c) un gen adenoviral ElA delecionado en el dominio CR2; y d) un gen exógeno de interés .1. - A recombinant conditioned replication adenovirus, characterized in that it comprises: a) a promoter operatively linked to the adenoviral ElA gene comprising at least one HRE hypoxia response element; b) a promoter operatively linked to the adenoviral gene E4 comprising at least one element of response to the E2F factor; c) an adenoviral ElA gene deleted in the CR2 domain; and d) an exogenous gene of interest.
2.- Un adenovirus recombinante según la reivindicación 1, caracterizado porque dicho promotor operativamente unido al gen adenoviral ElA comprende 9 copias en tándem de un elemento de respuesta a hipoxia.2. A recombinant adenovirus according to claim 1, characterized in that said promoter operatively linked to the adenoviral ElA gene comprises 9 tandem copies of a hypoxia response element.
3.- Un adenovirus recombinante según una cualquiera de las reivindicaciones 1 o 2, caracterizado porque dichos elementos de respuesta a hipoxia se derivan del promotor del factor de crecimiento del endotelio vascular (VEGF) humano .3. A recombinant adenovirus according to any one of claims 1 or 2, characterized in that said hypoxia response elements are derived from the human vascular endothelial growth factor (VEGF) promoter.
4.- Un adenovirus recombinante según una de las reivindicaciones 1 a 3, caracterizado porque dicho promotor operativamente unido al gen adenoviral ElA comprende la SEQ. ID. NO: 1.4. A recombinant adenovirus according to one of claims 1 to 3, characterized in that said promoter operatively linked to the adenoviral ElA gene comprises SEQ. ID. NO: 1.
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5.- Un adenovirus recombinante según una de las reivindicaciones 1 a 4, caracterizado porque dicho promotor de respuesta E2F-1 comprende la SEQ. ID. NO: 2.5. A recombinant adenovirus according to one of claims 1 to 4, characterized in that said E2F-1 response promoter comprises SEQ. ID. NO: 2.
6. - Un adenovirus recombinante según una cualquiera de las reivindicaciones 1 a 5, caracterizada porque dicha deleción en el dominio CR2 es una deleción Delta24.6. - A recombinant adenovirus according to any one of claims 1 to 5, characterized in that said deletion in the CR2 domain is a Delta24 deletion.
7.- Un adenovirus recombinante según una de las reivindicaciones 1 a 6, caracterizado porque comprende: a) un promotor operativamente unido al gen adenoviral ElA de secuencia SEQ ID NO: 1; b) un promotor de respuesta E2F-1 operativamente unido al gen adenoviral E4 de secuencia SEQ. ID. NO: 2; c) un gen adenoviral ElA delecionado en el dominio CR2 con la deleción Delta24; y d) un gen exógeno de interés.7. A recombinant adenovirus according to one of claims 1 to 6, characterized in that it comprises: a) a promoter operably linked to the adenoviral ElA gene of sequence SEQ ID NO: 1; b) an E2F-1 response promoter operably linked to the adenoviral gene E4 of sequence SEQ. ID. NO: 2; c) an adenoviral ElA gene deleted in the CR2 domain with the Delta24 deletion; and d) an exogenous gene of interest.
8.- Un adenovirus recombinante según una cualquiera de las reivindicaciones 1 a 1 , caracterizado porque dicho gen exógeno es un gen reportero.8. A recombinant adenovirus according to any one of claims 1 to 1, characterized in that said exogenous gene is a reporter gene.
9.- Un adenovirus recombinante según la reivindicación 8, caracterizado porque dicho gen reportero codifica la luciferasa.9. A recombinant adenovirus according to claim 8, characterized in that said reporter gene encodes luciferase.
10.- Un adenovirus recombinante según una cualquiera de las reivindicaciones 1 a 7, caracterizado porque dicho gen exógeno es un gen suicida.10. A recombinant adenovirus according to any one of claims 1 to 7, characterized in that said exogenous gene is a suicide gene.
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11.- Un adenovirus recombinante según la reivindicación 10, caracterizado porque dicho gen exógeno es el gen de la timidina-kinasa.11. A recombinant adenovirus according to claim 10, characterized in that said exogenous gene is the thymidine kinase gene.
12.- Un adenovirus recombinante según una cualquiera de las reivindicaciones 1 a 7, caracterizado porque dicho gen exógeno es un gen terapéutico.12. A recombinant adenovirus according to any one of claims 1 to 7, characterized in that said exogenous gene is a therapeutic gene.
13.- Un adenovirus recombinante según la reivindicación 12, caracterizado porque dicho gen exógeno es el gen de la interleuquina 12.13. A recombinant adenovirus according to claim 12, characterized in that said exogenous gene is the interleukin 12 gene.
14.- Una célula hospedadora que comprende un adenovirus recombinante descrito en una cualquiera de las reivindicaciones 1 a 13.14. A host cell comprising a recombinant adenovirus described in any one of claims 1 to 13.
15.- Un procedimiento para la propagación in vitro de un adenovirus recombinante según una de las reivindicaciones 1 a 13, caracterizado porque comprende: a) cultivar una célula hospedadora que contenga dicho adenovirus recombinante en condiciones que permitan la expresión del adenovirus; y b) aislar y purificar el adenovirus.15. A method for in vitro propagation of a recombinant adenovirus according to one of claims 1 to 13, characterized in that it comprises: a) culturing a host cell containing said recombinant adenovirus under conditions that allow adenovirus expression; and b) isolate and purify the adenovirus.
16.- Una composición caracterizada porque comprende un adenovirus recombinante descrito en una de las reivindicaciones 1 a 13 y un excipiente farmacéuticamente aceptable.16. A composition characterized in that it comprises a recombinant adenovirus described in one of claims 1 to 13 and a pharmaceutically acceptable excipient.
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US9345787B2 (en) | 2008-12-22 | 2016-05-24 | Targovax Oy | Adenoviral vectors and methods and uses related thereto |
CN102264760A (en) * | 2008-12-22 | 2011-11-30 | 昂克斯治疗有限公司 | Oncolytic adenoviral vectors and methods and uses related thereto |
RU2520823C2 (en) * | 2008-12-22 | 2014-06-27 | Онкос Терапьютикс Ой | Adenoviral vectors and related methods and applications |
CN102264760B (en) * | 2008-12-22 | 2017-03-22 | 昂克斯治疗有限公司 | Oncolytic adenoviral vectors and methods and uses related thereto |
AU2009332883B2 (en) * | 2008-12-22 | 2015-05-21 | Oncos Therapeutics Oy | Oncolytic adenoviral vectors and methods and uses related thereto |
WO2010072900A1 (en) * | 2008-12-22 | 2010-07-01 | Oncos Therapeutics | Oncolytic adenoviral vectors and methods and uses related thereto |
EP2682459A1 (en) * | 2011-03-02 | 2014-01-08 | Beijing Bio-Targeting Therapeutics Technology Inc. | Oncolytic adenovirus for target therapy of human tumor and use thereof |
JP2014509197A (en) * | 2011-03-02 | 2014-04-17 | 北京▲錘▼特生物科技有限公司 | Oncolytic adenovirus for targeted tumor therapy and its application |
EP2682459A4 (en) * | 2011-03-02 | 2014-12-10 | Beijing Bio Targeting Therapeutics Technology Inc | Oncolytic adenovirus for target therapy of human tumor and use thereof |
CN105307671A (en) * | 2013-04-18 | 2016-02-03 | 蒂尔坦生物制药有限公司 | Enhanced adoptive cell therapy |
WO2014170389A1 (en) * | 2013-04-18 | 2014-10-23 | Tilt Biotherapeutics Oy | Enhanced adoptive cell therapy |
AU2014255733B2 (en) * | 2013-04-18 | 2019-05-16 | Tilt Biotherapeutics Oy | Enhanced adoptive cell therapy |
US10647963B2 (en) | 2013-04-18 | 2020-05-12 | Tilt Biotherapeutics Oy | Enhanced adoptive cell therapy |
CN105307671B (en) * | 2013-04-18 | 2020-09-04 | 蒂尔坦生物制药有限公司 | Enhancing adoptive cell therapy |
CN111658670A (en) * | 2013-04-18 | 2020-09-15 | 蒂尔坦生物制药有限公司 | Oncolytic adenoviral vectors and adoptive T cell therapeutic compositions and uses thereof |
US10787645B2 (en) | 2013-04-18 | 2020-09-29 | Tilt Biotherapeutics Oy | Enhanced adoptive cell therapy |
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