MXPA99008515A - Recombinant adenoviral vectors and their utility in the treatment of various types of hepatic, renal, pulmonary fibrosis and hypertrophic scars - Google Patents

Abstract

The present invention relates to a recombinant adenoviral vector or plasmid containing an adenoviral genome with the deleted E1 and E3 regions, said vector or plasmid also containing a therapeutic gene or a DNA sequence of interest regulated by ubiquitous promoters and / or specific promoters. of tissue, which codes for a therapeutic protein consisting of the latent or active MMP-8 protein, thus the invention also relates to pharmaceutical compositions comprising the recombinant adenoviral vector or the plasmid and its pharmaceutical uses for the treatment of liver cirrhosis , pulmonary fibrosis and hypertrophic scars

Description

"RECOMBINANT ADENOVIRAL VECTORS AND THEIR UTILITY IN THE TREATMENT OF VARIOUS TYPES OF HEP TICA, RENAL, PULMONARY FIBROSIS AND HYPERTROPHIC SCARES" TECHNICAL FIELD OF THE INVENTION The present invention relates to the construction of recombinant adenoviral vectors carrying exogenous genes that encode therapeutic proteins useful in the treatment of liver cirrhosis and generalized fibrosis, such as renal fibrosis, pulmonary fibrosis, hypertrophic scars and keloids (fibrosis). of the skin), and / or in other white organs susceptible to suffer it. As well as a tissue-specific recognition mechanism of the affected cells by sending "therapeutic" genes to the cirrhotic organs. Also, the invention provides an effective route for the treatment of fibrosis by using the recombinant adenoviral vectors claimed herein, as well as the process for preparing said vectors, the pharmaceutical composition containing them, and their therapeutic uses in the treatment of fibrosis, which has a great commercial expectation in the pharmaceutical industry and also presents an important alternative as experimental gene therapy for the treatment of chronic-degenerative diseases that occur with fibrosis with great therapeutic application in the field of medicine. INTRODUCTION PHYSIOPATHOLOGY OF HEPATIC CIRRHOSIS Hepatic cirrhosis is a disease that results from chronic liver damage, it can be toxic (chronic alcohol intake), infectious (viral hepatitis, mainly hepatitis B virus and / or C), immune (primary biliary cirrhosis), biliary obstruction (secondary biliary cirrhosis), metabolic (Wilson's disease). All forms of cirrhosis have common characteristics: synthesis and excessive deposition of extracellular matrix proteins (MEC) (mainly collagen I and in smaller amounts, collagen type IV and III), and as a consequence hepatocyte nodule formation, abnormal vascularization and Portal hypertension (Antoni PP, Ishak KG, Nayak NC, Poulsen HE, Scheuer PJ, Sobin LH, The morphology of cirrhosis: definition, nomenclature, and classification, Bulletin of the World Health Organization, 1977; 55: 521-540 and Scott L Friedman The cellular basis of hepatic fibrosis: Mechanisms and treatment strategies, The New Ingland Journal of Medicine 1993, vol 328 No. 25: 1828-1835). These physiopathological processes lead to an alteration in the blood supply and therefore in the nutrition of the liver cell. Regardless of the etiological agent and of morphological differences, all forms of cirrhosis have as a common goal the liver failure _ and therefore the death of the patient. As a consequence of the excess in the deposition of collagenic proteins in the subendothelial space of the sinusoids (Disse Space), several changes arise in the hepatic microenvironment: loss of the villi of the hepatoai os, appearance of a basal membrane formed by collagen IV and I coating the sinusoids and loss of the fenestrations of the endothelial cells that form the sinusoids. This whole process is known as "capillarization" of sinusoids (Scott L. Friedman The cellular basis of hepatic fibrosis: Mechanisms and treatment strategies, The New Ingland Journal of Medicine 1993, vol 328 No. 25: 1828-1835) . Consequently, the liver is unable to maintain the physiological concentration of solutes in the terminal hepatic vein, in other words, hepatic insufficiency occurs. This capillarization, with the formation of a continuous endothelium (basement membrane collagen) and accumulation of other collagenic proteins, represents a barrier to the normal and bi-directional exchange of molecules between plasma and hepatocytes, as can be seen in figure 1, where liver cirrhosis is characterized by the accumulation of type I collagen in the liver. With the excess in the deposit of this protein prevents the free exchange of nutrients between the bloodstream and the liver, as well as the detoxification of harmful agents carried out by this organ, main causes of the pathophysiology of the disease. So far, no therapeutic agent has been described that reverses and / or prevents the progressive accumulation of hepatic collagen with 100% effectiveness. These physiopathological alterations that occur in liver cirrhosis are a constant in common for those organs that also suffer from it, such as, for example, the lung, heart, kidney, skin, among others, which should not be considered as limiting the scope of protection of the invention. So the methodology that is being presented here for the treatment of liver cirrhosis could also be applied to those organs that are susceptible to or that are affected by fibrosis.

VIRAL VECTORS AND HEPATIC GENE THERAPY This technology can be implemented with viral or non-viral vectors. Previous studies include plasmids and liposomes, (DOTMA) cationic and anionic, etc.

Among the methods that use viral vectors, the most commonly used have been retroviral and adenoviral.

In a good number of protocols retroviral vectors are used to introduce genes in hepatocytes (Douglas JT, and Curiel DT, Adenoviruses as Vectors for Gene Therapy, Science and Medicine March / April 1997 44-53). However, caution must be exercised because these vectors can potentially generate replication-competent viruses. Among the advantages of these vectors is their ability to integrate their genome stably into the genome of the host cell, which confers the possibility of expression, indefinitely, of the therapeutic transgene cloned in the retrovirus. On the other hand, to date, no study has reported incidences of mutagenesis due to insertion or activation of oncogenes due to the integration of retroviruses, provided that the viruses used are not replication-competent. Despite these considerations, the use of retroviral vectors to transduce genes to the liver is limited by the following considerations: 1) These vectors infect only actively dividing cells and 2) very low viral particle titers are obtained from packaging cell lines used to amplify these viruses (Graham FL, and Van Der Eb AJ A New Teqnique for the Assay of Infectivity of Human Adenovirus 5 DNA Virology 1973, 52: 456-467). These two limitations have been successfully overcome in other gene therapy protocols by inducing the proliferation of hepatocytes "in vivo" by the use of hepatic growth factors and by partial hepatectomy, a surgical procedure by which 70% of the liver is removed and in this way the division of the liver cells is stimulated "in vivo". The use of Lentiviral Vectors has allowed the partially significant overcoming of these limitations, since these can transduce cells that do not divide.

BACKGROUND OF THE INVENTION Hepatic cirrhosis is a chronic disease of the liver, where diffuse destruction and regeneration of hepatic parenchymal cells occurs and which results in the diffuse increase of the connective tissue, causing the distortion of the lobar architecture of the liver and inducing alterations. hemodynamics. Therefore, some strategies for the treatment of liver cirrhosis could include the prevention and / or reversion of fibrogenesis, stimulation of hepatic mitosis and reorganization of the architecture. liver tissue. That is why the documents of the state of the art related to the present invention are disclosed, which are mentioned below with the purpose of including them only as references. U.S. Patent No. 5,240,846, refers to the use of gene therapy to treat cystic fibrosis. It uses the sending and expression of a gene called CFTR, which induces a stable correction of the regulation of the chlorine channel. This defect is present in epithelial cells. In this invention, recombinant adenoviral vectors are used, as well as plasmid vectors. However, it has no relationship with the therapeutic genes of the present invention. Likewise, US Patent No. 5,910,487 describes the use of plasmid vectors for the delivery of therapeutic molecules, but there is no relationship with the delivery of the metalloprotease genes or of uPA or of Smad7 or of the truncated receptor of TGF-β ( Transforming Growth Factor-ß) as it is being presented here. US Pat. No. 5,827,703 relates to the use of adenoviral vectors and adenoviral vectors modified to send genes, however none of these vectors contain the genes used in this invention for the treatment of fibrosis. U.S. Patent No. 5770,442 claims the use of a recombinant adenovirus comprising a gene that directs the expression of a protein called "fiber" or a protein called "fiber chimera", however, does not mention in a specific manner which is the therapeutic gene. It also refers to the method of gene therapy, involving the use of such adenovirus and an adenoviral transfer vector for the generation of such recombinant adenoviruses. However, absolutely nothing is mentioned in relation to the use of cloned and inserted therapeutic genes in recombinant adenoviral vectors that in the present invention are used for delivery to fibrotic livers, as well as to other organs such as the kidney, lung and hypertrophic scars and others. Said therapeutic genes are the gene that codes for the latent and active metalloprotease 8, MMP-8, the plasminogen activator derived from the. urokinase (uPA), Smad7 and the truncated receptor of TGF-β, type 2, which are claimed in the present. By extension other members of the gene family represented are also included. U.S. Patent No. 5,166,320, relates to the use of a directed delivery system of genes to introduce exogenous genes into mammalian liver cells. But there is no relation to the genes - which are intended to be sent directly to cirrhotic livers or to kidneys or fibrotic lungs. US Patent No. 5,872,154, discloses a method for reducing the immune response induced by an adenoviral vector by co-administration of the recombinant adenoviral vector and a selected immune modulator, which works by inhibiting the formation of neutralizing antibodies and / or reducing the death of the virally infected cells. The North American Patent No.

No. 5,871,982, discloses a hybrid vector, which comprises a portion of an adenovirus, together with a portion of a viral adeno-associated vector that contains a selected transgene, also describes a hybrid virus by attaching a conjugate with a polycation to a gene adeno-associated network to form a simple particle. Unlike the present invention, in which hybrid viruses were not used, but only adenoviral vectors. In addition, the gene or transgene or the therapeutic gene used is not mentioned in the aforementioned patent. In US Pat. No. 5,856,152, the construction of a hybrid vector is disclosed, which contains the portion of an adenoviral vector in combination with an adeno-associated virus and a selected gene, whereby large quantities of vectors are produced recombinants, but which do not carry cloned therapeutic genes as described in this invention, where specific therapeutic genes are used for the treatment of hepatic fibrosis, renal fibrosis and hypertrophic scars. U.S. Patent 5,547,932, claims a composition of nucleic acid complexes to transfect eukaryotic cells. These complexes are formed by nucleic acids and another substance that has an affinity for the nucleic acid and optionally an internalizing factor, such as a virus or a component of the virus, which can be conjugated. They also use components of certain adenoviral vectors or certain viruses such as Ad2 or Ad5, but do not mention the genes they internalize in the cytoplasm and eventually in the nucleus of these eukaryotic cells. Similarly, US Pat. No. 5,521,291, relates to conjugated adenoviruses that are bound by an antibody to a substance that has an affinity for nucleic acids, in this way recombinant genes are transported to the interior of eukaryotic cells. These complex conjugates and nucleic acids are internalized in the cell, but the therapeutic genes that can be sent are not specifically mentioned. Said patent also does not mention the use of such adenoviruses to treat hepatic fibrosis or cirrhosis or any other type of fibrosis as described in the present invention. U.S. Patent No. 5,585,362, comprises an improved adenoviral vector and methods for making and using such vectors. Although the use of adenoviral vectors in said patent is not disclosed, the adenoviral vectors described in the present invention were used as vectors for the delivery of therapeutic genes. U.S. Patent No. 5,756,086, claims an adenovirus, which is represented by a protein called "fiber", in addition the adenovirus includes a ligand, which is specific for a receptor located in a certain cell type. This adenovirus can have at least a portion of this protein called "fiber" and can be removed and replaced with a ligand, which is specific for a receptor in certain cells of the economy, such as hepatocytes. These adenoviruses can include a gene that codes for a therapeutic agent. Based on the foregoing, the outstanding technical difference of the proposed invention with respect to what is disclosed in said patent is the specificity of the therapeutic agent as metalloproteases (active and latent MMP-8), uPA (urokinase Plasminogen Activator), the truncated receptor of TGF-β and Smad7 for the treatment of various fibrosis. U.S. Patent No. 5,895,759, claims a tissue-specific vector (liver) for gene therapy that can be used to send genes to a damaged liver. Said vector is coupled to a promoter chemically or enzymatically and can also be coupled to an antibody packaged in a polypeptide coat. In addition, the vector or virus to be tested is the hepatitis B virus, in such a way that the transmission of genes to damaged livers that are described in said patent, uses a completely different system from the one intended in this invention and there is no relationship with the process of fibrosis or cirrhosis to be treated. US Pat. No. 5,559,099, discloses an adenoviral recombinant vector comprising an adenovirus chimeric protein which is called a penton, which includes a non-penton sequence and a therapeutic gene to develop a gene therapy method, which involves the use of such adenovirus, adenoviral transfer vectors for the recombination of such adenoviral vectors containing a therapeutic gene. Also, US Patent No. 5,885,808, also claims the use of adenovirus with adenovirus binding molecules to cells of the economy, whose molecules have been modified. As in US Patents Nos. 5,846,782 and 5,712,136, where adenoviral vectors are used, which have been modified to contain different peptide domains. Finally, US Pat. No. 5,670,488, relates to vectors for gene therapy, which are especially useful for cystic fibrosis and also mentions the development of methods for using these vectors. The possible relation of the invention that is being claimed here, with respect to what is disclosed in the cited state of the art, lies in the use of adenoviral vectors, which can be modified, as well as the use of inducible promoters for the genes that go to be inserted in these adenoviral vectors, however, the technical characteristics of the present invention are directed to the specific use of therapeutic genes to treat fibrosis of different types such as hepatic, renal and pulmonary fibrosis, as well as hypertrophic scars. The importance of the present invention, unlike that described in the aforementioned documents of the state of the art, lies in the technical characteristics of the invention itself, as well as in the additional advantages derived therefrom, which are described in more detail. detail below.

ADENOVIRAL VECTORS In the present invention, it has been decided to use ADENOVIRAL VECTORS based on several considerations: 1) These vectors can be generated at very high titers of infectious particles per ml: (109-1010); 2) They infect a great variety of cells, however, when they are administered intravenously, most of them are located in the liver organ; 3) Efficiently transfer genes to cells not found in divisió.ny 4) Rarely, they are integrated into the host genome, which avoids the risk of cell transformation by insertional mutagenesis (Douglas JT, And Curiel DT. Adenoviruses as Vectors for Gene Therapy, Science and Medicine March / April 1997 44-53 and Zern AM, And Kresina TF, Hepatic drug delivery and gene therapy (Hepatology 1997 vol.25, No. 2, 484-491) Adenoviruses are probably the vehicles or These are the most promising vectors for gene delivery in gene therapy protocols in humans, since they have a unique attribute that provides them with great stability when administered to the bloodstream, which allows them to be used efficiently in clinical protocols with a comfortable administration to the patient intravenously (Douglas JT, And Curiel DT, Adenoviruses as Vectors for Gene Therapy, Science and Medicine March / April 1997 44-53). irus are double-stranded DNA viruses, have an icosahedral structure, which infect a wide variety of mammalian cell types, which may reflect the ubiquitous expression of a specific and as yet unidentified cell surface receptor. The binding to the cells is by the protein component of the adenovirus capsid and the virus enters the cell by receptor-mediated endocytosis. More than 40 different human serotypes of adenovirus have been identified, of which types 2 (Ad2) and 5 (Ad5) have been more extensively studied and, therefore, more used in adaptation as vectors for gene therapy. A very important feature of these 2 serotypes is that they have never been associated with human malignant processes. The strategy for the construction of recombinant adenoviruses is based on the organization of the adenoviral genome. The expression of adenoviral genes occurs in two phases, early and late, which are defined with respect to the replication time of the adenoviral genome. The early genes are encoded in four different transcriptional units. The, E2 and E4 code for essential regulatory proteins that induce the replication of the adenoviral DNA, the E3 gene is a non-essential gene. The products of the late genes include the major capsid proteins, which are transcribed from a single promoter (Graham FL, And Van Der Eb AJ, A New Technique for the Assay of Infectivity of Human Adenovirus 5 DNA, Virology 1973, 52 : 456-467). Recombinant adenoviruses are generated by the introduction of the exogenous gene or DNA sequence of interest in replacement of regions of the adenoviral genome required for virus replication. The recombinant adenoviral vectors undergo deletions in the El and E3 regions of their genome. The generation of recombinant adenoviruses is carried out both by the replacement of the El, E3 regions, or by insertion of the exogenous gene between the E4 region and the right end of the viral genome. Vectors based on the insertion of the exogenous gene at the right end of the adenoviral genome or by replacement of the E3 region retain the capacity for replication. On the contrary, the replacement of the early region produces a defective vector in its replication capacity, which can therefore be propagated only in a cell line that supplies "trans" to the absent functions of the replaced adenoviral region or in the presence of a collaborating virus. Of these, replication defective adenoviruses are the most commonly used as gene transfer vectors (Douglas JT, And Curiel DT, Adenoviruses as Vectors for Gene Therapy, Science and Medicine March / April 1997 44-53). The construction of the adenoviral vectors, as well as their application for the treatment of fibrosis, are shown in the examples that are described below.

OBJECTIVES OF THE INVENTION Next, the objectives and advantages deriving from this invention are disclosed. An objective of the present invention is to provide a process for preparing the recombinant adenoviral vectors pAdGFP-MMP-8, by cloning the reporter genes Lac-Z and GFP and the therapeutic gene of the collagenase MMP-8 (metalloprotease). Another objective of the invention is to provide a recombinant adenoviral vector with an exogenous gene or DNA sequence of interest that encodes therapeutic proteins useful in the treatment of generalized fibrosis in the target organs susceptible to suffering from it.

Pharmaceutical compositions containing the recombinant adenoviral vectors in therapeutically effective amounts of viral particles to treat generalized fibrosis are also provided in the present invention. Also, as its uses and therapeutic applications in the treatment of fibrosis. An advantage still of greater relevance in the treatment of generalized fibrosis, particularly that of liver cirrhosis, is that the delivery of the therapeutic genes is carried out through tissue-specific recognition by the administration route used. Another advantage of the therapeutic uses of the invention, which is directed in principle to the treatment of liver cirrhosis, is the treatment of generalized fibrosis in other white organs susceptible to suffering from it, which include, but are not limited to, the treatment of fibrosis in the lung, heart, skin, kidney, among others, in mammals, including man. Another objective is the design of a technology to efficiently send genes to the liver of animals that have cirrhosis, which resemble two types of cirrhosis that affect humans (alcohol cirrhosis and primary biliary cirrhosis).

Another advantage that results from the treatment of fibrosis is that the recombinant adenovirus does not induce lethal toxicity in any of the animals injected with the vectors. Likewise, another objective of the invention allows us to completely discriminate the modification of the staining reaction with X-Gal between the activity of the endogenous tissue β-galactosidase and the bacterial β-galactosidase induced by the infective action of the adenoviral vector. As well as, the use of the green fluorescent protein allows us to verify the in vivo transduction of the organs in the rats to verify if the administration of the vector was adequate, if the expression remains and besides that the animal is not sacrificed it can be Follow up after the surgery.

Finally, all this allows us to suggest that our system constitutes an efficient vehicle to send therapeutic genes such as MMP-8 metalloproteases; collagenases that degrade the excess of deposited collagen and / or genes that code for hepatic regenerative stimulating proteins such as uPA (urokinase Plasminogen Activator), the truncated receptor type II for TGF-β and Smad7 to livers of rats with cirrhosis for the purpose of restore the normal functions of the liver, or other organs affected by the same pathology. Thus, as in the present invention, the process of preparing recombinant adenoviral vectors, pharmaceutical compositions and therapeutic uses for the treatment of fibrosis, particularly in the treatment of liver cirrhosis, is disclosed.

BRIEF DESCRIPTION OF THE DRAWINGS Other features and advantages of the invention will be apparent from the following detailed description, of the objectives and preferred embodiments, of the appended claims and of the accompanying drawings or figures, wherein: Figure 1, shows the cellular pathophysiology of the Hepatic cirrhosis; Figure 2 shows a proof of concept of how gene therapy works by reversing the process of cirrhosis; Figure 3 is a schematic representation showing the cloning and production of the Ad5ß-gal adenoviralvector; Figure 4 shows the schematic development of the AdEasy system for the generation of Recombinant Adenoviruses, specifically pAdGFP-MMP-8; Figure 5 shows the analysis of β-galactosidase expression in cells in culture; Figure 6 shows the determination of the expression of the green fluorescent protein (GFP) in cells in culture; Figure 7 shows the expression of β-galactosidase in different organs after infusion with Ad5-ßgal through the iliac vein; Figure 8 shows the determination of the tropism of the vector Ad5-ßgal to different organs of the cirrhotic experimental animals by chronic poisoning with CC14, demonstrating that the main target organ is the liver; Figure 9 shows the determination of the tropism of the AdSßgal vector to different organs of the cirrhotic experimental animals by ligation of the bile duct, demonstrating that the main target organ is the liver; Figure 10 shows histological slices of images representative of the efficiency of "in vivo" transduction assays of the Ad5-ßgal vector in cirrhotic rats with chronic administration of CC1; Figure 11 shows histological sections of images representative of the efficiency of "in vivo" transduction assays of the .Ad5-ßgal vector in cirrhotic rats by ligation of the common bile duct; Figure 12 shows the "in vivo" determination of the green fluorescent protein expression; Figure 13 shows the cloning strategy of latent MMP-8 and active MMP-8; Figure 14 shows the mechanisms of complex formation with the DNA of MMP-8s for "in vitro" transfection assays in cells of hepatic origin (HepG2); Figure 15 shows the verification by agarose gel electrophoresis of the success of the cloning of the MMP-8 cDNAs in the appropriate plasmids; Figure 16 shows the efficiency of transfection in HepG2 cells (cells of hepatic origin) with the β-galactosidase and pcDNA-MMP-8 plasmids; Figure 17 shows the analysis by the reverse transcriptase-associated polymerase chain reaction (RT-PCR) of the messenger RNAs of MMP-8; Figure 18 shows the determination of the collagenolytic activity in the protein secreted into the culture medium by the HepG2 cells after being transfected with the cDNAs of latent MMP-8 and active MMP-8; Figure 19 shows the hormonal regulation of the expression of the MMP-8 gene under the transcriptional control of the regulator PEPCK and Figure 20 shows the dose-response assay of the different doses used to determine the optimal hepatic transduction response in I live with the reporter gene of ß-galactosidase.

DETAILED DESCRIPTION OF THE INVENTION There are many reports in which it is demonstrated that by administering systemically recombinant adenoviral vectors (AdR) in healthy experimental animals, the organ of predilection to which they are infected is the liver. To date, it was not known if the AdR were able to transduce the livers of rats with cirrhosis. As already mentioned, liver cirrhosis is characterized by an increase in fibrosis throughout the hepatic parenchyma mainly around the central and portal veins, forming a barrier that prevents free exchange between the sinusoid and the hepatocytes (Antoni PP, Ishak KG , Nayak NC, Poulsen HE, Scheuer PJ, Sobin LH The morphology of cirrhosis: definition, nomenclature, and classification Bulletin of the World Health Organization, 1977; 55,521-540 and Scott L. Friedman The cellular basis of hepatic fibrosis: Mechanisms and treatment strategies, The New Ingland Journal of Medicine 1993, vol 328 No. 25: 1828-1835), which is why this protocol was designed to verify if genes could be sent to the liver even with this barrier. Therefore, our hypothesis is: recombinant adenoviral vectors containing the reporter genes Lac-Z and GFP (Green Fluorescent Protein), are capable of transducing the livers of rats with cirrhosis even when the lobar architecture of the organ is altered In such a way that we could send to these livers, therapeutic genes such as collagenases (MMP-8), Urokinase-Flasminogen Activator (u-PA), the truncated receptor of TGF-ß type 2 and Smadl, which degrade excess collagen proteins deposited and / or that prevent the exacerbated synthesis of collagenic proteins, as shown in Figures 2 and 18 and / or genes coding for hepatic regenerative stimulating proteins such as uPA for the purpose of restoring normal functions of the liver as exemplified in Figure 2. With the development of the invention that is claimed in this, a line of research is initiated to perform gene therapy as an alternative for the treatment of chronic-degenerative diseases, specifically liver cirrhosis in humans, by establishing an efficient vehicle to send genes to the liver that produce therapeutic proteins that help to restore the normal functions of the liver, see figure 2, where it is shown as efficiently sending a therapeutic gene to the liver, in this case a collagenase (matrix metalloproteases, MMPs), the degradation of the collagen through the over-expression of these metalloproteases. In Figure 3, the strategy for the cloning and production of an adenoviral vector is outlined. Plasmid p? ElsplB containing Ad5 adenovirus sequences, to which the reporter gene was inserted. of bacterial origin lac-Z. This plasmid was recombined with the pBHGlO to obtain complete viral particles after co-transfecting into the 293 cell line. The pAdGFP vector was obtained as follows: The MMP-8 gene (from the PEPCK-MMP8 plasmid) was cloned in the delivery vector, pAdTrack-CMV, the resulting plasmid is linearized by directing with the restriction endonuclease Pme I, and then cotransformed in E. coli bacteria (BJ5183) with the plasmid pAdEasy-1, the recombinant colonies were selected by resistance to kanamycin, and recombination is confirmed by restriction analysis with endonucleases. Finally, the linearized recombinant plasmid is transfected into the packaging cell line (cells 293), recombinant adenoviruses are obtained within 7 to 12 days as outlined in figures 3 and 4 (TongChuan H., Shibin Z., Luis T., Jian Y., Kenneth W. and Vogelstein Bert: A simplified system for generating recombinant adenoviruses. Proc. Nati Acad. Sci. USA Vol. 95: 2509-2514, March 1998). To evaluate the degree of transduction in vi tro, the HepG2 cell line of hepatic origin and with mouse isolated perifonal macrophages was used. Figure 5 shows the expression of β-Galactosidase in cells in culture. A), B) and C) correspond to HepG2 cells (320X); D), E) and F) are mouse perifoneal macrophages (100X). In C and F the transduced cells are shown with 1 x 108 viral particles / ml of the Ad5ß-Gal vector. Three techniques were used to compare the degree of incorporation of the Lac-Z reporter gene, the plasmid pPGKßGal was administered to each culture box by precipitation with Calcium Phosphate (Chen C, And Okayama H. Calcium Phosphate-Mediated Gene Transfer, to Highly Efficient System for Stably Transforming Cells with Plasmid DNA, Biotechniques 1988, 6: 632-638), DNA-Polylysine-Lactose Complexes (Martinez-Fong D, Mullers an JE, Purchio AF, Armendariz-Borunda J, And Martinez-Hernandez A Nonenzimatic Glycosylation of Poly-L-lysine: A new Tool for Targeted Gene Delivery, Hepatology, Vol. 20, No. 6: 1602-1608), with the vectors Ad5-ßgal and pAdGFP-MMP8. The visualization of the ß-gal activity was verified with the X-gal reagent and the GFP in a fluorescence microscope. For the in vivo test, the development of ß-gal was standardized using different pHs of the development suspension with the X-gal reagent (Weiss DJ, Ligitt D, and Clark JG, In Situ Histochemical Detection of β-Galactosidase Activity in Lung : Assesment of X-gal Reagent in Distinguishing LacZ Gene Expression and Endogeneous ß-Galactosidase Activity, Human Gene Therapy September 1, 1997, 8: 1545-1554). The models of experimental hepatic cirrhosis that were used are: a) carbon tetrachloride intoxication (CC14), in which liver cirrhosis is established from the eighth week of the intraperitoneal administration of CC14 (Mion F, Geloen A, August E, And Minaire Y. Coal Tetrachoride-Induced Cirrhosis in Rats: Influence of the Acute Effects of the Toxin on Glucose Metabolism, Hepatology 1996, Vol 23, No. 2: 582-587); and b) bile duct ligation (BCL), in which frank cirrhosis is observed after week 4 of the surgical procedure (Lee SS, Girod C, Braillon A, Hadengue A, and Lebec D. Hemodynamic Characterization of Cronic Bile Duct -Ligated rats; effect of Pentobarbital Sodium Am Journal Physiol 1986; 251: 176-180; Nakano S, Harakane J, and Hasimoto H. Alteration in Bile ducts Peribiliary Microsirculation in Rats After Common Bile Duct Ligation, Hepatology, 1995, Vol. 21, No. 5 1380-1995, Dumaswala R, Berkowitz D, And Heubi JE, Adaptive Response of the Enterohepatic Circulation of Bile Acid to Extrahepatic, Cholestasis Hepatology, 1996, Vol. 23, No. 3: 623-629 and Pod JL , Estañes A., Pedraza-Chaverri J., Cruz C, Pérez C, Huberman A. and Uribe M.: Chronology of portal hypertension, decreased sodium excretion and activation of the Renin-Angiotensin system in experimental biliary cirrhosis Rev. Invest. Clin 49: 15-23, 1997). It was administered at the same time and with the same batch of Ad5ßgal to control rats without cirrhosis. Rats of 5 and 8 weeks of intoxication with CC14 and rats of 2 and 4 weeks of LCB were sacrificed, 72 hrs after administration of AdR for histological analysis and to determine the expression of the encoded β-galactosidase (β-gal) protein for the AdR, for this purpose liver, spleen, heart, lungs, kidneys and brain were extracted, sections of tissue were cut into cubes of 5-6 mm, which were embedded in the Tissue-Tek OCT freezing medium, the tissues were frozen at -30 ° C and cut with a cryostat to obtain 8μm sections. These cuts were placed on silanized glass slides and fixed with balanced formalin at pH 8.5, for 15-30 min and exposed to the X-gal reagent for 16-18 hrs, counterstaining with the neutral red dye (Weiss DJ, Ligitt D, and Clark JG.) In situ Hitichemical Detection of β-Galactosidase Activity in Lung: Assesment of X-gal Reagent in Distinguishing lacZ Gene Expression and Endogenus ß-Galactosidase Activity, Human Gene Therapy September 1, 1997, 8: 1545-1554). The percentage of positive cells was determined by morphometric analysis in multiple fields of the same size and calculating the average. Also cuttings were made from the livers of rats with cirrhosis, which were embedded in paraffin, cut and stained with Syrian red, Syrian red stained specifically collagenic proteins (Armendariz-Borunda J, and Rojkind M. A Simple Quantitative Method for Collagen Typing in Tissue Samples: Its Application to Human Liver with Schistosomiasis. Collagen Reí. Res. 1984, Vol. 4: 35-47). With this technique we can verify the degree of fibrosis and the increase of bile ducts in a clear way in the hepatic parenchyma. To verify the transduction of cells in vivo with GFP, we used healthy Wistar rats, which were administered the vector pAdGFP-MMP8, 72 hrs, then a medium laparatomy was performed and the exposed organs were visualized in the fluorescence microscope, closing the wound later to keep the animal alive. The previous results presented here regarding the study of the pathophysiology of experimental hepatic cirrhosis can be summarized in figure 2. This figure outlines the participation of pro-inflammatory and pro-fibrogenic cytokines produced in vivo by Kupffer cells, which will activate in turn the hepatic stellar cells to produce excess collagen and be deposited in the subendothelial space, obstructing the exchange between hepatocytes and sinusoids (Armendáriz-Borunda J., Katayama K. and Seyer JM: Transcriptional mechanisms of type I collagen gene expression are differentially regulated by IL-lß, TNFa and TGFβ into cells, J. Biol. Chem. 267: 14316-14321, 1992, Armendáriz-Borunda J., Katai H., Jones CM, Seyer JM, Kang AH and Raghow R.: Transforming growth factor B is transiently enhanced at a critical stage during liver regeneration following CC14 treatment, Laboratory Investigation 69: 283294, 1993 and Armendáriz- Borunda J., Roy N., Simkewich C, Raghow R., Seyer J.M. and Kang A.H .: Activation of Ito cells involves regulation of API binding proteins and induction of type I collagen gene expression. Biochemical Journal 304: 817-824, 1994). The degree of incorporation of the Lac-Z gene in cells in culture visibly contrasted when comparing the Calcium phosphate techniques, DNA-Polylysine-Lactose complexes. and with the recombinant adenoviral vector in HepG2 and MPR (mouse perifoneal macrophages) the degree of transduction with adenovirus being 100% and with the other two techniques approximately 1% as shown in figure 5. Figure 6 shows the Expression of green fluorescent protein (GFP) in cells in culture. A) Peripheral mouse macrophages transduced with the adenoviral vector pAdGFP-MMP8, 72 hrs after its administration (50X), B) HepG2 cells transduced with the adenoviral vector pAdGFP-MMP8, 72 hrs after its administration (50X) and C) HepG2 cells without the adenoviral vector. All images were taken in a fluorescence stereomicroscope. It should be noted that in the development of β-galactosidase activity, the cells have to be fixed and die, in the GFP assay, the cells remain intact and alive.

In figure 7, the expression of ß-gal in different organs after the infusion put Ad5ßgal via the iliac vein. Fixation, washing and X-gal solutions were used at different pHs to discriminate between the endogenous expression and the exogenous bacterial β-galactosidase. In figure A a pH of 7.0 was used and in figure B the pH was 8.5, this summarizes the results of the tests of the different experimental conditions and it can be seen that the exposure of tissues to the X-Gal solution at pH 8.5, allowed us to eliminate the expression of endogenous β-galactosidase. We performed freezing cuts of the following organs: liver, kidney, lung, heart, brain and spleen of rats healthy and intoxicated with CCI4 for five and eight weeks, as shown in figure 8, the graphs clearly show us as the main organ White is the liver, both in healthy rats and in rats with chronic administration of CCI4. A), five weeks of administration of the CCI4 and B), eight weeks of administration of the CCI4. The spleen and the lung have a degree of transduction of less than 1%, so in the graphs it is very little known; to which they were administered a dose of 3 x 1011 PV / ml (viral particles per milliliter) of the vector Ad5ßGal. The healthy control rats present a total of 70% of transduced hepatocytes, in addition to the liver, the spleen and the lung have less than 1% transduction, in the other organs no transduction was found. Freezing cuts were also made of the same organs of rats, sanas and rats with two and four weeks of LCB, see in figure 9, the graphic representations that clearly show us how the main target organ is the liver, both in healthy rats as in rats with bile duct ligation (XCB¡L.A), two weeks of LCB and B), four weeks of LCB. The spleen and the lung have a degree of transduction of less than 1%, so in the graphs it is not very noticeable. With a dose of 3 x 1011 PV / ml of the AddßGal vector. The rats with LCB present a total of 10% of transduced hepatocytes, in addition to the liver, the spleen and the lung present less than 1% transduction, in the other organs no transduction was found. Figure 10 shows the histological results with the hepatic cirrhosis model induced by chronic administration of CC1, where A) represents a liver cut of a normal rat 72 hrs after administration of the Ad5ßGal via the iliac vein (a cut representative of the experiments of a total of five rats). More than 70% of hepatocytes are positive for the expression of ß-gal (200X); D) the same liver as in Figure A) but were stained with Sirius Red to observe the synthesis and deposit of collagen (200X); B) liver with five weeks of chronic poisoning with CC14. Approximately 30-40% of the hepatocytes were transduced successfully; E) the same livers as in B) but stained with Sirius Red, the increase of the present quantity of collagen is remarkable and the architecture begins to distort (200X); C) liver of rats after eight weeks of chronic poisoning with CCI4 where a typical cirrhosis has been established, again, more than 40% of liver cells were positive for the expression of ß-gal and F) the same livers as in C ) but dyed with Sirius Red. A very important characteristic is the presence of thick collagen septa that form between the central veins and the portal tract (200X). In Figure 11, the results obtained in the model of cirrhosis induced by bile duct ligation (BCL) are shown. A) shows a liver cut from a normal rat 72 hrs after administration of the Ad5ßGal via the iliac vein (a cut representative of the experiments of a total of five rats), more than 70% of the hepatocytes are positive to the expression of ß -gal (200X); D) the same liver as in A) but were stained with Sirius Red to observe the collagen (200X amplification); B) rat liver after two weeks of LCB, the ß-gal test was performed 72 hrs after administration of the Ad5ßGal via the iliac vein. Approximately 10% of the hepatocytes were transduced successfully with the reporter gene; E) the same livers as in B) but dyed with Sirius Red. The architecture begins to distort due to the fibrosis induced by the invention. The preparation of a pharmaceutical composition that includes the recombinant adenoviral vectors of the invention, can be carried out by the use of standard techniques well known to those skilled in the art. combination with any of the pharmaceutically acceptable carriers described in the state of the art, including but not limited to starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, magnesium stearate, stearate of sodium, talc of glyceryl monostearate, sodium chloride, glycerol, propylene glycol, water, ethanol and the like. These compositions take the pharmaceutical form of solutions, suspensions, tablets, lozenges, capsules, powders and prolonged release formulation and the like. The above description and the following examples are intended to illustrate particular modes of carrying out the invention and should not be considered as limiting the scope of protection thereof.

EXAMPLES Example 1 METHODOLOGY to demonstrate the activity of Metalloprotease or Collagenase (MMP-8) and also regulate its function a) Cell culture HepG2 cells, a cell line cholestasis, were cultured as well as the considerable increase in the number of bile ducts (200X ); C) rat liver after four weeks of LCB to produce cirrhosis, the ß-gal test was performed 72 hrs after administration of the Ad5ßGal via the iliac vein. Again, 10% of the hepatocytes were transduced successfully and F) the same livers of C) but stained with Sirius Red. Note the abundant deposit of collagen proteins and the great proliferation of bile ducts (200X). Figure 12 shows a mean laparatomy of a healthy Wistar rat that was administered the vector pAdGFP-MMP8, the expression of GFP in the liver and in insignificant amounts in the spleen is clearly observed. An important fact is that the injection of the adenoviral vectors did NOT induce lethal toxicity in any of the experimental animals both healthy and controls. The preferred way of applying the present invention is by intravenous delivery of the recombinant adenoviral vectors of the invention or of the pharmaceutical composition containing them, wherein a therapeutically effective amount with a convenient unit dose rate is provided to an individual. who suffers from fibrosis, said regimen can be adjusted according to the degree of affliction. Generally, a unit dose of approximately 107-1014 viral particles per individual is employed. of parenchymal origin from human hepatoma, in culture boxes of 60 mm diameter at 37 ° C in a humid atmosphere with 95% air and C02 5% in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 2 mM L-glutamax and antibiotics (100 U / ml penicillin and 100 μg / ml streptomycin). b) Expression vectors of the latent and active genes of MMP-8 Plasmids with two types of MMP-8 genes were used to transfect hepatic cells: the plasmid pcDNA-MMP8 containing the cDNA encoding the MMP -8 latent (pro-MMP-8) together with the strong cytomegalovirus viral promoter (CMV) and the plasmid pcDNA3-MMP8 containing the cDNA encoding the active MMP-8 together with the CMV promoter. The latter was created by subcloning from the plasmids pcDNA3 and PETIIa-HNC by cutting with the restriction enzymes BamHl and Xbal and inserting the PCR product coding for the catalytic domain of MMP-8 (which lacks the propeptide and carboxyl fragments). terminal), as shown in Figure 13, the sending of latent and active MMP8 genes. Two types of plasmids with the MMP8 gene were used for delivery to hepatic cells in culture: 1) pcDNA3-MMP8, plasmid with the strong cytomegalovirus (CMV) viral promoter and the cDNA coding for collagenase in its shape.

As a reporter gene, the plasmid pSV2-βgal was used, which has inserted the gene coding for the β-galactosidase enzyme adjacent to the SV40 virus promoter. c) Transformation, amplification and purification of plasmids To obtain a sufficient quantity of each of the plasmids to be used in the different assays, E. coli bacteria of the strain DH5a ™ (process known as transformation) were introduced as indicated in the protocol of the provider of these competent cells (Life Technologies, Gaithersburg, MD): in a reaction tube, 50 μl of the competent strain DH5a was used, to which 2 μl of plasmid (1-10 ng DNA) was added, then of mixing was incubated on ice for 30 min and then a thermal shock was applied at 37 ° C for 20 sec and immediately placed on ice for 2 min, after this time 0.95 ml of bacterial culture medium Luria was added. Base (LB) and stirred at 225 rpm for 1 h at 37 ° C for the expression of the plasmid. After the expression, 50 μl of the reaction mixture was taken to seed an LB agar culture plate with 100 μg / ml ampicillin and incubated at 37% ° C overnight. The colonies that grow after this period are those that contain the plasmid of interest which gives them the ability to resist the antibiotic.

To amplify the amount of the plasmid, a palin of cellosia was taken from the agar plate to grow in 1 L of LB medium containing 100 μg / ml of ampicillin for 24 h at 37 ° C with constant agitation at 225 rpm. Once the optical density of the culture is > 0.6 is centrifuged at 6,000 rpm for 10 min to collect the bacterial pellet. From this bacterial pellet, plasmid DNA was isolated from the genomic DNA of the bacteria using a plasmid purification kit (Monster-prep, BIO101, Vista, CA) which is based on the alkaline lysis of the bacterial wall, the release of the plasmid of its interior and the separation of this DNA by means of a particular resin. The plasmid DNA quantification was performed by spectrophotometrically measuring the resulting absorbance at λ = 260 nm. d) Transfer of cells in culture One of the most widely used methods for the introduction of genes into eukaryotic cells is the transfection of DNA with calcium phosphate, in which the exogenous DNA is deposited as a fine precipitate on the surface of the cell. the cell, to later be incorporated by it and transiently integrated into the chromosomal DNA. In order to direct the DNA with greater selectivity towards the liver cells, the DNA is sent in the form of a complex with polylysine-lactose, given the characteristic that these cells have a specific galactose receptor on their surface. To do this, the HepG2 cells were cultured at approximately 70-80% confluency for transfection with the plasmids pcDNA-MMP8, pcDNA3-MMP8 and pSV2-β-galactosidase. Transfection was done both by the calcium phosphate precipitation method (Graham and Van derEb, 1973; Chen and Okayama, 1988) as well as that of complex formation with polylysine-lactose (Martínez-Fong et al., 1994). In brief: the newly formed precipitate is added to the cells in culture, product of the addition to the plasmid DNA of a 2M CaCl solution and a HEPES pH 7.12 buffer solution for the case of calcium phosphate transfection, or the DNA complex is added with polylysine-lactose. The cells are incubated 4 to 16 hours to allow the precipitate to adhere to the cell surface and then the DNA is endocyted and transiently enter the nucleus, at the end of this time the culture medium is changed to a fresh one, see figure 14 , where the HepG2 hepatic cell line was cultured in DMEM medium with 10% fetal bovine serum. When presenting a 60-80% confluence, 10 mg of the plasmid with the MMP-8 gene was added to the culture medium, both in its latent form and in the active or mature form. At the same time, the prokaryotic β-galactosidase (ß-gal) gene was sent to monitor the efficiency of incorporation and expression. The MMP-8 gene was sent in various forms: naked, in complex with CaP0 or in complex with polylysine-lactose. e) Formation of the polylysine-lactose and DNA: polylysine-lactose complexes (DNA: PL) The polylysine-lactose complex is formed by reacting 14.8 mg of poly-L-lysine (0.1 N) with 200 μl of a-lactose 0.5 N (lactose-polylysine ratio: 1.0N), then 20 mg of the reducing agent 3M sodium cyanoborohydride is added and incubated at 37 ° C for 48 h with constant agitation at 225 rpm. At the end of this time the reaction mixture is passed through a desalination column (Biorad 10-DG) previously conditioned with phosphate buffer solution (PBS pH 7.2) to which it is eluted with this same buffer. The carbohydrate content is determined by the DuBois method (1956) to analyze the degree of lactosylation of the complex and the polylysine content according to the method of Shen et al., (1984), which is taken as the basis for the eluted fractions. to evaluate the final concentration of the PL complex. The fraction with the highest concentration of PL is used for its subsequent reaction with the plasmid DNA with the gene of interest as shown in figures 14 and 16. To evaluate the optimal molar ratio of DNA: PL to be used in transfection assays the DNA with different concentrations of PL. At the end of 1 h of incubation, the samples were applied to a 1.0% agarose retardation gel and subjected to DNA electrophoresis (60mV, 1.5h), in which the DNA: PL complex with the highest PL content crossed a smaller distance compared to that covered by the free plasmid (0% retardation). The DNA: PL ratio causing 80-90% of migration retardation was used, as shown in figure 16, the incorporation and expression of the exogenous genes of β-galactosidase and pcDNA-MMP8 sent to the cells in complexes with CaP04 and polylysine-lactose. f) Transient expression assays using the reporter system of β-galactosidase (β-gal) This system determines the activity of the β-galactosidase enzyme as a measure of the level of expression of the gene of interest transfected together with the gene coding for this enzyme. Β-galactosidase is a bacterial enzyme that catalyzes the conversion of the colorless substrate X-gal to a blue coloration product. Due to this, the activity of β-galactosidase observed in eukaryotic cells subjected to transfection will indicate the successful incorporation of the gene of interest associated with the bacterial gene.

The ß-gal assay for staining cells in culture boxes consists of fixing the cells at 4 ° C for 5 min with 2% p-formaldehyde, the subsequent washing with PBS (3X) and the addition of 1 ml of a PBS staining solution containing 20mM potassium ferricyanide, 20mM potassium ferrocyanide and 2mM magnesium chloride, followed by the addition of the X-gal substrate to a final concentration of 0.5 mg / ml, after incubation at 4 ° C during the whole night (18 h) the cells of blue coloration are identified under the microscope (Ausubel, 1995). g) RNA extraction 48 hrs after transfection, the cells were harvested to perform the extraction of the RNA by the method of Chomczynski and Sacchi (1987) using the Trizol reagent (fi) as described below: to each of the boxes was added 1 ml of PBS solution and the cells were scraped from the box to transfer them to an eppendorf tube; Later, it was centrifuged at 1,000 rpm for 1 min and 500 μl of Trizol was added to the cell pellet, followed by homogenization and immediately after incubation for 5 min at 4 ° C. 100 μl of chloroform was added and incubated for 5 min at 4 ° C. After this time, it was centrifuged at 12,000 g for 15 min at 4 ° C and the aqueous phase (upper) was transferred to another tube to which the same volume by volume of isopropanol was added and incubated at -70 ° C during 15 min to precipitate the extracted RNA. Subsequently, it was centrifuged at 12,000 g for 15 min at 4 ° C and the supernatant was removed by decanting and drying the tube with clean and sterile paper. 500 μl of 75% ethanol was added and centrifuged at 12,000 g for 10 min at 40 ° C.

Finally, the tablet was resuspended with 20-50 μl of deionized water treated with diethylcarbonate and the concentration of RNA obtained in a spectrophotometer was quantified at a wavelength? of 260 mn. h) Analysis of the expression of the MMP-8 gene by the polymerase chain reaction (PCR) associated with the reverse transcriptase reaction (RT) To determine the degree of expression of the exogenous gene of MMP-8 incorporated in the cells were obtained obtaining the complementary DNA (cDNA) from the previously extracted RNA and subsequently the amplification of the expression signal was carried out through the polymerase chain reaction. To obtain the cDNA the following procedure was used: 2 μg of total RNA were taken to a volume of 8 μl with sterile deionized water and incubated at 70 ° C for minutes. Following this, the sample was stirred in ice water for 5 min and on ice, the following reagents were added: 4 μl 5X buffer for the RT enzyme, 4 μl 2.5 mM dNTP's mix, 1 μl random primers (1 μg / μl) , 1 μl inhibitor RNase (lU / μl) and finally 2 μl of the reverse transcriptase enzyme (200 U / μl). The reaction mixture was incubated at room temperature for 10 min and subsequently at 37 ° C for 1 hr. At the end of this time, it was placed immediately at a temperature of 95 ° C for 10 min and finally placed on ice with water for 5 min with constant agitation and stored at -70 ° C until its later use. To analyze the specific expression of the MMP-8 gene, the polymerase chain reaction was performed using the primers or oligonucleotides. specific for this gene according to the experimental conditions described below: in a reaction tube with 2 μl of cDNA, 5 μl 2.5 mM MgCl2, 5 μl 5X buffer for the polymerase enzyme from Moloney murine leukemia virus (MMLV) was added ), 2 μl 2.5 mM dNTPs, 5 μl of the 3 μM sense oligonucleotide, 5 μl of the 3 μM anti-sense, 1 μl of the polymerase enzyme (1 U / μl) and brought to a final volume of 50 μl with deionized water (Innis et al., 1990). The sense oligonucleotide specific for MMP-8 is 5'-AGCTGTCAGAGGCTGGAGGTAGAAA-3 ', and the antisense is 5'-CCTGAAAGCATAGTT GGGATACAT-3' (Cole et al., 1996). After the addition of these reagents, the mixture was placed in a thermal cycler for 30 cycles according to the following program: denaturation (94 ° C, 5 min), alignment (60 ° C, 1 min) and extension (72 ° C) , 1.5 min). At the end of this time, the PCR products are subjected to electrophoresis (60mV, 1.5 h) on a 1.5% agarose gel. i) Collagenase activity assay The analysis of the enzymatic activity of the collagenase was carried out to determine the functionality of the enzyme produced, since this protein could be found enzymatically inactive, which could not be known through the unique determination of the RNA that encodes it After 24 hours of maintaining the cells in culture with serum-free medium, the culture medium is harvested and the collagenase activity secreted by the cells is determined therein by a modified method of Hasty et al., (1986). electrophoresis in an 8% polyacrylamide gel to identify the degradation products. Briefly: cellular supernatants containing 1-1.5 μg of protein were incubated at 27 ° C for 18 hrs with 5 μg of native type I collagen and 60 μl of incubation buffer: 50mM Tris-HCl, 5mM CaCl2, 0.02% NaN3, 50mM arginine, Triton X-100 1% and in the absence or presence of lmM APMA, pH 7.6. Finally, 30 μl of the reaction product was mixed with 30 μl of sample buffer for proteins and 7.5% denaturing polyacrylamide electrophoresis was run to identify the alA and a2A degradation products of type I collagen.

Example 2 RESULTS to demonstrate the activity of Metalloprotease or Collagenase (MMP-8) and also regulate its function The subcloning allowed us to incorporate the complementary DNA of MMP-8 coding for the fully functional enzyme into a vector appropriate to our needs. Thus, in Figure 15, the electrophoresis of the DNA fragments released by cutting the plasmids of MMP-8 with restriction enzymes is shown. Lane: A) 1 kb ladder DNA base marker (Gibco BRL); B) Perfect DNA marker (Novagen, Inc.); 1) pcDNA-MMP8 cut with BamHl and Xbal; 2) pcDNA3-MMP8 cut with BamHI and Xbal; C) fX174 marker (Gibco BRL); ? HindIII marker (Gibco BRL), where latent MMP-8 cDNAs (lane 1) and mature MMP (lane 2) were successfully subcloned into pcDNA and pcDNA3 expression vectors. The inserts released with restriction enzymes BamHl and Xbal are clearly observed. The bands stained with ethidium bromide correspond to each of the cDNAs comprised between approximately 506 and 560 base pairs (bp), for the mature and latent MMP cDNAs, respectively. To evaluate the efficiency of the incorporation of the cDNA of MMP-8 sent to HepG2 cells as a complex with CaP04 and with polylysine-lactose, the co-transfection of this plasmid was carried out together with that of the reporter gene of β-galactosidase. In this way, the cells that are observed under the microscope with blue coloration indirectly indicate that they have also incorporated the plasmid of interest. Figure 16 shows the expression of β-galactosidase in HepG2 cells co-transfected with the free plasmid, in the form of a CaP0 complex or in its complex form with polylysine-lactose. This figure shows that the coupling of DNA with polylysine-lactose was carried out, given that a higher concentration of polylysine showed a clear retardation of the β-galactosidase plasmid. The selected ratio to transfect the cells was that which retarded 80% of the migration of the plasmid. Once it has been demonstrated that the cells in culture are capable of incorporating and expressing genes that have been transfected, it was necessary to corroborate that said genes were transcribed by the machinery of the host cells by the RT-PCR assays. Figure 17 shows an analysis by the reverse transcriptase-associated polymerase chain reaction (RT-PCR) of the mRNAs of MMP8 and MMP13. (This plasmid was used as another positive transfection control); wherein a DNA electrophoresis of the ified PCR products of the MMP-8 cDNA sent in its complex form with CaP 4 and polysine lactose has been transcribed for both cases in the transfected HepG 2 cells. It is observed that the signal of the PCR products of the MMP8 (359 bp) was more intense when the plasmid was sent in complex with polylysine-lactose. To demonstrate that the transcript of MMP-8 expressed by HepG2 cells was translated into a functional protein, the enzyme activity assay was performed using type I collagen as a substrate. Figure 18 shows the enzymatic degradation activity of collagen type I of the protein secreted into the medium, which was observed in the cells transfected with the latent MMP8 gene, after activation with the APMA mercurial agent (lane 7) and with the MMP8 gene active in its complex with CaP04 (lane 9) and with polylysine-lactose (lane 10), and its specific inhibition with 2mM EDTA. Negative controls: type I collagen without the addition of cell supernatants (lane 1) and with the addition of trypsin (lane 3), collagen with supernatants of untransfected cells (lane 2). Positive lanes: collagen type I with supernatant of human leukocytes (lane 3), collagen type I with addition of 0.015% bacterial collagenase (lane 4); as well as the degradation products of the native type I collagen separated in an 8% polyacrylamide gel after being incubated with the supernatant of the cells transfected with the latent and active MMP-8 gene. It is observed that in both cases the collagenolytic activity is present in the presence of the organomercurial agent APMA for the case of the former and its inhibition by EDTA for both cases, which indicates that this proteolytic activity corresponds to an interstitial matrix metalloprotease. Incubation of native collagen type I with trypsin showed no degradation. Therefore, this experiment clearly demonstrates that the action of MMP-8 was specific given the intact nature of the collagen molecule. Figure 19 shows the evidence that the activity of enzymes that specifically degrade collagen can be controlled (switched off and / or turned on) by cloning their respective cDNAs which in turn are under the transcriptional control of regulatable genes such as PEPCK (phosphoenol-pyruvate carboxikinase). It is clear to see that both the stimulation of the cells in culture with Glucagon (lanes 5 and 6) and cAMP (lanes 7 and 8) overstimulate the production of the messenger RNA that codes for MMP-8, as well as that Insulin decreases this production ( lanes 9 and 10). Observations on endogenous β-galactosidase activity suggest that this activity tends. to be granular and weaker in color than the dense blue coloring that is the result of the activity of the exogenous enzyme (Shimohama S., Rosenbergh MB, Fagan AM, Wolff JA, Short MP, Brakfielf XO, Friedman T. and Gage FH : Genetically modified cells into the mouse brain: Characteristics of E. coli-galactosidase as a reporrter gene Brain Res. 5: 271-278, 1989). Many modifications have been described to increase the specificity in the assay for the determination of the exogenous lac-Z gene. Thus, according to the data previously reported by Weiss DJ, Ligitt D, And Clark JG. In Situ Histochemical Detection of ß-Galactosidase Activity in Lung. Assessment of X-gal Reagent in Distinguishing lacZ gene Expression and Endogenous ß-Galactosidase Activity. Human Gene Therapy September 1, 1997, 8: 1545-1554; In the present invention an X-Gal solution was used at a stable pH of 8.5, thus, the activity of the exogenous β-Gal was demonstrated while the endogenous activity was minimized in vivo. One of the indicators that are currently used to monitor in vivo the efficiency and localization of cells transduced with recombinant adenoviruses is the detection in the expression of the green fluorescent protein.

(GFP) For this, the gene coding for this protein is subcloned in the adenoviral vectors in such a way that by using a fluorescent microscope, the fluorescence can be observed directly without sacrificing the experimental animal, to which the vector was administered.

(Rojas-Martinez A., Wyde P.R., Montgomery C.A., Chem S-H., Woo SLC and Aguilar-Cordova E .: Distribution toxicity and lack of replication of an ElA-recombinant adenoviral vector after systemic delivery in the cotton rat. Cancer Gene Ther. 1998 and TongChuan H., Shibin Z., Luis T., Jian Y., Kenneth W. and Vogelstein Bert: A simplified system for generating recombinant adenoviruses. Proc. Nati Acad. Sci. USA Vol. 95: 2509-2514, March 1998). Many data have been obtained in which it is demonstrated that after the administration of the adenoviruses in an intravenous form in healthy animals, the main target cells were hepatocytes. This observation has been shown in mice, rabbits, dogs and primates (Zern AM, And Kresina TF, Hepatic drug delivery and gene therapy, Hepatology 1997 Vol 25, No. 2, 484-491), but not in rats with cirrhosis. The portal vein injection is likely to be more effective in reaching target cells in the liver by providing a favorable inoculum of viral particles to the entire liver before it is diluted in the peripheral circulation. It has been shown that this route is very effective, but the great disadvantage is that it is required to do a laparatomy. On the other hand, peritoneal administration is a faster and simpler infusion, but does not favor the transduction of hepatocytes. The results of the present invention show that the injection of 3 x 1011 viral particles by iliac vein in Wistar rats of approximately 200 grams of weight, produces a very high level of expression (70% transduction of hepatocytes). Our results are similar to a recent report showing liver-directed transmission of the primate reporter gene via saphenous vein, in which almost the same level of transduction and expression of the transgene occurs in the liver compared to infusion through the portal vein (Marie-Jean TFD, V Poeters, Lieber A, Perkins J, and Kay MA.Multiple Portal for Vein Infusions in Mice: Quantitation of Adenovirus-Mediated Hepatic Gene Transfer, Biotechniques February 1996, 20: 278-285 and Zhu G, Nicolson AG, Zheng X, Strom TB, and Sukhame VP Adenovirus-Mediated ß-Galactosidase Gene Delivery to the Liver Leads to Protein Deposition in Kidney Glomeruli, Kidney International, 1997, Vol. 52, 992-999). Furthermore, the expression of the reporter gene in our animals with cirrhosis induced by chronic administration of CCI4 was surprisingly almost as high as normal (40% of transduced hepatocytes). These results are very exciting since our animals with cirrhosis would hardly survive the surgical procedure required to be able to administer the adenovirus by portal vein. This is because the hapática functional tests, as well as the Prothrombin Times, are very high and the bleeding would appear in an important way. Although rats with bile duct ligation showed a substantial reduction in the number of transduced hepatocytes (5-10%), the number of hepatocytes is also important, which can be transduced with therapeutic genes, such as metalloproteases (MMP-8). ) and / or genes that code for hepatic regenerative stimulating proteins such as uPA (urokinase Plasminogen Activator) and Smad7. It should be apparent to those skilled in the art that other variations not specifically disclosed in the present invention, but which, however, are proposed by the present and which are considered to be within the scope and spirit of this invention. Therefore, the invention should not be limited by the description of the specific modalities disclosed, but only by the following:

Claims (21)

1. CLAIMS 1.- A recombinant adenoviral vector comprising an adenoviral genome from which the El and / or E3 open reading frames have been deleted, but which retains sufficient sequence for said adenoviral vector to be able to replicate in vitro. Additionally said vector contains a therapeutic gene or an AND sequence of interest regulated by ubiquitous promoters and / or tissue-specific promoters, which codes for therapeutic proteins useful in the treatment of fibrosis.
2. 2. The recombinant adenoviral vector according to claim 1, wherein the tissue-specific promoter is PEPCK.
3. 3. The recombinant adenoviral vector according to claim 1, wherein the therapeutic gene or the DNA sequence cloned in said adenoviral vector is selected from the latent and active metalloprotease MMP-8 gene; urokinase-derived plasminogen activator gene (uPA), Smad7 and truncated receptor gene type II of TGF-β, which encode therapeutic proteins that degrade the excess of collagenic proteins deposited in the cirrhotic organs.
4. 4. The recombinant adenoviral vector according to claim 3, wherein the therapeutic gene is a DNA sequence that is selected from the gene of the growth factor for hepatocytes HGF, which codes for stimulating proteins of hepatic regeneration with the purpose of restoring the normal functions of the liver.
5. 5. The recombinant adenoviral vector according to claim 1, wherein the therapeutic proteins for the treatment of fibrosis are the latent and / or active MMP-8 protein; uPA or mutant thereof; TGF-β; betaglycan; HGF and Smad7.
6. 6. The recombinant adenoviral vector according to claim 1, further comprising the delivery of therapeutic genes or DNA sequences that encode therapeutic proteins for the treatment of fibrosis in livers with cirrhosis.
7. 7. The replacement adenoviral vector according to claim 6, wherein the delivery of the therapeutic genes is carried out in other organs with generalized fibrosis.
8. 8. The recombinant adenoviral vector according to claim 7, wherein the tissue-specific recognition of the therapeutic genes to organs with fibrosis is carried out by the administration route used.
9. 9. The recombinant adenoviral vector according to claim 8, wherein the route of administration is intravenous.
10. 10. - The recombinant adenoviral vector according to claim 6, wherein the organs with fibrosis are selected from liver, lung, heart, kidney, skin and hypertrophic scars.
11. 11. The recombinant adenoviral vector according to claim 10, wherein the main target organ is the liver.
12. 12. Recombinant vectors according to claims 1 to 11, wherein the delivery of therapeutic genes is carried out by the use of viral or non-viral vectors.
13. 13. The recombinant vector according to claim 12, wherein the non-viral vectors are selected from cationic and anionic plasmids and liposomes.
14. 14. The recombinant adenoviral vector according to claim 6, wherein the efficient delivery of the MMP-8 collagenase gene to the cirrhotic liver can achieve the degradation of the collagen through the over-expression of metalloproteases.
15. 15. The recombinant adenoviral vector according to claim 1, characterized in that it is used for the treatment of hepatic, pulmonary, renal, heart, keloid and hypertrophic scars, which does not induce lethal toxicity.
16. 16.- A process for preparing recombinant adenoviral vectors by cloning Lac-Z and GFP reporter genes and the therapeutic gene, which codes for therapeutic proteins for the treatment of hepatic, pulmonary, renal, heart, keloid and hypertrophic scars .
17. 17.- The process in accordance with the claim 16, wherein the therapeutic gene is selected from the latent and active metalloprotease MMP-8 gene; urokinase-derived plasminogen activator gene (uPA); Smad7 and truncated receptor gene type II TGF-β.
18. 18. The process according to claim 16, wherein the recombinant adenoviral vector is pAdGFP-MMP-8.
19. 19. A pharmaceutical composition containing a therapeutically effective amount with a unit dose regimen of viral particles of the recombinant adenoviral vectors according to claim 1, for the treatment of hepatic, pulmonary, renal, heart, keloid fibrosis and hypertrophic scars, in combination with a pharmaceutically acceptable carrier.
20. 20. The pharmaceutical composition according to claim 19, wherein the unit dose is about 107-1014 viral particles per individual suffering from fibrosis.
21. 21. The use of recombinant adenoviral vectors according to claim 1 for the preparation of a medicament for the treatment of hepatic, pulmonary, renal, heart, keloid and hypertrophic scars. Next, the definitions of the symbols used in the figures corresponding to the present invention are described, wherein: Figure 1: CEH = STELL CELL HEP TICA CES = ENDOTHELIAL CELL SINUSOIDAL CK = CELL OF KUPFFER ESET = SUBENDOTELIAL SPACE HE = HIPATOCITOS HIDC = LIVER WITH CHRONIC DAMAGE HIN = NORMAL LIVER SINU = SINUSOID Figure 2: COLASE = COLAGENASE DCA = COLLAGEN DEGRADATION TGE = EXPERIMENTAL GENE THERAPY MMPs = METALOPROTEASES Figure 3: CT 293 = COTRANSFECTION IN CELLS 293 PG CsCl = PURIFICATION WITH GRADIENTS OF CsCl Figure 4: BD = RIGHT ARM Bl = LEFT ARM CTBK = COTRANSFECTAR IN BACTERIA AND SELECTION IN KANAMICINA CUL = CULTIVAR Ll Pací = LINEARIZE WITH Pací Ll Pmel = LINEARIZE WITH Pmel PV = VIRAL PARTICLES T 293 = TRANSFECT IN CELLS 293 GENADR = GENERATION OF RECOMBINANT ADENOVIRUS Figure 7: B = CE BRAKE = BRAIN i CO = HEART% CT =% OF TRANSDUCED CELLS H = LIVER P = LUNG R = KIDNEY SAdß-Gal = WITHOUT THE VECTOR Adß-Gal CAdß-Gal = WITH THE VECTOR Adß-Gal X-GAL7 = REAGENT X-GAL pH 7.0 X-GAL8.5 = REAGENT X-GAL pH 8.5 Figure 8: B = BACON CC145 = 5 WEEKS OF INTOXICATION WITH CC14 CC148 = 8 WEEKS OF INTOXICATION WITH CC14 CE = BRAIN CO = HEART% CT =% OF TRANSDUCED CELLS H = LIVER P = LUNG R = KID PV = VIRAL PARTICLES HN = NORMAL LIVER Figure 9: B = CE BRAIN = BRAIN CO = HEART% CT =% OF TRANSDUCED CELLS H = LIVER LCB2S = 2 WEEKS OF BILIARY DUCK LINK LCB4S = 4 WEEKS OF BILIARY DUCK LINK P = LUNG R = KID PV = VIRAL PARTICLES HN = NORMAL LIVER Figure 13: Prot = PROTEIN APMA = MERCURY AMINOPHENIL ACETATE Figure 14: ACTß-gal = ACTIVITY OF ß-GALACTOSIDASE CES = CELLS EAC = ACTIVITY ENZYMATIC PL = POLYLYSIN PROT = PROTEIN RGAL = RESIDUES OF GALACTOSE SNAD = SUPERNATANT Figure 16: ADND = NAKED DNA GELRADN-PL = RETARDING GEL FOR POLYLYSINE Figure 18: CA = WITH APMA CACE = WITH APMA AND WITH EDTA CaP04 = CALCIUM PHOSPHATE CE = WITH EDTA COB = BACTERIAL COLAGENASE COL1 = COLLAGEN TYPE I PL = POLYLYSIN SA = NO APMA SNL = LEUKOCYTE SUPERNER ST = NO TRANSFER TRIP = TRIPSINA Figure 20:% CT =% OF TRANSDUCED CELLS PV = VIRAL PARTICLES SUMMARY OF THE INVENTION The use of gene therapy is proposed for its application in the treatment of diverse fibrosis in humans. The objective is the use of "therapeutic" genes specifically directed to target organs to reverse and / or prevent the development of the process that is fibrous. The potential application of gene therapy to patients with fibrosis and / or cirrhosis will largely depend on the successful delivery of genes coding for therapeutic proteins to livers with extensive fibrosis and that these genes encoding MMP-8 proteins, uPA (or its truncated version), truncated receptor type II of TGF-β and Smad7 are directed by adenoviruses and / or other recombinant vectors that do not transduce (infect) other organs of the economy. Recombinant adenoviruses (AdR) are highly efficient vectors for the transduction of therapeutic genes to different target cells. We have proven that genes can be carried to cirrhotic livers. The delivery of therapeutic genes by said adenoviral vectors and other recombinant vectors may be performed using cationic and anionic liposomes (DOTMA). Likewise, we propose the use of this patent so that it is applied equally to: * Renal fibrosis, * Pulmonary fibrosis, * Hypertrophic and keloid scars (Fibrosis of the skin) and * Other types of fibrosis.