PATENT DOCUMENT

Abstract:
Hepatic cirrhosis is considered a severe health problem in Mexico, since it is the third mortality cause in working-age people and there is no 100% effective treatment. Cirrhosis is characterized by an exacerbated increase of collagen in liver parenchyma, replacing the hepatocytes and thus provoking liver failure. This is one of the reasons why we have used a gene therapy through specific delivery to cirrhotic livers of the gene of human urokinase plasminogen activator (huPA), which activates mechanisms that induce the degradation of excess cellular matrix and stimulate hepatocyte proliferation, obtaining thus a fast re-establishment of the liver function. In the instant invention, the modified human uPA gene was inserted in the adenoviral vector (pAd-ΔhuPA), because it is not secreted and does not provoke hypercoagulation or spontaneous internal bleedings. Moreover, data from the bio-distribution essay with an adenoviral vector with reporter gene β-gal have shown liver specificity as the target organ of the vector. Using ELISA, huPa protein was detected in liver homogenates (4500 pg/ml) in animals treated with pAd-ΔhuPA and was also intracellularly detected through immunochemistry in liver cuts (80% positive cells). huPa induced a dramatic fibrosis reduction (85%) on day 10 of vector administration, compared to control cirrhotic rats and 55% hepatocyte proliferation increase. Liver function tests (ALT, AST, alkaline phosphatase and bilirubin) dropped to nearly normal levels and hepatocyte proliferation was observed. Because of the two beneficial event cascades, gene therapy with modified huPA can be developed as a definite potential treatment for patients with liver cirrhosis.

Full Description:
TECHNICAL FIELD OF THE INVENTION  
         [0001]    The present invention relates to the creation of recombinant viral, non viral, plasmidic and synthetic vectors having the human gene of urokinase derived plasminogen activator cloned, hereinafter huPA cDNA (complementary DNA). It is also related to the utilization of the TGF-beta type II truncated receptor gene. The viral vectors used can be, without limitation, first, second, third and/or fourth generation adenoviral vectors, “gutless” adenoviral vectors, retroviral vectors, adeno-associated vectors. Non-viral vectors can be constituted of phospholipid components, liposomes of various structures and combined through different ligands for specific receptors. Synthetic vectors can consist of the construction and coupling of huPA cDNA and TGF-beta type II truncated receptor with plasmids from different origins and regulatable promoters. huPA cDNA encodes for therapeutic proteins useful in the treatment of hepatic cirrhosis and generalized fibrosis, such as renal fibrosis, pulmonary fibrosis, pancreatic fibrosis, heart fibrosis, hypertrophic scars and keloids (skin scars) and/or in other target organs susceptible of suffering from it. It also relates to a mechanism of tissue-specific recognition of the affected cells in vivo by means of delivery of therapeutic huPA gene to organs chronically affected by fibrosis.  
           [0002]    Moreover, the invention provides an effective way for the treatment of fibrosis through the use of recombinant vectors which are claimed here, as well as the process involve in the preparation of these vectors, the pharmaceutical composition containing them, the treatment methods and their therapeutic uses in fibrosis treatment, which has great commercial potential in the pharmaceutical industry and also presents an important alternative as experimental gene therapy for the treatment of chronic-degenerative diseases characterized by fibrosis, with important therapeutic application in the field of modern medicine.  
         INTRODUCTION  
       Epidemiology and Physiopathology of Hepatic Cirrhosis  
         [0003]    Hepatic cirrhosis represents a world health problem because it is an important mortality cause. It is a terminal illness usually caused by chronic alcohol ingestion and hepatitis C infection and there is no definite available treatment when it affects adults. About 14 million people are affected by alcoholism in the USA and the figure is similar in Mexico (Mariani, S., Birmingham, K. and Novak, K. Knocking out Alcohol Damage. Nature Med. 1999:5(11):1243). Hepatic cirrhosis is considered a severe health problem in Mexico, since it is the fourth cause of death in working-age people and there is no 100% effective treatment. Moreover, hepatic cirrhosis is also a cause of death in children due to the consequences of biliary obstruction. However, in this case, the incidence is much lower and the surgical approach through Kasai shunt mitigates the illness during a few months. However, 50% of the children who undergo said surgery die during the following months and the rest of them who partially respond to the shunt effects are channeled to a possible liver transplantation. Currently, there are gene therapy protocols for other chronic-degenerative illnesses, but up to now no protocol has been reported in which a gene therapy is used to cure cirrhosis. Thus, in the instant invention, this gene therapy protocol has been developed at preclinical level. Its further use with human beings will depend on the successful and safe sending of genes coding for therapeutic proteins in livers with extensive fibrosis, using as the main sending strategy, viral, non-viral, plasmidic and synthetic vectors. For this purpose, a preclinic study was previously conducted to determine the safety, transduction efficacy, toxicity and bio-distribution of an adenoviral vector bearing as reporter gene the gene lacZ in cirrhotic Wistar rats (Mexican Patent Application # 998515, pending). Once the potential use of adenoviral vectors has been shown for sending exogenous genes to damaged livers without notably worsening its function, the effect of “therapeutic genes” on cirrhotic livers was evaluated.  
           [0004]    Hepatic cirrhosis is characterized by a fibrosis increase where there is an accumulation of extracellular matrix proteins (especially I, IV and III type collagen) synthesized by hepatic stellate cells (Arthur, M. J. P. Collagenases and liver fibrosis, J. Hepatology 1995:22:43-48; Kim, T. H. Mars, W. M., Stolz, D. B., Petersen, B. E., and Michalopoulos, G. K. Extracellular matrix remodeling at the early stages of liver regeneration in the rat. Hepatology 1997:26:896-904; and Olaso, E. and Friedman, S. L. Molecular regulation of hepatic fibrogenesis. J. Hepatology 1998:29:836-847) throughout liver parenchyma, mainly around the central and portal veins, forming a barrier blocking the free exchange of nutrients between the sinusoid and the hepatocytes leading to function deterioration (FIG. 1A) (Arthur, M. J. P. Collagenases and liver fibrosis, J. Hepatology 1995:22:43-48; and Kim, T. H. Mars, W. M., Stolz, D. B., Petersen, B. E., and Michalopoulos, G. K. Extracellular matrix remodeling at the early stages of liver regeneration in the rat. Hepatology 1997:26:896-904). Thus, the inventors of the instant invention suggest as a theory that recombinant viral and non viral vectors could be used in gene therapy to revert exacerbated fibrosis, the cardinal characteristic of cirrhotic livers, and, in turn, with the induction of genes promoting liver cell proliferation, enhance the rapid reestablishment of functional liver mass. For this purpose, in said vectors, the modified DNA of human urokinase plasminogen activator (huPA), that activates a cascade (lower part of FIG. 1B), wherein, on the one hand it activates latent liver collagenases or matrix metalloproteases (MMPs) to promote the degradation in situ of excess extracellular matrix (ECM) in the perisinusoidal space or space of Disse. The deposited excess extracellular matrix blocks nutrient exchanges between hepatocytes and circulating blood and thus the liver function is affected. Concomitantly, huPA restores the functional liver mass inducing the replication of the remaining hepatocytes and thus repopulate liver parenchyma (FIG. 1B). The reason to use said recombinant vectors is because of the additional advantage that because they do not secrete significant amount of huPA, they do not cause hypocoagulation or spontaneous bleeding which is the main disadvantage in cirrhotic animals. In this way, inducing two different cascades of beneficial events, the modified huPA cADN therapy can be very useful for the treatment of liver cirrhosis. It is important to observe that up to date no therapeutic agent reverting and/or preventing with 100% effectiveness the progressive accumulation of liver collagen has been described.  
           [0005]    Such physiopathological alterations occurring in hepatic cirrhosis are constant and common for the other organs that also undergo fibrosis, such as, lung, heart, kidney, pancreas and skin, among others, which should be not considered as limitations of the scope of protection of this invention. Therefore, the methodology presented here for the treatment of hepatic cirrhosis could be applied also to those organs that are susceptible to, or are affected by fibrosis.  
         Viral and Non-viral Vectors in Hepatic Gene Therapy  
         [0006]    The viral vectors used in this invention to implement this technology can be, without limitation, first, second, third and/or fourth generation adenoviral vectors, “gutless” adenoviral vectors, retroviral vectors and adeno-associated vectors. The non-viral vectors can be constituted by plasmids, phospholipids, liposomes (cationic and anionic) of different structures and combined with different ligands for specific receptors. The synthetic vectors consist of the construction and coupling huPA cDNA and TGF-beta Type II truncated receptor cDNA with plasmids from different origins and with regulatable and/or inducible promoters. In many protocols, retroviral vectors have been used to introduce genes in hepatocytes (Douglas J. T., and Curiel D. T., Adenoviruses as Vectors for Gene Therapy. Science and Medicine March/April 1997 44-53). However, precautions have to be taken since these vectors can generate potential replication-competent viruses. Among the advantages of these vectors is their ability to integrate their genome in a stable way in the chromosomes of the guest cell, which confers the possibility of expression, in an indefinite way, of the therapeutic transgene cloned in the retrovirus. On the other hand, up to date, no study has reported incidences of mutagenesis by insertion or activation of oncogenes through retrovirus integration if the viruses used are not replication-competent. Nevertheless, the use of retroviral vectors to transduce genes to the liver is limited for the following considerations: 1) these vectors infect only cells which actively divide and 2) very low viral particles titers are obtained in the packing cell lines used to amplify these viruses (Graham F. L., and Van Der Eb A J. A New Technique 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 through the induction of hepatocytes proliferation in vivo, through the use of hepatic growth factors and through partial hepatectomy, surgical procedure through which the removal of 70% of liver mass induces division of the remaining hepatic cells in vivo. The use of lentiviral vectors has permitted to overcome partially said limitations, because they are able to transduce cells which are not actually dividing.  
         BACKGROUND OF THE INVENTION  
         [0007]    Hepatic cirrhosis is a chronic illness of the liver, where diffuse cell necrosis and a limited regeneration of parenchymal hepatic cells result in diffuse percentage increase of connective tissue, causing the distortion of lobular hepatic architecture and inducing hemodynamic alterations. Therefore, some strategies for the treatment of hepatic cirrhosis could include the prevention and/or reversion of the fibrogenesis, stimulation of hepatic mitosis and re-arrangement of the architecture of hepatic tissue. The documents of the state of the art related to the present invention are mentioned hereinafter only as references.  
           [0008]    U.S. Pat. No. 5,240,846 relates to the use of gene therapy called “CFTR”, which induces a stable correction of the regulation of the chlorine channel. This defect is present in epithelial cells. In said invention, adenoviral recombinant vectors are used as well as plasmidic vectors. However, it does not have any association with the therapeutics genes of the present invention. Likewise, U.S. Pat. No. 5,910,487 describes the use of plasmidic vectors for sending therapeutic molecules, but there is no association with the delivery of wild and/or modified huPA genes or of the TGF-beta (Transforming Growth Factor-beta) Type II truncated receptor genes as presented here. U.S. Pat. No. 5,827,703 refers to the use of adenoviral vector and modified adenoviral vector to send genes, however, none of these vectors contain the genes used in the present invention for the treatment of fibrosis.  
           [0009]    U.S. Pat. No. 5,770,442 claims the use of a recombinant adenovirus that contains one gene directing the expression of a protein called “fiber” or a protein called “fiber-chimera”, however said patent does not specifically mention, which one is the therapeutic gene. Moreover, a method of gene therapy involving the use of such adenovirus and a vector of transference for the generation of such recombinant adenovirus is presented. However, nothing is mentioned with regard to the use of therapeutic genes cloned arid inserted in recombinant adenoviral vectors used in this invention in fibrotic livers, or to other target organs such as kidney, lung, and hypertrophic scars and others. These therapeutic genes are the gene that codes for the wild and/or modified huPA activator and the TGF-beta (Transforming Growth Factor-beta) type II truncated receptor, claimed in the instant invention. Other members of the family of genes represented are also included.  
           [0010]    U.S. Pat. No. 5,166,320 refers to the use of a targeted delivery system to introduce exogenous genes in mammalian hepatic cells. But there is no association with putative genes directly sent to fibrotic livers, kidneys, lungs or other fibrotic organs.  
           [0011]    U.S. Pat. No. 5,872,154 describes a method to reduce the immune response induced by an adenoviral recombinant vector through co-administration of recombinant adenoviral vector and a selected immune modulator, which functions by inhibiting the formation of neutralizing antibodies and/or reducing the death of the virally infected cells. U.S. Pat. No. 5,871,982 is directed to a hybrid vector, in which a portion of an adenovirus is included, together with a portion of an adeno-associated viral vector that contains a selected transgene. A hybrid virus consisting of the union of a conjugate with a polycation to a gene mesh of the adeno-associated viral vector to form a simple particle is also described. This is contrary to the present invention in which no hybrid viruses are employed, only adenoviral vectors. Besides, in the above-mentioned patent the gene, transgene or therapeutic gene used is not stated. U.S. Pat. No. 5,856,152 is directed to the creation of a hybrid vector that contains the portion of an adenoviral vector in combination with an adeno-associated virus and a selected gene, through which large quantities of recombinant vectors are produced, but they are not carrying cloned therapeutic genes as described in this invention, in which specific therapeutic genes for the treatment of liver, kidney, pancreas, heart fibrosis as well as keloids and hypertrophic scars are used. U.S. Pat. No. 5,547,932 claims a compound of nucleic acid complexes for transfecting eucaryotic cells. These complexes are formed by nucleic acids and another substance with affinity for nucleic acids and optionally an internalizing factor, such as a virus or a component of the virus that can be conjugated. It also uses components of specific adenoviral vectors or specific viruses such as Ad2 or Ad5, but does not mention the genes that are internalized in the cell cytoplasm and eventually in the nucleus of these eucaryotic cells. Similarly, U.S. Pat. No. 5,521,291 relates to conjugated adenovirus bound through an antibody to a substance with affinity for nucleic acids. In this way recombinant genes are transported to the interior of eucaryotic cells. These conjugated complexes and nucleic acids are internalized in the cell, but the genes that can be sent are not specifically mentioned. In said patent, contrary to what is described in the instant invention, the use of such adenovirus to treat liver fibrosis or cirrhosis or any another type of fibrosis is not mentioned.  
           [0012]    U.S. Pat. No. 5,585,362 relates to an improved adenoviral vector and methods to obtain and use such vectors. Although the use of adenoviral vectors is not mentioned in said patent, the adenoviral vectors described in the present invention were used as vectors for sending therapeutic genes.  
           [0013]    U.S. Pat. No. 5,756,086 claims an adenovirus, which is represented by a protein called “fiber”. The adenovirus also includes a ligand, that is specific for a receptor located on a specific cell type. This adenovirus can have at least a portion of this protein called “fiber” and it can be removed and replaced with a ligand, which is specific for a receptor on specific cells, such as hepatocytes. These adenoviruses can include a gene that codes for a therapeutic agent. Based on the previous statement, the outstanding technical difference of the instant invention, compared to the state of the art, is the specificity of the therapeutic agent as wild and/or modified huPA and the TGF-beta type II truncated receptor for the treatment of various fibrosis.  
           [0014]    U.S. Pat. No. 5,895,759 claims a tissue-specific vector (liver) for gene therapy that can be used to send genes to a damaged liver. These vectors are chemically or enzyme coupled to a promoter and can also be coupled to an antibody packaged in a polypeptidic envelope. Besides, the vector or the virus to be assayed is the hepatitis B virus. Thus the sending of genes to damaged livers described in this patent makes use of a system completely different from the one of this invention, and there is no relation with the process of fibrosis or cirrhosis to be treated. U.S. Pat. No. 5, 559,099 describes an adenoviral recombinant vector that contains a chimeric protein from the adenovirus called pentona, which includes a non-pentona sequence and a therapeutic gene to develop a gene therapy method involving the use of such adenovirus, transference adenoviral vectors for the recombination of such adenoviral vectors containing a therapeutic gene. Moreover, U.S. Pat. No. 5,885,808 claims also the use of adenovirus with bonding molecules of adenovirus to different cells, the molecules of which have been modified, as in U.S. Pat. Nos. 5,846,782 and 5,712,136, in which adenoviral vectors are employed, which have been modified to contain different peptidic domains.  
           [0015]    Finally, U.S. 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 the use of these vectors. The possible relation of the instant invention to the mentioned state of the art refers to the use of adenoviral vectors, that can be modified, as well as the use of inducible promoters driving the expression of genes to be inserted in these adenoviral vectors. However, the technical characteristics of the present invention are focused on the specific use of therapeutic genes to treat different types of fibrosis such as liver, kidney, lung, pancreas, heart fibrosis, keloids, as well as hypertrophic scars.  
           [0016]    The importance of the present invention, contrary to the state of the art described in the above-mentioned documents, is based on the technical characteristics of the invention itself, as well as on the additional advantages derived from the same, which are described with more details below.  
         Adenoviral Vectors  
         [0017]    In the instant invention the decision has been made to use adenoviral vectors, although it is important to stress that the viral vectors used to implement this technology can be, not restrictively, first, second, third and/or fourth generation adenoviral vectors, “gutless” adenoviral vectors, retroviral vectors, adeno-associated vectors. The non-viral vectors can be constituted by plasmids, phospholipidic components, (cationic and anionic) liposomes of different structures and combined with different ligands for specific receptors. The synthetic vectors are prepared through the construction and coupling of huPA cDNA and the TGF-beta type II truncated receptor with plasmids from different origins and with regulatable and/or inducible promoters.  
           [0018]    The adenoviral vectors were initially selected based on several considerations: 1) these vectors can be generated to very high titers of infectious particles per ml.: (10 9 -10 10 ); 2) they infect a great variety of cells, however, when they are administered i.v., most of them are located in the hepatic organ; 3) they transfer efficiently genes to cells that are not dividing, and 4) they are seldom integrated in the guest genome, which avoids the risk of cellular transformation by insertional mutagenesis (Douglas J. T., and Curiel D. T. Adenoviruses as Vectors for Gene Therapy. Science and Medicine, March/April 1997. 44-53 and Zern A M, and Kresina T F. Hepatic Drug delivery and Gene Therapy (Hepatology 1997, Vol. 25, No. 2, 484-491).  
           [0019]    Adenovirus are probably the most promising vehicles or vectors for the delivery of genes in the protocols of gene therapy in human beings, since they possess a unique attribute that provides them great stability when they are administered into the blood stream. This specific characteristic permits them to be efficiently used in clinical trials with a comfortable i.v. administration for the patient. (Douglas J. T., and Curiel D. T. Adenoviruses as vectors for Gene Therapy. Science and Medicine, March/April, 1997, 44-53).  
           [0020]    Adenoviruses are double stranded DNA viruses. They have an icosahaedric structure, infect a great variety of mammalian cell types, and support the ubiquitous expression of a specific receptor in the cell surface not yet identified. Their union to cells occurs by means of the protein component of the adenovirus capside and the virus enters into the cell by receptor-mediated endocytosis.  
           [0021]    More than 40 different human serotypes of adenovirus have been identified, of which type 2 (Ad2) and 5(Ad5) have been more extensively studied and, therefore, more widely used as vectors for gene therapy. A very important characteristic of these two Ad serotypes is that they have never been associated with malignant human processes.  
           [0022]    The strategy for the creation of recombinant adenovirus is based on the organization of the adenoviral genome. The expression of the adenoviral genes occurs in two phases, early and late, that are defined by the time of replication of the adenoviral genome. The early genes encode themselves in 4 distinct transcriptional units. E1, E2 and E4 encode for essential regulatory proteins that induce the replication of the adenoviral DNA. The gene E3 is a non-essential gene. The products of the late genes include the main proteins of the capside, which are transcribed from a unique promoter. (Graham F. L., and Van Der Eb A J. A New Technique for the Assay of Infectivity of Human Adenovirus 5 DNA. Virology 1973, 52:456-467).  
           [0023]    The recombinant adenoviruses are generated by introduction of the exogenous gene or sequence of DNA of interest in substitution of the adenoviral genome regions required for the replication of the virus. The adenoviral recombinant vectors present deletions in E1 and E3 genome regions. Recombinant adenovirus generation is conducted both through the replacement of E1 or E3 regions or through the insertion of the exogenous gene between the E4 region and the extreme right part of the adenoviral genome. Vectors based on the insertion of the exogenous gene at the extreme right part of the adenoviral genome or by the replacement of the E3 region keep their replication capability. On the contrary, the substitution of early region E1 produces a vector which is faulty with regard to its replication capability, that can spread only in a cell line that supplies in “trans” the absent functions of the replaced adenoviral region, or in presence of a collaborator virus. Among them, the most commonly used as gene transference vectors are the replication-deficient adenovirus (Douglas J. T., and Curiel D. T. Adenoviruses as Vectors for Gene Therapy. Science and Medicine, March/April, 1997, 44-53).  
           [0024]    The creation of adenoviral vectors, as well as their application for the treatment of fibrosis, are shown in the examples described hereinafter.  
         OBJECTS OF THE INVENTION  
         [0025]    Hereinafter, the objects and advantages derived from this invention are presented.  
           [0026]    An object of the present invention is to provide a procedure to prepare recombinant adenoviral vectors AdΔhuPA, by means of the cloning of huPA modified cDNA in appropriate adenoviral vectors; besides, huPa cloning in “gutless”, adeno-associated vectors and the formation of liposome and phospholipid component complexes.  
           [0027]    Another object of the invention is to provide adenoviral recombinant vectors (all the previously mentioned in the instant invention), with an exogenous gene or DNA sequence of interest that encodes for therapeutic proteins useful in the treatment of the generalized fibrosis in target organs susceptible to suffer from it. Such gene is, but not restrictively, a wild and/or modified huPA gene or the TGF-beta (Transforming Growth Factor-beta) type II truncated receptor gene.  
           [0028]    Also, in the present invention, pharmaceutical compositions (or other compositions) are provided which contain the recombinant viral, non-viral, plasmidic vectors in quantities therapeutically effective of viral particles for the treatment of generalized fibrosis; as well as therapeutic treatment methods, their uses and therapeutic applications in the treatment of fibrosis.  
           [0029]    An advantage of greater importance in the treatment of the generalized fibrosis, particularly of hepatic cirrhosis, is that the delivery of therapeutic gene(s) is carried out through tissue-specific recognition by the route of administration employed and by the natural tropism to the cirrhotic liver of the recombinant vectors used.  
           [0030]    Another advantage of the therapeutic uses of the invention, which is directed initially to the treatment of hepatic cirrhosis, is the treatment of generalized fibrosis in other target organs susceptible to suffer from it, including, not restrictively, the treatment of liver, lung, heart, skin, kidney, pancreas fibrosis, among others, in mammalian animals, including human beings.  
           [0031]    Another object is the design of a technology to deliver genes efficiently first to livers of cirrhotic animals affected by cirrhosis induced by carbon tetrachloride (CCl 4 ) and common biliary obstruction. This type of cirrhosis experimental models is very similar to the two types of liver cirrhosis that usually affect human beings in Mexico and in the rest of the world (alcoholic cirrhosis, chronic infection caused by hepatitis C virus, and secondary biliary cirrhosis).  
           [0032]    Another advantage resulting from the fibrosis treatment is that the recombinant adenovirus used (and none of the other proposed adenoviral vectors) does not induce lethal toxicity in the animals injected with the vectors.  
           [0033]    Another object of the invention allows us to conclude that liver fibrosis is totally resolved, and that the proliferation of liver cells is dramatically stimulated, obtaining thus the reestablishment of liver function in cirrhotic animals.  
           [0034]    Another advantage of the design of this technology is the fact that it is possible to detect the expression of human uPA therapeutic gene delivered to cirrhotic animals through the expression of the corresponding human protein through ELISA essays and through immunohistochemistry. We can thus discriminate between the endogenous rat protein and the therapeutically induced protein production. Thus, this allows us to check the transduction in vivo of the different organs of the rat to see if the vector administration was adequate and if the expression remains only in the target organ.  
           [0035]    Finally, all this evidence allows us to suggest that our system comprises an efficient and adequate vehicle to deliver therapeutic genes such as wild and/or modified huPA and the TGF-beta (Transforming Growth Factor-beta) type II truncated receptor that degrade the excess collagen and/or prevent its deposition; and that produce protein stimulating liver regeneration, to cirrhotic rat livers in order to restore normal functions of the liver or other organs affected by the same pathology.  
           [0036]    Thus, in the present invention a process of preparation is given for recombinant adenoviral vectors and other viral and non-viral above-mentioned vectors, pharmaceutical compounds, therapeutic treatment methods, uses and therapeutic applications for the treatment of fibrosis, especially for the treatment of hepatic cirrhosis. 
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0037]    Other particularities and advantages of this invention will be evident from the following detailed description of the preferred objects and embodiments, from the enclosed claims and from the drawings or figures attached, in which:  
         [0038]    [0038]FIG. 1 shows the cellular physiopathology of hepatic cirrhosis (A); and the concept evidence regarding how the gene therapy works to revert the fibrosis process, as well as the cascade of events induced by huPA cDNA leading to fibrosis reversion and liver cell regeneration (B);  
         [0039]    [0039]FIG. 2 shows the scheme of the adenoviral vector map containing the reporter gene LacZ (A) used to determine dose-response, safety, toxicity, and bio-distribution of an exogenous gene in several normal and cirrhotic laboratory rat organs (B-I);  
         [0040]    [0040]FIG. 3 is a scheme representation showing the genetic modification of human uPA cDNA and the formation of Ad-ΔNΔC-huPA (therapeutic) corresponding adenoviral vectors, the administration through the iliac vein (A-D). The determination of human huPA therapeutic protein expression levels in cirrhotic rats is also shown through corresponding immunohistochemistry and ELISA essays (E-G);  
         [0041]    [0041]FIG. 4 shows liver histological cuts from rats treated with CCl 4  during 6 weeks obtained on different days after the injection of Ad-GFP (A), irrelevant vector, and Ad-ΔNΔC-huPA (B), therapeutic vector, the quantitative determination through morphometrical analysis of fibrosis grade, in each case, and the concomitant induction of metalloprotease 2 (MMP-2) activity is also shown (C and C′). The expression of the protein product of fluorescent green gene is also shown as an adequate control of in vivo transduction;  
         [0042]    [0042]FIG. 5 shows the semi-quantitative analysis through reverse transcriptase polimerase chain reaction (RT-PCR) of the messengers RNA for collagens types I, III, and IV of the livers of cirrhotic rats treated with the adenoviral vector Ad-ΔNΔC-huPA, Ad-GFP or only with saline in which amplified PCR products are shown corresponding to said genes (A), and their corresponding densitometrical determination compared to the expression of a constitutive expression gene (B);  
         [0043]    [0043]FIG. 6 shows the immunohistochemical staining of rat cirrhotic livers with anti α-SMA (smooth muscle actin) antibody after the delivery of Ad-ΔNΔC-huPA (A) therapeutic vector, as well as the quantitative computing through a computer assisted morphometrical analysis of the percentage of α-SMA positive cells (B);  
         [0044]    [0044]FIG. 7 shows the immunohistochemical staining of cirrhotic rat liver sections with anti-PCNA (Proliferating Cell Antigen) antibody to evaluate hepatocytes proliferation after the administration of Ad-ΔNΔC-huPA adenoviral vector, Ad-GFP o saline only (A); (B) and (C) show the percentage of immunostained cells in the periportal and lobular region, respectively, through an automated image analyzer. (D) shows the semi-quantitative RT-PCR determination of hepatocyte growth factor (HGF), c-met and huPA after the administration of different vectors in which the PCR amplified products corresponding these genes are shown; and  
         [0045]    [0045]FIG. 8, (A) shows the prothrombrin times of rats sacrificed on different days after the administration of Ad-ΔNΔC-huPA vector o treated only with saline. Besides (B) shows the evaluation of liver weight/body weight x-100 ratio to detect a possible liver hyperplasia and/or hypertrophy in these livers. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0046]    There are many reports showing that through systemic administration of recombinant adenoviral vectors (AdR) into healthy experiment animals, a specific homing and highly preferential tropism of these vectors into the liver is observed. The inventors of the instant invention have shown that the cirrhotic liver is also a favorite target of adenoviral vectors, even though the organ lobular architecture is altered because of the fibrosis established in the entire liver parenchyma, mainly around the central and portal veins.  
         [0047]    Therefore, in the instant invention the hypothesis has been established according to which, using a modified cDNA of human urokinase derivate plasmid gene activator (huPA), it could be possible to promote, in damaged livers, the in situ degradation of excess collagen components of the extracellular matrix through latent MMPs activation, and to reestablish the free exchange of macromolecules between the sinusoid and the hepatocytes, as well as the functionality of the hepatocytes affected upon inducing them to proliferate and in this way, repopulate the liver parenchyma and obtain the regeneration and cure of the damaged liver.  
         [0048]    With the development of the invention claimed herein, a research line is started to conduct gene therapy as an alternative for the treatment of chronic-degenerative illnesses, specifically liver cirrhosis in human beings, upon establishing an efficient vehicle to send genes to the liver, said genes producing therapeutic proteins that help reestablish the normal functions of the liver (see FIGS. 1 and 4), where it is shown how to deliver effectively and in an adequate way huPA gene to obtain the degradation of the excess collagens deposited through the over-expression of huPA protein.  
         [0049]    In the left panel of FIG. 2B, frozen cuts are shown which are stained with X-gal. In the left panel of FIG. 2B tissues of the same livers on which a X-gal determination was conducted are shown, but said tissues were soaked in paraffin before being cut and were stained with Sirius Red in order to visualize the fibrosis grade. Transduction grade was about 40% in cirrhotic rats, compared to 84% in normal rats (FIG. 2C). Because the dose-response essay establishes a dose of 3×10 11  total viral particles (FIGS. 2D, E, and F), in FIG. 2H the adenoviral vector bio-distribution is shown and in the graph it can be seen that the main target organ is the liver, both in healthy rats as well as in rats receiving a chronic administration of CCL 4 . Spleen and kidney presented a transduction grade under 1%. The adenoviral vector bio-distribution was corroborated by PCR, the primers use amplify a region of adenovirus and a region of reporter gene Lac-Z and the bands obtained (FIG. 2-I) in the different organs correspond to the distribution found with X-gal reaction. Finally, using the 3×10 12  viral particle dose, we found that about 50% of the animals died through disseminated intravascular coagulopathy.  
       The Modified huPA Produced is Intracellularly Located  
       [0050]    Taking into account the well known uPA function as one of the main primers of the extracellular matrix proteolysis (ECM), and as plasmid activator and hepatocyte growth factor (HGF) (Kim, T. H., Mars, W. M., Stolz, D. B., Peterson, B. E., and Michalopoulos, G. K. Extracellular matrix remodeling at the early stages of liver regeneration in the art. Hepatology 1997:26:896-904; Mars, W. M. Zarnegar, R., Michalopoulos, G. K. Activation of hepatocyte growth factor by the plasminogen activators uPA and tPA. Am. J. pathol. 1993:143:949-958. (35); and Roselli, H T., Su, M., Washington, K., Kerins, D. M., Vaughan, D. E. and Russel W. E. Liver regeneration is transiently impaired in urokinase-deficient mice. Am. J. Phisiol. 1998:275:G1472-G1479), it was decided to use human uPA to induce enzyme activity specifically in cirrhotic livers and to test its effect in fibrosis reversion. However, to avoid bleeding risk as a consequence of huPA secretion, we used a non secreted huPA form with a modification in the amino-terminus and carboxi-terminus ends to prevent to the greatest possible extent it secretion in the blood stream (FIG. 3A and 3B).  
         [0051]    To induce cirrhosis, the rats received CCl 4 , intraperitoneally, 3 times per week during six to eight weeks (FIG. 3D). After a six-week administration of CCl 4 , various livers sections of cirrhotic animals injected with pAd-GFP and saline used as control were analyzed, and nodes of various sizes similar to human cirrhosis were observed. Thick and thin fibrosis bands were observed around the nodes with obvious parenchyma collapse and the typical collagen bridge pattern linking the portal tracts and central veins. Necrotic and swollen hepatocytes were frequently associated with the fibrosis bands (FIG. 4A). Thus, the pAd.PGK-ΔNΔC-huPA was used to treat the animals and revert severe fibrosis as well as stimulate hepatocytes regeneration. Based in the previous observation (García-Bañuelos J, Siller Lopéz, F, Aguilar-Córdova, E and Armendáriz-Borunda J. Adenovirus-Mediated gene delivery to cirrhotic rat livers: potential tool for gene therapy. Gene Ther. And Mol. Biol. Accepted 2000), the dose of 6×10 11  viral particles/kg of pAd.PGK-ΔNΔC-huPA adenoviral vector was established, administrated through the iliac vein in one dose at the end of the sixth week of CCl 4  chronic intoxication (Armendáriz-Borunda, J. Katai H., Jones, C. M. , Seyer J. M. , Kang J. M. and Ragliow R. Transforming growth factor β is transiently enhanced at a critical stage during liver regeneration following CCl 4  treatment. Lab Invest. 1993:69:283-294). It is important to observe that CCl 4  damage continued in rats sacrificed two days after the administration of adenoviral vector (FIG. 3D). In other words, rats sacrificed on days 8 and 10 received three additional injections of hepatotoxic agent compared to the rest of the animals. After only one pAd.PGK-ΔNΔC-huPA injection and ELISA essay was conducted to determine the presence of huPA protein in liver homogenate and serum (FIG. 3G). The results showed that a large quantity of modified huPA protein was detected in the tissue extract, compared to the concentration detected in the sera, which can be explained by the fact that the produced protein leaves the damage hepatocytes. Importantly, huPA levels produced in said experiments were very high (˜5 ng/ml) [100 times higher compared to other protein induction systems in similar experimental models (Schirmacher, P., Geerts, A., Jung., W., Pietrangelo, A., Rogler, C. E., Dienes, H. P. The role of Ito cells in the biosynthesis of HGF-SF in the liver. EXS 1993:65:28.5-299). (43)]. huPA levels increased by day 2, diminishing afterwards but showing detectable levels, compared to the lack of expression in control animals injected with irrelevant adenovirus, pAd-GFP (FIG. 3G). Besides, there are evidences that the modified huPA was located intracellularly when the detection was made by huPA immunohistochemistry with specific antibodies, showing and confirming the induction of said protein in cirrhotic livers (FIGS. 3E and F). Normal and cirrhotic rat liver tissues used as controls show occasionally hepatocytes stained with huPA antibody (˜3.5%). Contrasting with this finding, the animals treated with pAd.PGK-ΔNΔC-huPA showed by day 2 a significant increase of huPA immunostained hepatocytes (46%) (FIGS. 3E and F), reaching a peak after day 6 on which over 80% of the cells were positives. Besides, Kupffer cells and biliary epithelium cells also showed immunoreactivity.  
         [0052]    Even though the vector application induced a histological appearance of degenerate hepatocytes and there was an important serum transaminase increase in the first days after pAd-ΔNΔC-huPA treatment (table 1), said increase was transient and none of the animals died of liver failure as a consequence are overlain adenoviral hepatitis. Compared to cirrhotic control animals that received saline (n=5) and an irrelevant adenovirus preparation (pAd-GFP) (n=5), the animals injected with pAd.PGK-ΔNΔC-huPA showed a remarkable improvement in liver function tests (table 1). Globally, the rats sacrificed in the last days showed prothrombin times (PT) of 14 seconds (very similar to normal values) 8 and 10 days after the adenovirus administration compared to 24 seconds on days 2, 4 and 6 (FIG. 8A). Importantly, no anemia was observed, but a transitory small decrease of platelets (table 2) in animals that received pAd.PGK-ΔNΔC-huPA could be seen. With regard to this point, it has previously been reported that the injection of adenoviral vectors in normal animals induces a transitory platelet decrease (Brann, T., Kayda, D., Lyons, R. M., Shirley, P., Ruy, S., Kaleko, M., and Smith, T. Adenoviral vector-mediated expression of physiologic levels of human factor VIII nonhuman primates. Hum. Gene Ther. 1999:10:2999-3011).  
                                                                                     TABLE 1                           SUMMARY OF GROUP AVERAGES OF LIVER       FUNCTION TEST RESULTS                DAYS                GROUP   2   4   6   8   10                        ALT   Normal   73.6 ± 10.9                       (IU/L)   pAd-ΔNΔC-huPA    1563 ± 948.8   443.75 ± 308     345 ± 60    88.5 ± 24.7   684 ± 50            pAd-GFP     890 ± 260.2   337.5 ± 54.4    426 ± 48    410 ± 45    715 ± 60        AST(IU/L)   Normal   162.3 ± 30.9            pAd-ΔNΔC-huPA     1590 ± 957.09   676.5 ± 576.3   395 ± 70     137 ± 29.7   599 ± 30            pAd-GFP     737 ± 156.98   627.5 ± 389.6   2100 ± 114    2250 ± 55.6     2315 ± 115.3       Alcaline   Normal   159.7 ± 34.7        phosphatase   pAd-ΔNΔC-huPA   525.7 ± 11.3    385.2 ± 221.7   336 ± 40     205 ± 54.5    310 ± 28.5       (IU/L)   pAd-GFP   382.5 ± 40.3    715 ± 181    577 ± 63.4    454 ± 46.5    485 ± 45.3       Bilirrubine   Normal   0.55 ± 0.07       total   pAd-ΔNΔC-huPA   0.97 ± 0.6    0.825 ± 0.43    0.6 ± 0.2   0.94 ± 0.3    0.7 ± 0.3       (mg/dL)   pAd-GFP   0.35 ± 0.07   0.75 ± 0.6    2.3 ± 0.4   1.4 ± 0.3   1.2 ± 0.2                  
 
         [0053]    [0053]                                                                     TABLE 2                           SUMMARY OF THE AVERAGES PER GROUPS       OF HEMATOLOGY RESULTS                DAYS                GROUP   2   4   6                        Hemo-   Normal   13.5 ± 0.64               globin   pAd-ΔNΔC-   13.5 ± 1.77   15.5 ± 0.8    16.5 ± 0.14       (g/dL)   huPA   14.9 ± 1.13     14 ± 1.12   14.1 ± 0.99           pAd-GFP       Hema-   Normal   40.6 ± 0.85       tocrite   pAd-ΔNΔC-   40.1 ± 5.87   46.5 ± 0.7    49.5 ± 0.25       (%)   huPA   40.3 ± 0.07   41.9 ± 2.3    42.4 ± 2.83           pAd-GFP       Eryth-   Normal    6.95 ± 0.226       rocytes   pAd-ΔNΔC-   7.1 ± 1.2   6.99 ± 0.56   7.5 ± 0.7       (10 6 /   huPA    7.14 ± 0.226    7.7 ± 0.93   7.3 ± 0.51       mm 3 )   pAd-GFP       Leuco-   Normal   2.47 ± 1.79       cyte   pAd-ΔNΔC-    5.6 ± 2.08   4.96 ± 2.9    8.9 ± 2.2       (10 3 /   huPA    6.8 ± 0.99   9.43 ± 1.2    11.3 ± 6.7        mm 3 )   pAd-GFP       Lymph-   Normal   45.5 ± 12.6       ocyte   pAd-ΔNΔC-   41.7 ± 0.99   55.4 ± 1.3    49.8 ± 2.8        (%)   huPA   44.9 ± 1.01   61.7 ± 4.5    50.7 ± 27.08           pAd-GFP       Neutro-   Normal   46.2 ± 7.99       philes   pAd-ΔNΔC-   53.9 ± 3.18   41.4 ± 2.5    49.2 ± 3.2        (%)   huPA   49.8 ± 2.5     37 ± 3.5   47.1 ± 27             pAd-GFP       Mono-   Normal    1.5 ± 1.75       cytes   pAd-ΔNΔC-    3.1 ± 4.23   1.33 ± 1.22    0.2 ± 0.02       (% + −)   huPA    3.9 ± 5.24   0.24 ± 2.3     0.3 ± 0.35           pAd-GFP       Plate-   Normal   1050 ± 45.6        lets   pAd-ΔNΔC-   959.5 ± 78.5     859 ± 52.3   1020.5 ± 38.9        (10 3 /   huPA    896 ± 80.6   961 ± 79   693 ± 127       mm 3 )   pAd-GFP                    
         [0054]    huPA Over-expression in Liver Induces Fibrosis Reversion  
         [0055]    Besides its plasminogen activation role, huPA is considered one of the main primers leading to the activation of the cascade of metalloproteases associated to the extracellular matrix degradation (Kim, T. H., Mars, W. M., Stolz, D. E., Petersen, B. E., and Michalopoulos, G. K. Extracellular matrix remodeling at the early stages of liver regeneration in the rat. Hepatology 1997: 26:896-904). After activating plasminogen in plasmin, uPA also activates procolagenases and possibility other metalloproteases (MMPs) converting them into the active form at early stages (Steler-Stevenson, W. G. Dynamics of matrix turnover during pathologic remodeling of the extracellar matrix. Am. J. Pathol. 1996:148:1345-1350. (33)). Thus, we determined first the fibrosis grade in cirrhotic livers stained with Masson. Computer assisted morphometrical analysis of multiple fields showed that rats treated with pAd.PGK-ΔNΔC-huPA presented a dramatic fibrosis reduction by day ten (85%) (FIGS.  4 B and B′), compared to fibrosis levels at the beginning of the treatment, as well as compared to cirrhotic animals receiving pAd.GFP (FIGS.  4 A and A′) and only saline. Said findings are consistent with the data showed on FIGS.  4 C and C′ where metalloprotease-2 (MMP-2) activity in liver homogenates was quantitatively determined through a specific ELISA essay. Said essay detects active MMP-2 levels (Verheijen, J H, Nieuwenbroek, N M, Beekman B, Hanemaijer, R, Verspaget H W, Ronday H K, Bakker A H. Modified proenzymes as artificial substrates for proteolytic enzymes: colorimetric assay of bacterial collagenase and matrix metalloproteinase activity using modified pro-urokinase. Biochem J. 1997:323 (Pt3): 603-609), which specifically degrades type IV collagen and, to a lesser extent, other collagens (Corcoran, M L, Hewitt, R E, Kleiner, D E Jr., Stetler-Stevenson, W G. MMP-2: expression, activation and inhibition, Enzyme protein 1996:49(1-3):7-19; Nogase, H., Ogata, Y, Suzuki, K, Enghild, J J, Salvensen G. Substrate specific and activation mechanisms of matrix metalloproteinases. Biochem. Soc. Trans. 1991(3)715-8). A six-fold MMP-2 increase was found (above 11.5 ng/ml) four days after huPA administration compared to normal rats and a three-fold increase was observed compared to control cirrhotic animals. On the other hand, and independently from the induction of specific huPA collagens and the physiological importance of collagen degradation activity, the expression of type I, II and IV endogenous collagen genes was evaluated through semi-quantitative RT-PCR, which permitted to detect specific changes in the baseline levels of said genes. The expression of type IV collagen gene presented ˜5-fold decrease by day six and the expression of type I collagen gene presented a four-fold decrease by day 10 in rats that received pAd.PGK-ΔNΔC-huPA compared to the rats that received an irrelevant adenovirus, pAd-GFP. Type III collagen expression diminished ˜2 times on day 10 of injection of huPA adenoviral vector compared to pAd-GFP (FIGS. 5A and B). Said data strongly suggest that the over-expression of human huPA induces the turning off of said genes that code for ECM proteins. In cirrhotic livers with severe fibrosis, hepatic stellate cells (HSC, main collagen producing cells) are increased in fibrotic areas, and most of them change their phenotype to activated HSC that specifically express alfa-smooth muscle actin (α-SMA) (Nyberg-Hoffman, C., Shabram, P., Li, W., Giroux, D. and Aguilar-Cordova, E. Sensitivity and reproducibility in adenoviral infectious titer determination. Nature Med. 1997:3(7):808-11). α-SMA expression was examined in rat livers through immunohistochemistry and it was discovered that it correlates to the distribution of excess ECM deposited throughout liver parenchyma, results that are consistent with the % of fibrotic tissue. There is a significant reduction in the number of α-SMA positive cells (8.9% on day ten compared to 43.2% on day 2 after the treatment) (FIGS. 6 A and B). Besides, a representative fibrosis area is magnified, where the HSC are clearly visible (FIG. 6C).  
         [0056]    HuPA Induces a Vigorous Cirrhotic Liver Cell Regeneration  
         [0057]    Having solved the first half of the process, we proceeded to determine the liver regeneration level “necessary to refill the empty spaces” after conducting extracellular matrix degradation (ECM). FIG. 7 represents mitosis kinetics in livers, probably stimulated by in situ HGF activation (Locaputo, S., Carrick, T. L and Bezerra, J. A. Zona Regulation of gene expression during liver regeneration of urokinase transgenic mice. Hepatology 1999:291106-1113). Hepatocyte proliferation was measured as the percentage of stained cells with a specific antibody against proliferation cellular nuclear antigen (PCNA). The number of PCNA positive hepatocytes was dramatically higher (10 times) in livers of rats treated with pAd.PGK-ΔNΔC-huPA compared to livers of rats treated with pAd-GFP and normal livers (FIG. 7A). The marking index showed that about 55% of hepatocytes were positively stained with anti-PCNA antibodies both in periportal areas (FIG. 7B) and in centrilobular areas (FIG. 7C) (Hattori, N., Sisson, T. H., Xu, Y., and Simon R. H. Upregulation of fibrinolysis by adenovirus-mediated transfer of urokinase-type plasminogen activator genes to lung cells in vitro-and in vivo. Hum. Gene Ther. 1999:10:215-222) 2 days after pAd.PGK-ΔNΔC-huPA injection together with numerous mitotic figures and binucleated hepatocytes (FIG. 7A). Marking indexes of 40% were detected even 8 days after the adenoviral vector administration (FIGS. 7B and C). The positive cells detected by day 10 indicate a re-establishment of the liver functional mass, which was confirmed through the clear normalization trend (Table 1 and FIG. 8A) shown by means of livers function tests (AST, ALT, Bilirubin and prothrombin times).  
         [0058]    HGF is one of the most potent hepatocytes mitogens (Michalopoulos, G. K. HGF in liver regeneration and tumor promotion. Prog Clin. Biol. Res. 1995:391:179-185) and c-met is its corresponding tirosine kinase receptor transducing its signal. FIG. 7D shows an over-expression of HGF gene in animals treated with huPA and only a minor expression in cirrhotic control animals and pAd-GFP treated animals. Said over-expression of this gene is most notable in c-met 2 days after huPA administration which suggest that liver cell proliferation is controlled through this mechanism.  
         [0059]    Finally, the liver weight related to body weight of the animals was measured at the time when the animals were sacrificed to determine a possible uncontrolled growth (FIG. 8B). As can be observed, there was only a very small increase in the weight of the organ by day 10, suggesting that no “abnormal” cell growth regulating mechanism is involved.  
         [0060]    The preferred way to apply the present invention is through endovenous administration of the recombinant adenoviral vectors (or any previously mentioned vector containing the therapeutic genes) of the instant invention, in which therapeutically effective amount is administered with a unitary dose regimen convenient to a fibrotic individual. This regimen can be adjusted according to the affliction degree. Generally, unitary doses of about 10 7  to 10 14  viral particles per individual are employed.  
         [0061]    The preparation of a pharmaceutical compound including the adenoviral recombinant vectors of this invention can be made through the employment of standard techniques very well known by the persons skilled in the art, in combination with any of the pharmaceutically acceptable carriers described in the state of the art, including without restriction, starch, glucose, lactose, saccharose, gel, malt, rice, wheat flour, chalk, silica-gel, magnesium stearate, sodium stearate, glyceril monostearate powder, NaCl, glycerol, propilene glycol, water, ethanol, and similar. These compounds can take the pharmaceutical form of solutions, suspensions, pills, tablets, capsules, powders and slow release formula, and similar.  
         [0062]    The above description and the following examples have the purpose to illustrate particular embodiments of the invention and they should not be considered as limitations of the scope of this patent.  
       EXAMPLES  
     Methodology to Demonstrate huPA Activity on Fibrosis Reversion and Liver Regeneration Stimulation  
       [0063]    a) Experiment Animals Mimicking Human Liver Cirrhosis  
         [0064]    The model consisted of animals submitted to CCl 4  chronic intoxication (Armendáriz-Borunda, J. Seyes, J. M., Kang, A. H. and Ranghow, R. Regulation of TGF gene expression in rat liver intoxicated with carbon tetrachloride. FASEB J. 1990:4:215-221) in which liver cirrhosis is established since the sixth week of CCl 4  intraperitoneal administration (FIG. 3D) and that resembles human liver cirrhosis induced by alcohol abuse or chronic hepatitis C virus infection. Animals weighting 80 g received 3 intraperitoneal weekly doses of CCL 4 -mineral oil mixture 1:6 during the first week, 1:5 during the second week, 1:4 during the third week and 1:3 during the weeks four to eight. Rats were paired to be used as control injecting them similarly only with the carrier. All the experimental methods have been previously described (García- Buñuelos J., Siller-López, F, Aguilar-Córdova, E. and Armendáriz-Borunda J. Adenovirus-mediated gene delivery to cirrhotic rat livers: potential tool for gene therapy. Gene Ther. and Mol. Biol. Accepted 2000), and all the adenovirus applications were conducted through the iliac vein (FIG. 3C).  
         [0065]    b) Expression Vectors Containing Reporter Genes  
         [0066]    Ad5-βGal adenoviral vector (FIG. 2A) comes from pΔElsplB, to which bacterial β-galactosidase gene (lac-Z) was inserted. β-Gal activity visualization was obtained with X-gal reagent which, in presence of β-galactosidase enzyme changes from colorless to blue. At the same time, the same Ad5-βGal batch was administrated to healthy rats (n=5) and cirrhotic rats. (n=5), after 5 and, 8 weeks of CCl 4  intoxication. The animals were sacrificed 72 hours after Ad5-βGal. For histological analysis and the determination of the expression of β-galactosidase (β-gal) protein coded for by the recombinant adenovirus, different organs were extracted: liver, spleen, heart, lungs, kidneys, brain, testes and ileum. Said organs were frozen at −30° C. and cut with a cryostat to obtain 8 μm sections. The cuts were exposed to X-gal reagent during 16-18 hours, counterstaining with Neutral Red. The percentage of transduced cells was determined through computerized image analysis in several fields. Cirrhotic rat liver cuts were also made, said cuts were soaked in paraffin, cut and stained with Sirius Red, which specifically stains collagen.  
         [0067]    c) Adenoviral Vectors Containing huPA Therapeutic Gene  
         [0068]    An adenoviral vector was constructed with the insertion of the cDNA coding for the totally functional non-secreted human urokinase plasminogen activator (pAd.PGK-ΔNΔC-huPA). Retention signals were added to the protein in endoplasmic reticulum (RE) in the amino-terminal and carboxi-terminal end in order to prevent the hemorrhage risk secondary to huPA secretion. Generally, proteins secreted are first translocated through the endoplasmic reticulum membrane and then are carried in vesicles to the Golgi apparatus. In the case of uPA, the traslocation through the endoplasmic reticulum membrane is activated by a characteristic signal in the amino-terminus of the precursor protein. Said signal peptide is cut by signal peptidases during polipeptide transfer through the membrane. To inhibit or diminish uPA secretion in systemic circulation, the protein was modified in such a way that it was necessary to avoid it export from the endoplasmic reticulum. Human uPA cDNA (1,326 bp) cloned in pGEM3 in Xba I/Asp 718 sites (FIG. 3A) was modified in the carboxi-terminal end adding a sequence coding for KDEL signal (a highly conserved sequence characteristic of soluble proteins residing in RE lumen) besides residues upstream through the cloning of 75 nucleotides generated by PCR (FIG. 3B). To modify the amino-terminus, the 25 amino-terminal amino acids including the pre-uPA peptidic signal were replaced by an amino-terminal retention signal (RR) together with the “anchor” in the transmembrane region (TM) separated by a peptide spacer (31 a.a.) coming from the transmembrane II protein Iip33 obtained through PCR (FIG. 3B). The RR sequence is constituted by arginin residues MHRRRSR located near the transmembrane anchor region (TM) and are present in type II transmembrane protein as the invariable chain protein Iip33. This resulting modified huPA gene was cloned in the adenoviral plasmid pAd.PGK-ΔNΔC-huPA, for the subsequent production of recombinant adenoviral vectors. The preparation of said adenoviral vector was monitored to discard endotoxin in microplasm contamination. The reason for the use of this vector is the advantage that, because it does not secrete huPA, it does not cause hypocoagulation, or spontaneous bleeding, which is the main this disadvantage in cirrhotic animals. The PAd-GFP vector used here is an adenoviral vector deleted in E1 and E3 regions replication deficient previously described (Nyberg-Hoffman, C., Shabram, P., Li, W., Giroux, D. and Aguilar-Cordova, E. Sensivity and reproducibility in adenoviral infectious titer determination. Nature Med. 1997:3(7):808-11). The two vectors were produced under sterile laboratory conditions implemented to obtain totally characterized vectors. The vectors were titled and characterized (Nyberg-Hoffman, C., Shabram, P., Li, W., Giroux, D. and Aguilar-Cordova, E. Sensivity and reproducibility in adenoviral infectious titer determination. Nature Med. 1997:3(7):808-11) and batches were obtained with viral particle title (vp) to infection units (IU) ratio ≧30.  
         [0069]    d) Liver Homogenate Preparation for the Detection of huPA Therapeutic Protein Production and Metalloprotease Activity  
         [0070]    The rats were sacrificed as indicated in FIG. 3D and liver homogenates were prepared from 150 mg of tissue as described (Gao, C., Jokers, R., Gondipalli, P., Cai, S. H., Kennedy S. and Ponder K. P. Intramuscular of an hepatic transduction with a retroviral vector in mice. Hum. Gene Ther. 1999:10:911-922) and kept at −70° C. At the same time, serum samples were obtained and kept at −20° C. In summary, for huPA and in presence of protease inhibitors, 150 mg of liver were obtained and macerated in 400 μl homogenate buffer (0.05 M Tris-HCl, 0.15 M NaCl, 0.01 M HEPES, 2mM CaCl 2 , 0.01% Tween 80, 1 mM phenylmethylsulfonyl fluoride (PMSF), pH 8.5). The homogenate was centrifuged at 12,000 ×g during 15 minutes and huPA levels were determined through ELISA in supernatant using a commercially available kit (Biopool, Sweden) consisting of a specific enzyme immunoessay for human uPA quantitative determination. The essay sensitivity detects uPA antigen baseline levels in 0-5 ng/mL linear range. The total protein levels were determined using Bradford technique for protein quantification (Bradford, M. M A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976:72:248-54). For MMP-2 essay the samples were homogenized using a homogenizer (Politron PT 3000, Kinematica AG, Brinkman, Switzerland) during 5 minutes at 8,000 ×g in 4 ml 0.15 M NaCl, at 4° C. After 3 cycles of freezing-thawing they were sonicated twice at 21 kilocycles per second during 1 minute at 4° C. and centrifuged at 8,000 ×g during 10 minutes at 4° C., aliquoted and kept at −70° C. MMP-2 levels were determined in said supernatants through ELISA using a commercial kit (Biotrak, Amersham).  
         [0071]    e) Biochemical Evaluation of Liver and Blood Functional Tests  
         [0072]    Blood was taken from the animals at specific times and liver functional tests were conducted in serum (ALT, AST, alkalinephosphatase and bilurubin) in an automated apparatus (Synchron Cx7). Prothrombrin times were analyzed on plasma with an automated device (ACL-3000) and blood analyses were conducted with another automated device (Cell-Dyn 3500R).  
         [0073]    f) Histological and Immunohistochemical Evaluation of Liver Sections  
         [0074]    The rats were sacrificed on days 2, 4, 6, 8 and 10 after pAd.PGK-ΔNΔC-huPA administration (FIG. 3D). A group of cirrhotic animals that received pAd-GFP (irrelevant adenovirus) and a group of cirrhotic animals that received a saline were used as control groups. At least five rats were included in each group. For the histological study, the livers obtained were immediately fixed through immersion in 10% formaldehyde in phosphate buffer (PBS), dehydrated in ethyl alcohol and soaked in paraffin. The 5μ thick cuts were stained with hematoxylin/eosin, Sirius Red and Masson trichromic stain and in this last experiment fibrosis percentage was determined in the affected livers using a computerized image analyzer (Qwin Leica) through the random analysis of 10 fields per slide and calculating the connective tissue ratio to total liver area. For the immunohistochemistry, liver sections were mounted on silanized slides, paraffin was removed and the endogenous peroxidase activity was blocked with 0.03% H 2 O 2  in absolute methanol. They were incubated overnight at room temperature with monoclonal antibodies against PCNA (Proliferation cellular nuclear antigen) and smooth muscle α-actin (Boehringer Mannheim, Ger) diluted with PBS 1/20 and 1/50, respectively, and with antihuman uPA goat polyclonal antibody (Chemicon International, USA) diluted in PBS 1/400. The reaction was detected with peroxidase marked rabbit or goat polyclonal antibodies, developed with diaminobenzidine, and counterstained with hematoxyllin. Four fields were randomly evaluated for quantification purposes from intralobular and periportal areas. Positive and negative cells were counted with an automated image analyzer (Qwin, Leica) and data were expressed as positive cell percentage. The histopathological analysis was interpreted by two independent certified pathologists with a 5% difference margin.  
         [0075]    g) RNA Extraction and Semi-quantitative RT-PCR  
         [0076]    Total RNBA was immediately isolated after obtaining the livers in the defined times, according to Chomczynski and Sacchi&#39;s method (Chomczynski, P. and Sacchi, N. Single-step method of RNA isolation by acid guanidinium thiocyanato-phenol chloroform extraction. Anal. Biochem. 1987:162:156-159). Liver tissue was homogenized through a Politron device in presence of Trizol and then chloroform was added obtaining an aqueous phase and precipitating RNA with isopropanol. RNA quantity was determined through spectrophotometry at 260/280 nm. The quality was verified through 1% agarose gel and formaldehyde electrophoresis.  
         [0077]    Analysis of HGF (Hepatocyte Growth Factor) gene expression, c-met (HGF cellular receptor) and collagens were conducted through semi-quantitative RT-PCR. We used a methodology developed in our laboratory and described in Delgado-Rizo et al, (Delgado-Rizo, V., Salazr, A, A. Panduro, A., Armendáriz-Borunda, J. Treatment with anti-transforming grown factor β antibodies influences an altered pattern of cytokines gene expression in injured rat liver. Biochim. Biophys, Acta 1998: 1442:20-27). Briefly, the livers of at least 3 animals in each group were processed. In the same way, 3 different RT-PCR reactions were conducted on each liver, and quantitative densometrical results of their averages are shown. The amplified genes were:  
                                                                         AMPLIFIED           GENE   PRIMERS   FRAGMENT                                HGF   (sense)   5′ATGCTCATTGGACCCTGGT3′    700 bp               (antisense)   5′GCCTGGCAAGCTTCATTA3′               c-met   (sense)   5′CAGTGATGATCTCAATGGGCAAT3′    726 bp           (antisense)   5′AATGCCCTCTTCCTATGACTTC3′               Collagen I   (sense)   5′CAAGAATGGCGACCGTGGTGA3′   1043 bp           (antisense)   5′GGTGTGACTCGTGCAGCCATC3′               Collagen III   (sense)   5′AGATGGATCAAGTGGACATC3    449 bp           (antisense)   5′CATGTTTCTCCGGTTTCCAT3′                  
 
         [0078]    The previously isolated RNA was underwent reverse transcription through enzyme (M-MLV) and the obtained cDNAs were submitted to amplification in a thermocycler under the following conditions: 5 minutes at 94° C., 1 minute at 60° C. and 1.5 minutes at 72° C. during 30 cycles. The expression levels of all the transcripts were normalized with a HPRT constitutive expression gene.  
         [0079]    The hepatocyte growth factor (HGF) has multifunctional activities including cell proliferation, migration and differentiation (Kim, T. H., Mars, W. M., Stolz, D. B., Petersen B. E. and Michalopoulos, G. K. Extracellular matrix remodeling at the early stages of liver regeneration in the rat. Hepatology 1997:26:896-904; and Michalopoulos, G. k., DeFrances M. C. liver regeneration. Science 1997:276:60-66). In the normal liver, HGF is produced by hepatic stellate cells (HSC) (Schirmacher, P., Geerts, A., Jung, W., Pietrangelo, A., Rogler, C. E., Dienes, H. P. The role of Ito cells in the biosynthesis of HGF-SF in the liver. EXS 1993:65:285-299) and is sequestered in the extracellular matrix (ECM) (Liu, M. L. Mars, W. M., Zarnegar, R., Michalopoulos, G. K., Uptake and distribution of hepatocyte growth factor in normal and regenerating adult rat liver. Am. J. Pathol. 1994:144:129-140). Moreover, HGF is also produced by the placenta, lung and brain (Schirmacher, P., Geerts, A., Jung, W., Pietrangelo, A., Rogler, C. E., Dienes, H. P. The role of Ito cells in the biosynthesis of HGF-SF in the liver. EXS 1993:65:285-299 and Wolf, H. K., Zarnegar, R. Michalopoulos, G. K. Localization of hepatocyte growth factor in human and rat tissues: an immunohistochemical study. Hepatology 1991:14488-494). It has been reported that uPA can activate single chain HGF (scHGF, inactive form), twin chain HGF (tcHGF, active form) (Schirmacher, P., Geerts, A., Jung, W., Pietrangelo, A., Rogler, C. E., Dienes, H. P. The role of Ito cells in the biosynthesis of HGF-SF in the liver. EXS 1993:65:285-299 and Wolf, H. K., Zarnegar, R. Michalopoulos, G. K. Localization of hepatocyte growth factor in human and rat tissues: an immunohistochemical study. Hepatology 1991:14488-494). In this way, in our experimental model, the impressive human uPA production modified in remaining functional hepatocytes led strongly to the in situ HGF activation which binds to its c-met receptor and induces early cell proliferation in liver, both in total parenchyma and in periportal areas (FIGS. 7A, B, C and D). Besides, it is known that HGF application in normal rat liver stimulates hepatocyte proliferation only in periportal hepatocytes (Liu, M. L. Mars, W. M., Zarnegar, R., Michalopoulos, G. K. Collagenase pretreatment and the mitogenic effects of hepatocyte growth factor and transforming growth factor-alpha in adult rat liver. Hepatology 1994:19:1521-152 and Liu, M. L. Mars, W. M., Zarnegar, R., Michalopoulos, G. K., Uptake and distribution of hepatocyte growth factor in normal and regenerating adult rat liver. Am. J. Pathol. 1994:144:129-140), but a dramatic increase in DNA synthesis has been observed in hepatocytes of lobular areas, when normal livers were previously treated with collagenases (Liu, M. L. Mars, W. M., Zarnegar, R., Michalopoulos, G. K. Collagenase pretreatment and the mitogenic effects of hepatocyte growth factor and transforming growth factor-alpha in adult rat liver. Hepatology 1994:19:1521-152). In summary, the combined evidences presented here suggest that the active ECM degradation together with c-met increased regulation and HGF synchronous activation and binding to its corresponding receptor, activate hepatocyte proliferation. The strong proliferation response observed in cirrhotic animals injected woith uPA (about 60%) could reflect the additive effect of a direct action of HGF inducible on hepatocytes and MMPs effect on hepatocyte preparation in such a way that they can offer a more effective response to growth factors (Kim, T. H., Mars, W. M., Stolz, D. B., Peterson, B. E., and Michalopoulos, G. K. Extracellular matrix remodeling at the early stages of liver regeneration in the rat. Hepatology 1997:26:896-904).  
         [0080]    Besides its function in plasminogen activation, it is considered that uPA is one of the main primers leading to the activation of metalloprotease cascade associated with matrix degradation (Kim, T. H., Mars, W. M., Stolz, D. B., Peterson, B. E., and Michalopoulos, G. K. Extracellular matrix remodeling at the early stages of liver regeneration in the rat. Hepatology 1997:26:896-904). After plasminogen activation into plasmin through uPA, said plasmin, in turn, activates procolagenases and possibly other metalloproteases (MMPs) to their active form, in early stages (Kim, T. H., Mars, W. M., Stolz, D. B., Peterson, B. E., and Michalopoulos, G. K. Extracellular matrix remodeling at the early stages of liver regeneration in the rat. Hepatology 1997:26:896-904). The in situ action of said enzymes brought about the ECM specific component degradation, remodeling the altered organ architecture and the appearance of newly formed blood vessels (angiogenesis) (data not shown). Even though it is clear that modified uPA overproduction activates the rapid ECM reorganization observed here, this could be regulated through mechanisms not yet elucidated, but the specific MMP-2 induction is part of said mechanisms.  
         [0081]    Thus, besides the uPA/plasmin system initiating matrix remodeling, membrane type matrix metalloproteases (MT-MMPs) could be other inducible proteinases that have to be identified in ECM remodeling in our system, since it has been reported that MT1-MMPs could initiate pro-MMP2 activation. Thus, a collateral object of the study conducted at our laboratory is to elucidate MMPs function in huPA-induced liver fibrosis reversion.  
         [0082]    Finally, the application of this type of strategies is being evaluated to be used on cirrhotic human beings prior determination and evaluation in animals superior to the rat. We are in the process of integrating the corresponding protocol for its evaluation in Beagle dogs and/or non-human primates.  
         [0083]    It must be obvious for those skilled in the art that other embodiments of the present invention not shown in this description are possible and within the scope and spirit of this invention. Thus the invention is not limited to the embodiments presented in this description, and the invention is only limited by following claims and their equivalent.  
     
       
       
         1 
         
           
             13  
           
           
             1  
             35935  
             DNA  
             Human adenovirus type 5  
           
            1 

catcatcaat aatatacctt attttggatt gaagccaata tgataatgag ggggtggagt     60 

ttgtgacgtg gcgcggggcg tgggaacggg gcgggtgacg tagtagtgtg gcggaagtgt    120 

gatgttgcaa gtgtggcgga acacatgtaa gcgacggatg tggcaaaagt gacgtttttg    180 

gtgtgcgccg gtgtacacag gaagtgacaa ttttcgcgcg gttttaggcg gatgttgtag    240 

taaatttggg cgtaaccgag taagatttgg ccattttcgc gggaaaactg aataagagga    300 

agtgaaatct gaataatttt gtgttactca tagcgcgtaa tatttgtcta gggccgcggg    360 

gactttgacc gtttacgtgg agactcgccc aggtgttttt ctcaggtgtt ttccgcgttc    420 

cgggtcaaag ttggcgtttt attattatag tcagctgacg tgtagtgtat ttatacccgg    480 

tgagttcctc aagaggccac tcttgagtgc cagcgagtag agttttctcc tccgagccgc    540 

tccgacaccg ggactgaaaa tgagacatat tatctgccac ggaggtgtta ttaccgaaga    600 

aatggccgcc agtcttttgg accagctgat cgaagaggta ctggctgata atcttccacc    660 

tcctagccat tttgaaccac ctacccttca cgaactgtat gatttagacg tgacggcccc    720 

cgaagatccc aacgaggagg cggtttcgca gatttttccc gactctgtaa tgttggcggt    780 

gcaggaaggg attgacttac tcacttttcc gccggcgccc ggttctccgg agccgcctca    840 

cctttcccgg cagcccgagc agccggagca gagagccttg ggtccggttt ctatgccaaa    900 

ccttgtaccg gaggtgatcg atcttacctg ccacgaggct ggctttccac ccagtgacga    960 

cgaggatgaa gagggtgagg agtttgtgtt agattatgtg gagcaccccg ggcacggttg   1020 

caggtcttgt cattatcacc ggaggaatac gggggaccca gatattatgt gttcgctttg   1080 

ctatatgagg acctgtggca tgtttgtcta cagtaagtga aaattatggg cagtgggtga   1140 

tagagtggtg ggtttggtgt ggtaattttt tttttaattt ttacagtttt gtggtttaaa   1200 

gaattttgta ttgtgatttt tttaaaaggt cctgtgtctg aacctgagcc tgagcccgag   1260 

ccagaaccgg agcctgcaag acctacccgc cgtcctaaaa tggcgcctgc tatcctgaga   1320 

cgcccgacat cacctgtgtc tagagaatgc aatagtagta cggatagctg tgactccggt   1380 

ccttctaaca cacctcctga gatacacccg gtggtcccgc tgtgccccat taaaccagtt   1440 

gccgtgagag ttggtgggcg tcgccaggct gtggaatgta tcgaggactt gcttaacgag   1500 

cctgggcaac ctttggactt gagctgtaaa cgccccaggc cataaggtgt aaacctgtga   1560 

ttgcgtgtgt ggttaacgcc tttgtttgct gaatgagttg atgtaagttt aataaagggt   1620 

gagataatgt ttaacttgca tggcgtgtta aatggggcgg ggcttaaagg gtatataatg   1680 

cgccgtgggc taatcttggt tacatctgac ctcatggagg cttgggagtg tttggaagat   1740 

ttttctgctg tgcgtaactt gctggaacag agctctaaca gtacctcttg gttttggagg   1800 

tttctgtggg gctcatccca ggcaaagtta gtctgcagaa ttaaggagga ttacaagtgg   1860 

gaatttgaag agcttttgaa atcctgtggt gagctgtttg attctttgaa tctgggtcac   1920 

caggcgcttt tccaagagaa ggtcatcaag actttggatt tttccacacc ggggcgcgct   1980 

gcggctgctg ttgctttttt gagttttata aaggataaat ggagcgaaga aacccatctg   2040 

agcggggggt acctgctgga ttttctggcc atgcatctgt ggagagcggt tgtgagacac   2100 

aagaatcgcc tgctactgtt gtcttccgtc cgcccggcga taataccgac ggaggagcag   2160 

cagcagcagc aggaggaagc caggcggcgg cggcaggagc agagcccatg gaacccgaga   2220 

gccggcctgg accctcggga atgaatgttg tacaggtggc tgaactgtat ccagaactga   2280 

gacgcatttt gacaattaca gaggatgggc aggggctaaa gggggtaaag agggagcggg   2340 

gggcttgtga ggctacagag gaggctagga atctagcttt tagcttaatg accagacacc   2400 

gtcctgagtg tattactttt caacagatca aggataattg cgctaatgag cttgatctgc   2460 

tggcgcagaa gtattccata gagcagctga ccacttactg gctgcagcca ggggatgatt   2520 

ttgaggaggc tattagggta tatgcaaagg tggcacttag gccagattgc aagtacaaga   2580 

tcagcaaact tgtaaatatc aggaattgtt gctacatttc tgggaacggg gccgaggtgg   2640 

agatagatac ggaggatagg gtggccttta gatgtagcat gataaatatg tggccggggg   2700 

tgcttggcat ggacggggtg gttattatga atgtaaggtt tactggcccc aattttagcg   2760 

gtacggtttt cctggccaat accaacctta tcctacacgg tgtaagcttc tatgggttta   2820 

acaatacctg tgtggaagcc tggaccgatg taagggttcg gggctgtgcc ttttactgct   2880 

gctggaaggg ggtggtgtgt cgccccaaaa gcagggcttc aattaagaaa tgcctctttg   2940 

aaaggtgtac cttgggtatc ctgtctgagg gtaactccag ggtgcgccac aatgtggcct   3000 

ccgactgtgg ttgcttcatg ctagtgaaaa gcgtggctgt gattaagcat aacatggtat   3060 

gtggcaactg cgaggacagg gcctctcaga tgctgacctg ctcggacggc aactgtcacc   3120 

tgctgaagac cattcacgta gccagccact ctcgcaaggc ctggccagtg tttgagcata   3180 

acatactgac ccgctgttcc ttgcatttgg gtaacaggag gggggtgttc ctaccttacc   3240 

aatgcaattt gagtcacact aagatattgc ttgagcccga gagcatgtcc aaggtgaacc   3300 

tgaacggggt gtttgacatg accatgaaga tctggaaggt gctgaggtac gatgagaccc   3360 

gcaccaggtg cagaccctgc gagtgtggcg gtaaacatat taggaaccag cctgtgatgc   3420 

tggatgtgac cgaggagctg aggcccgatc acttggtgct ggcctgcacc cgcgctgagt   3480 

ttggctctag cgatgaagat acagattgag gtactgaaat gtgtgggcgt ggcttaaggg   3540 

tgggaaagaa tatataaggt gggggtctta tgtagttttg tatctgtttt gcagcagccg   3600 

ccgccgccat gagcaccaac tcgtttgatg gaagcattgt gagctcatat ttgacaacgc   3660 

gcatgccccc atgggccggg gtgcgtcaga atgtgatggg ctccagcatt gatggtcgcc   3720 

ccgtcctgcc cgcaaactct actaccttga cctacgagac cgtgtctgga acgccgttgg   3780 

agactgcagc ctccgccgcc gcttcagccg ctgcagccac cgcccgcggg attgtgactg   3840 

actttgcttt cctgagcccg cttgcaagca gtgcagcttc ccgttcatcc gcccgcgatg   3900 

acaagttgac ggctcttttg gcacaattgg attctttgac ccgggaactt aatgtcgttt   3960 

ctcagcagct gttggatctg cgccagcagg tttctgccct gaaggcttcc tcccctccca   4020 

atgcggttta aaacataaat aaaaaaccag actctgtttg gatttggatc aagcaagtgt   4080 

cttgctgtct ttatttaggg gttttgcgcg cgcggtaggc ccgggaccag cggtctcggt   4140 

cgttgagggt cctgtgtatt ttttccagga cgtggtaaag gtgactctgg atgttcagat   4200 

acatgggcat aagcccgtct ctggggtgga ggtagcacca ctgcagagct tcatgctgcg   4260 

gggtggtgtt gtagatgatc cagtcgtagc aggagcgctg ggcgtggtgc ctaaaaatgt   4320 

ctttcagtag caagctgatt gccaggggca ggcccttggt gtaagtgttt acaaagcggt   4380 

taagctggga tgggtgcata cgtggggata tgagatgcat cttggactgt atttttaggt   4440 

tggctatgtt cccagccata tccctccggg gattcatgtt gtgcagaacc accagcacag   4500 

tgtatccggt gcacttggga aatttgtcat gtagcttaga aggaaatgcg tggaagaact   4560 

tggagacgcc cttgtgacct ccaagatttt ccatgcattc gtccataatg atggcaatgg   4620 

gcccacgggc ggcggcctgg gcgaagatat ttctgggatc actaacgtca tagttgtgtt   4680 

ccaggatgag atcgtcatag gccattttta caaagcgcgg gcggagggtg ccagactgcg   4740 

gtataatggt tccatccggc ccaggggcgt agttaccctc acagatttgc atttcccacg   4800 

ctttgagttc agatgggggg atcatgtcta cctgcggggc gatgaagaaa acggtttccg   4860 

gggtagggga gatcagctgg gaagaaagca ggttcctgag cagctgcgac ttaccgcagc   4920 

cggtgggccc gtaaatcaca cctattaccg ggtgcaactg gtagttaaga gagctgcagc   4980 

tgccgtcatc cctgagcagg ggggccactt cgttaagcat gtccctgact cgcatgtttt   5040 

ccctgaccaa atccgccaga aggcgctcgc cgcccagcga tagcagttct tgcaaggaag   5100 

caaagttttt caacggtttg agaccgtccg ccgtaggcat gcttttgagc gtttgaccaa   5160 

gcagttccag gcggtcccac agctcggtca cctgctctac ggcatctcga tccagcatat   5220 

ctcctcgttt cgcgggttgg ggcggctttc gctgtacggc agtagtcggt gctcgtccag   5280 

acgggccagg gtcatgtctt tccacgggcg cagggtcctc gtcagcgtag tctgggtcac   5340 

ggtgaagggg tgcgctccgg gctgcgcgct ggccagggtg cgcttgaggc tggtcctgct   5400 

ggtgctgaag cgctgccggt cttcgccctg cgcgtcggcc aggtagcatt tgaccatggt   5460 

gtcatagtcc agcccctccg cggcgtggcc cttggcgcgc agcttgccct tggaggaggc   5520 

gccgcacgag gggcagtgca gacttttgag ggcgtagagc ttgggcgcga gaaataccga   5580 

ttccggggag taggcatccg cgccgcaggc cccgcagacg gtctcgcatt ccacgagcca   5640 

ggtgagctct ggccgttcgg ggtcaaaaac caggtttccc ccatgctttt tgatgcgttt   5700 

cttacctctg gtttccatga gccggtgtcc acgctcggtg acgaaaaggc tgtccgtgtc   5760 

cccgtataca gacttgagag gcctgtcctc gagcggtgtt ccgcggtcct cctcgtatag   5820 

aaactcggac cactctgaga caaaggctcg cgtccaggcc agcacgaagg aggctaagtg   5880 

ggaggggtag cggtcgttgt ccactagggg gtccactcgc tccagggtgt gaagacacat   5940 

gtcgccctct tcggcatcaa ggaaggtgat tggtttgtag gtgtaggcca cgtgaccggg   6000 

tgttcctgaa ggggggctat aaaagggggt gggggcgcgt tcgtcctcac tctcttccgc   6060 

atcgctgtct gcgagggcca gctgttgggg tgagtactcc ctctgaaaag cgggcatgac   6120 

ttctgcgcta agattgtcag tttccaaaaa cgaggaggat ttgatattca cctggcccgc   6180 

ggtgatgcct ttgagggtgg ccgcatccat ctggtcagaa aagacaatct ttttgttgtc   6240 

aagcttggtg gcaaacgacc cgtagagggc gttggacagc aacttggcga tggagcgcag   6300 

ggtttggttt ttgtcgcgat cggcgcgctc cttggccgcg atgtttagct gcacgtattc   6360 

gcgcgcaacg caccgccatt cgggaaagac ggtggtgcgc tcgtcgggca ccaggtgcac   6420 

gcgccaaccg cggttgtgca gggtgacaag gtcaacgctg gtggctacct ctccgcgtag   6480 

gcgctcgttg gtccagcaga ggcggccgcc cttgcgcgag cagaatggcg gtagggggtc   6540 

tagctgcgtc tcgtccgggg ggtctgcgtc cacggtaaag accccgggca gcaggcgcgc   6600 

gtcgaagtag tctatcttgc atccttgcaa gtctagcgcc tgctgccatg cgcgggcggc   6660 

aagcgcgcgc tcgtatgggt tgagtggggg accccatggc atggggtggg tgagcgcgga   6720 

ggcgtacatg ccgcaaatgt cgtaaacgta gaggggctct ctgagtattc caagatatgt   6780 

agggtagcat cttccaccgc ggatgctggc gcgcacgtaa tcgtatagtt cgtgcgaggg   6840 

agcgaggagg tcgggaccga ggttgctacg ggcgggctgc tctgctcgga agactatctg   6900 

cctgaagatg gcatgtgagt tggatgatat ggttggacgc tggaagacgt tgaagctggc   6960 

gtctgtgaga cctaccgcgt cacgcacgaa ggaggcgtag gagtcgcgca gcttgttgac   7020 

cagctcggcg gtgacctgca cgtctagggc gcagtagtcc agggtttcct tgatgatgtc   7080 

atacttatcc tgtccctttt ttttccacag ctcgcggttg aggacaaact cttcgcggtc   7140 

tttccagtac tcttggatcg gaaacccgtc ggcctccgaa cggtaagagc ctagcatgta   7200 

gaactggttg acggcctggt aggcgcagca tcccttttct acgggtagcg cgtatgcctg   7260 

cgcggccttc cggagcgagg tgtgggtgag cgcaaaggtg tccctgacca tgactttgag   7320 

gtactggtat ttgaagtcag tgtcgtcgca tccgccctgc tcccagagca aaaagtccgt   7380 

gcgctttttg gaacgcggat ttggcagggc gaaggtgaca tcgttgaaga gtatctttcc   7440 

cgcgcgaggc ataaagttgc gtgtgatgcg gaagggtccc ggcacctcgg aacggttgtt   7500 

aattacctgg gcggcgagca cgatctcgtc aaagccgttg atgttgtggc ccacaatgta   7560 

aagttccaag aagcgcggga tgcccttgat ggaaggcaat tttttaagtt cctcgtaggt   7620 

gagctcttca ggggagctga gcccgtgctc tgaaagggcc cagtctgcaa gatgagggtt   7680 

ggaagcgacg aatgagctcc acaggtcacg ggccattagc atttgcaggt ggtcgcgaaa   7740 

ggtcctaaac tggcgaccta tggccatttt ttctggggtg atgcagtaga aggtaagcgg   7800 

gtcttgttcc cagcggtccc atccaaggtt cgcggctagg tctcgcgcgg cagtcactag   7860 

aggctcatct ccgccgaact tcatgaccag catgaagggc acgagctgct tcccaaaggc   7920 

ccccatccaa gtataggtct ctacatcgta ggtgacaaag agacgctcgg tgcgaggatg   7980 

cgagccgatc gggaagaact ggatctcccg ccaccaattg gaggagtggc tattgatgtg   8040 

gtgaaagtag aagtccctgc gacgggccga acactcgtgc tggcttttgt aaaaacgtgc   8100 

gcagtactgg cagcggtgca cgggctgtac atcctgcacg aggttgacct gacgaccgcg   8160 

cacaaggaag cagagtggga atttgagccc ctcgcctggc gggtttggct ggtggtcttc   8220 

tacttcggct gcttgtcctt gaccgtctgg ctgctcgagg ggagttacgg tggatcggac   8280 

caccacgccg cgcgagccca aagtccagat gtccgcgcgc ggcggtcgga gcttgatgac   8340 

aacatcgcgc agatgggagc tgtccatggt ctggagctcc cgcggcgtca ggtcaggcgg   8400 

gagctcctgc aggtttacct cgcatagacg ggtcagggcg cgggctagat ccaggtgata   8460 

cctaatttcc aggggctggt tggtggcggc gtcgatggct tgcaagaggc cgcatccccg   8520 

cggcgcgact acggtaccgc gcggcgggcg gtgggccgcg ggggtgtcct tggatgatgc   8580 

atctaaaagc ggtgacgcgg gcgagccccc ggaggtaggg ggggctccgg acccgccggg   8640 

agagggggca ggggcacgtc ggcgccgcgc gcgggcagga gctggtgctg cgcgcgtagg   8700 

ttgctggcga acgcgacgac gcggcggttg atctcctgaa tctggcgcct ctgcgtgaag   8760 

acgacgggcc cggtgagctt gagcctgaaa gagagttcga cagaatcaat ttcggtgtcg   8820 

ttgacggcgg cctggcgcaa aatctcctgc acgtctcctg agttgtcttg ataggcgatc   8880 

tcggccatga actgctcgat ctcttcctcc tggagatctc cgcgtccggc tcgctccacg   8940 

gtggcggcga ggtcgttgga aatgcgggcc atgagctgcg agaaggcgtt gaggcctccc   9000 

tcgttccaga cgcggctgta gaccacgccc ccttcggcat cgcgggcgcg catgaccacc   9060 

tgcgcgagat tgagctccac gtgccgggcg aagacggcgt agtttcgcag gcgctgaaag   9120 

aggtagttga gggtggtggc ggtgtgttct gccacgaaga agtacataac ccagcgtcgc   9180 

aacgtggatt cgttgatatc ccccaaggcc tcaaggcgct ccatggcctc gtagaagtcc   9240 

acggcgaagt tgaaaaactg ggagttgcgc gccgacacgg ttaactcctc ctccagaaga   9300 

cggatgagct cggcgacagt gtcgcgcacc tcgcgctcaa aggctacagg ggcctcttct   9360 

tcttcttcaa tctcctcttc cataagggcc tccccttctt cttcttctgg cggcggtggg   9420 

ggagggggga cacggcggcg acgacggcgc accgggaggc ggtcgacaaa gcgctcgatc   9480 

atctccccgc ggcgacggcg catggtctcg gtgacggcgc ggccgttctc gcgggggcgc   9540 

agttggaaga cgccgcccgt catgtcccgg ttatgggttg gcggggggct gccatgcggc   9600 

agggatacgg cgctaacgat gcatctcaac aattgttgtg taggtactcc gccgccgagg   9660 

gacctgagcg agtccgcatc gaccggatcg gaaaacctct cgagaaaggc gtctaaccag   9720 

tcacagtcgc aaggtaggct gagcaccgtg gcgggcggca gcgggcggcg gtcggggttg   9780 

tttctggcgg aggtgctgct gatgatgtaa ttaaagtagg cggtcttgag acggcggatg   9840 

gtcgacagaa gcaccatgtc cttgggtccg gcctgctgaa tgcgcaggcg gtcggccatg   9900 

ccccaggctt cgttttgaca tcggcgcagg tctttgtagt agtcttgcat gagcctttct   9960 

accggcactt cttcttctcc ttcctcttgt cctgcatctc ttgcatctat cgctgcggcg  10020 

gcggcggagt ttggccgtag gtggcgccct cttcctccca tgcgtgtgac cccgaagccc  10080 

ctcatcggct gaagcagggc taggtcggcg acaacgcgct cggctaatat ggcctgctgc  10140 

acctgcgtga gggtagactg gaagtcatcc atgtccacaa agcggtggta tgcgcccgtg  10200 

ttgatggtgt aagtgcagtt ggccataacg gaccagttaa cggtctggtg acccggctgc  10260 

gagagctcgg tgtacctgag acgcgagtaa gccctcgagt caaatacgta gtcgttgcaa  10320 

gtccgcacca ggtactggta tcccaccaaa aagtgcggcg gcggctggcg gtagaggggc  10380 

cagcgtaggg tggccggggc tccgggggcg agatcttcca acataaggcg atgatatccg  10440 

tagatgtacc tggacatcca ggtgatgccg gcggcggtgg tggaggcgcg cggaaagtcg  10500 

cggacgcggt tccagatgtt gcgcagcggc aaaaagtgct ccatggtcgg gacgctctgg  10560 

ccggtcaggc gcgcgcaatc gttgacgctc tagaccgtgc aaaaggagag cctgtaagcg  10620 

ggcactcttc cgtggtctgg tggataaatt cgcaagggta tcatggcgga cgaccggggt  10680 

tcgagccccg tatccggccg tccgccgtga tccatgcggt taccgcccgc gtgtcgaacc  10740 

caggtgtgcg acgtcagaca acgggggagt gctccttttg gcttccttcc aggcgcggcg  10800 

gctgctgcgc tagctttttt ggccactggc cgcgcgcagc gtaagcggtt aggctggaaa  10860 

gcgaaagcat taagtggctc gctccctgta gccggagggt tattttccaa gggttgagtc  10920 

gcgggacccc cggttcgagt ctcggaccgg ccggactgcg gcgaacgggg gtttgcctcc  10980 

ccgtcatgca agaccccgct tgcaaattcc tccggaaaca gggacgagcc ccttttttgc  11040 

ttttcccaga tgcatccggt gctgcggcag atgcgccccc ctcctcagca gcggcaagag  11100 

caagagcagc ggcagacatg cagggcaccc tcccctcctc ctaccgcgtc aggaggggcg  11160 

acatccgcgg ttgacgcggc agcagatggt gattacgaac ccccgcggcg ccgggcccgg  11220 

cactacctgg acttggagga gggcgagggc ctggcgcggc taggagcgcc ctctcctgag  11280 

cggtacccaa gggtgcagct gaagcgtgat acgcgtgagg cgtacgtgcc gcggcagaac  11340 

ctgtttcgcg accgcgaggg agaggagccc gaggagatgc gggatcgaaa gttccacgca  11400 

gggcgcgagc tgcggcatgg cctgaatcgc gagcggttgc tgcgcgagga ggactttgag  11460 

cccgacgcgc gaaccgggat tagtcccgcg cgcgcacacg tggcggccgc cgacctggta  11520 

accgcatacg agcagacggt gaaccaggag attaactttc aaaaaagctt taacaaccac  11580 

gtgcgtacgc ttgtggcgcg cgaggaggtg gctataggac tgatgcatct gtgggacttt  11640 

gtaagcgcgc tggagcaaaa cccaaatagc aagccgctca tggcgcagct gttccttata  11700 

gtgcagcaca gcagggacaa cgaggcattc agggatgcgc tgctaaacat agtagagccc  11760 

gagggccgct ggctgctcga tttgataaac atcctgcaga gcatagtggt gcaggagcgc  11820 

agcttgagcc tggctgacaa ggtggccgcc atcaactatt ccatgcttag cctgggcaag  11880 

ttttacgccc gcaagatata ccatacccct tacgttccca tagacaagga ggtaaagatc  11940 

gaggggttct acatgcgcat ggcgctgaag gtgcttacct tgagcgacga cctgggcgtt  12000 

tatcgcaacg agcgcatcca caaggccgtg agcgtgagcc ggcggcgcga gctcagcgac  12060 

cgcgagctga tgcacagcct gcaaagggcc ctggctggca cgggcagcgg cgatagagag  12120 

gccgagtcct actttgacgc gggcgctgac ctgcgctggg ccccaagccg acgcgccctg  12180 

gaggcagctg gggccggacc tgggctggcg gtggcacccg cgcgcgctgg caacgtcggc  12240 

ggcgtggagg aatatgacga ggacgatgag tacgagccag aggacggcga gtactaagcg  12300 

gtgatgtttc tgatcagatg atgcaagacg caacggaccc ggcggtgcgg gcggcgctgc  12360 

agagccagcc gtccggcctt aactccacgg acgactggcg ccaggtcatg gaccgcatca  12420 

tgtcgctgac tgcgcgcaat cctgacgcgt tccggcagca gccgcaggcc aaccggctct  12480 

ccgcaattct ggaagcggtg gtcccggcgc gcgcaaaccc cacgcacgag aaggtgctgg  12540 

cgatcgtaaa cgcgctggcc gaaaacaggg ccatccggcc cgacgaggcc ggcctggtct  12600 

acgacgcgct gcttcagcgc gtggctcgtt acaacagcgg caacgtgcag accaacctgg  12660 

accggctggt gggggatgtg cgcgaggccg tggcgcagcg tgagcgcgcg cagcagcagg  12720 

gcaacctggg ctccatggtt gcactaaacg ccttcctgag tacacagccc gccaacgtgc  12780 

cgcggggaca ggaggactac accaactttg tgagcgcact gcggctaatg gtgactgaga  12840 

caccgcaaag tgaggtgtac cagtctgggc cagactattt tttccagacc agtagacaag  12900 

gcctgcagac cgtaaacctg agccaggctt tcaaaaactt gcaggggctg tggggggtgc  12960 

gggctcccac aggcgaccgc gcgaccgtgt ctagcttgct gacgcccaac tcgcgcctgt  13020 

tgctgctgct aatagcgccc ttcacggaca gtggcagcgt gtcccgggac acatacctag  13080 

gtcacttgct gacactgtac cgcgaggcca taggtcaggc gcatgtggac gagcatactt  13140 

tccaggagat tacaagtgtc agccgcgcgc tggggcagga ggacacgggc agcctggagg  13200 

caaccctaaa ctacctgctg accaaccggc ggcagaagat cccctcgttg cacagtttaa  13260 

acagcgagga ggagcgcatt ttgcgctacg tgcagcagag cgtgagcctt aacctgatgc  13320 

gcgacggggt aacgcccagc gtggcgctgg acatgaccgc gcgcaacatg gaaccgggca  13380 

tgtatgcctc aaaccggccg tttatcaacc gcctaatgga ctacttgcat cgcgcggccg  13440 

ccgtgaaccc cgagtatttc accaatgcca tcttgaaccc gcactggcta ccgccccctg  13500 

gtttctacac cgggggattc gaggtgcccg agggtaacga tggattcctc tgggacgaca  13560 

tagacgacag cgtgttttcc ccgcaaccgc agaccctgct agagttgcaa cagcgcgagc  13620 

aggcagaggc ggcgctgcga aaggaaagct tccgcaggcc aagcagcttg tccgatctag  13680 

gcgctgcggc cccgcggtca gatgctagta gcccatttcc aagcttgata gggtctctta  13740 

ccagcactcg caccacccgc ccgcgcctgc tgggcgagga ggagtaccta aacaactcgc  13800 

tgctgcagcc gcagcgcgaa aaaaacctgc ctccggcatt tcccaacaac gggatagaga  13860 

gcctagtgga caagatgagt agatggaaga cgtacgcgca ggagcacagg gacgtgccag  13920 

gcccgcgccc gcccacccgt cgtcaaaggc acgaccgtca gcggggtctg gtgtgggagg  13980 

acgatgactc ggcagacgac agcagcgtcc tggatttggg agggagtggc aacccgtttg  14040 

cgcaccttcg ccccaggctg gggagaatgt tttaaaaaaa aaaaagcatg atgcaaaata  14100 

aaaaactcac caaggccatg gcaccgagcg ttggttttct tgtattcccc ttagtatgcg  14160 

gcgcgcggcg atgtatgagg aaggtcctcc tccctcctac gagagtgtgg tgagcgcggc  14220 

gccagtggcg gcggcgctgg gttctccctt cgatgctccc ctggacccgc cgtttgtgcc  14280 

tccgcggtac ctgcggccta ccggggggag aaacagcatc cgttactctg agttggcacc  14340 

cctattcgac accacccgtg tgtacctggt ggacaacaag tcaacggatg tggcatccct  14400 

gaactaccag aacgaccaca gcaactttct gaccacggtc attcaaaaca atgactacag  14460 

cccgggggag gcaagcacac agaccatcaa tcttgacgac cggtcgcact ggggcggcga  14520 

cctgaaaacc atcctgcata ccaacatgcc aaatgtgaac gagttcatgt ttaccaataa  14580 

gtttaaggcg cgggtgatgg tgtcgcgctt gcctactaag gacaatcagg tggagctgaa  14640 

atacgagtgg gtggagttca cgctgcccga gggcaactac tccgagacca tgaccataga  14700 

ccttatgaac aacgcgatcg tggagcacta cttgaaagtg ggcagacaga acggggttct  14760 

ggaaagcgac atcggggtaa agtttgacac ccgcaacttc agactggggt ttgaccccgt  14820 

cactggtctt gtcatgcctg gggtatatac aaacgaagcc ttccatccag acatcatttt  14880 

gctgccagga tgcggggtgg acttcaccca cagccgcctg agcaacttgt tgggcatccg  14940 

caagcggcaa cccttccagg agggctttag gatcacctac gatgatctgg agggtggtaa  15000 

cattcccgca ctgttggatg tggacgccta ccaggcgagc ttgaaagatg acaccgaaca  15060 

gggcgggggt ggcgcaggcg gcagcaacag cagtggcagc ggcgcggaag agaactccaa  15120 

cgcggcagcc gcggcaatgc agccggtgga ggacatgaac gatcatgcca ttcgcggcga  15180 

cacctttgcc acacgggctg aggagaagcg cgctgaggcc gaagcagcgg ccgaagctgc  15240 

cgcccccgct gcgcaacccg aggtcgagaa gcctcagaag aaaccggtga tcaaacccct  15300 

gacagaggac agcaagaaac gcagttacaa cctaataagc aatgacagca ccttcaccca  15360 

gtaccgcagc tggtaccttg catacaacta cggcgaccct cagaccggaa tccgctcatg  15420 

gaccctgctt tgcactcctg acgtaacctg cggctcggag caggtctact ggtcgttgcc  15480 

agacatgatg caagaccccg tgaccttccg ctccacgcgc cagatcagca actttccggt  15540 

ggtgggcgcc gagctgttgc ccgtgcactc caagagcttc tacaacgacc aggccgtcta  15600 

ctcccaactc atccgccagt ttacctctct gacccacgtg ttcaatcgct ttcccgagaa  15660 

ccagattttg gcgcgcccgc cagcccccac catcaccacc gtcagtgaaa acgttcctgc  15720 

tctcacagat cacgggacgc taccgctgcg caacagcatc ggaggagtcc agcgagtgac  15780 

cattactgac gccagacgcc gcacctgccc ctacgtttac aaggccctgg gcatagtctc  15840 

gccgcgcgtc ctatcgagcc gcactttttg agcaagcatg tccatcctta tatcgcccag  15900 

caataacaca ggctggggcc tgcgcttccc aagcaagatg tttggcgggg ccaagaagcg  15960 

ctccgaccaa cacccagtgc gcgtgcgcgg gcactaccgc gcgccctggg gcgcgcacaa  16020 

acgcggccgc actgggcgca ccaccgtcga tgacgccatc gacgcggtgg tggaggaggc  16080 

gcgcaactac acgcccacgc cgccaccagt gtccacagtg gacgcggcca ttcagaccgt  16140 

ggtgcgcgga gcccggcgct atgctaaaat gaagagacgg cggaggcgcg tagcacgtcg  16200 

ccaccgccgc cgacccggca ctgccgccca acgcgcggcg gcggccctgc ttaaccgcgc  16260 

acgtcgcacc ggccgacggg cggccatgcg ggccgctcga aggctggccg cgggtattgt  16320 

cactgtgccc cccaggtcca ggcgacgagc ggccgccgca gcagccgcgg ccattagtgc  16380 

tatgactcag ggtcgcaggg gcaacgtgta ttgggtgcgc gactcggtta gcggcctgcg  16440 

cgtgcccgtg cgcacccgcc ccccgcgcaa ctagattgca agaaaaaact acttagactc  16500 

gtactgttgt atgtatccag cggcggcggc gcgcaacgaa gctatgtcca agcgcaaaat  16560 

caaagaagag atgctccagg tcatcgcgcc ggagatctat ggccccccga agaaggaaga  16620 

gcaggattac aagccccgaa agctaaagcg ggtcaaaaag aaaaagaaag atgatgatga  16680 

tgaacttgac gacgaggtgg aactgctgca cgctaccgcg cccaggcgac gggtacagtg  16740 

gaaaggtcga cgcgtaaaac gtgttttgcg acccggcacc accgtagtct ttacgcccgg  16800 

tgagcgctcc acccgcacct acaagcgcgt gtatgatgag gtgtacggcg acgaggacct  16860 

gcttgagcag gccaacgagc gcctcgggga gtttgcctac ggaaagcggc ataaggacat  16920 

gctggcgttg ccgctggacg agggcaaccc aacacctagc ctaaagcccg taacactgca  16980 

gcaggtgctg cccgcgcttg caccgtccga agaaaagcgc ggcctaaagc gcgagtctgg  17040 

tgacttggca cccaccgtgc agctgatggt acccaagcgc cagcgactgg aagatgtctt  17100 

ggaaaaaatg accgtggaac ctgggctgga gcccgaggtc cgcgtgcggc caatcaagca  17160 

ggtggcgccg ggactgggcg tgcagaccgt ggacgttcag atacccacta ccagtagcac  17220 

cagtattgcc accgccacag agggcatgga gacacaaacg tccccggttg cctcagcggt  17280 

ggcggatgcc gcggtgcagg cggtcgctgc ggccgcgtcc aagacctcta cggaggtgca  17340 

aacggacccg tggatgtttc gcgtttcagc cccccggcgc ccgcgcggtt cgaggaagta  17400 

cggcgccgcc agcgcgctac tgcccgaata tgccctacat ccttccattg cgcctacccc  17460 

cggctatcgt ggctacacct accgccccag aagacgagca actacccgac gccgaaccac  17520 

cactggaacc cgccgccgcc gtcgccgtcg ccagcccgtg ctggccccga tttccgtgcg  17580 

cagggtggct cgcgaaggag gcaggaccct ggtgctgcca acagcgcgct accaccccag  17640 

catcgtttaa aagccggtct ttgtggttct tgcagatatg gccctcacct gccgcctccg  17700 

tttcccggtg ccgggattcc gaggaagaat gcaccgtagg aggggcatgg ccggccacgg  17760 

cctgacgggc ggcatgcgtc gtgcgcacca ccggcggcgg cgcgcgtcgc accgtcgcat  17820 

gcgcggcggt atcctgcccc tccttattcc actgatcgcc gcggcgattg gcgccgtgcc  17880 

cggaattgca tccgtggcct tgcaggcgca gagacactga ttaaaaacaa gttgcatgtg  17940 

gaaaaatcaa aataaaaagt ctggactctc acgctcgctt ggtcctgtaa ctattttgta  18000 

gaatggaaga catcaacttt gcgtctctgg ccccgcgaca cggctcgcgc ccgttcatgg  18060 

gaaactggca agatatcggc accagcaata tgagcggtgg cgccttcagc tggggctcgc  18120 

tgtggagcgg cattaaaaat ttcggttcca ccgttaagaa ctatggcagc aaggcctgga  18180 

acagcagcac aggccagatg ctgagggata agttgaaaga gcaaaatttc caacaaaagg  18240 

tggtagatgg cctggcctct ggcattagcg gggtggtgga cctggccaac caggcagtgc  18300 

aaaataagat taacagtaag cttgatcccc gccctcccgt agaggagcct ccaccggccg  18360 

tggagacagt gtctccagag gggcgtggcg aaaagcgtcc gcgccccgac agggaagaaa  18420 

ctctggtgac gcaaatagac gagcctccct cgtacgagga ggcactaaag caaggcctgc  18480 

ccaccacccg tcccatcgcg cccatggcta ccggagtgct gggccagcac acacccgtaa  18540 

cgctggacct gcctcccccc gccgacaccc agcagaaacc tgtgctgcca ggcccgaccg  18600 

ccgttgttgt aacccgtcct agccgcgcgt ccctgcgccg cgccgccagc ggtccgcgat  18660 

cgttgcggcc cgtagccagt ggcaactggc aaagcacact gaacagcatc gtgggtctgg  18720 

gggtgcaatc cctgaagcgc cgacgatgct tctgaatagc taacgtgtcg tatgtgtgtc  18780 

atgtatgcgt ccatgtcgcc gccagaggag ctgctgagcc gccgcgcgcc cgctttccaa  18840 

gatggctacc ccttcgatga tgccgcagtg gtcttacatg cacatctcgg gccaggacgc  18900 

ctcggagtac ctgagccccg ggctggtgca gtttgcccgc gccaccgaga cgtacttcag  18960 

cctgaataac aagtttagaa accccacggt ggcgcctacg cacgacgtga ccacagaccg  19020 

gtcccagcgt ttgacgctgc ggttcatccc tgtggaccgt gaggatactg cgtactcgta  19080 

caaggcgcgg ttcaccctag ctgtgggtga taaccgtgtg ctggacatgg cttccacgta  19140 

ctttgacatc cgcggcgtgc tggacagggg ccctactttt aagccctact ctggcactgc  19200 

ctacaacgcc ctggctccca agggtgcccc aaatccttgc gaatgggatg aagctgctac  19260 

tgctcttgaa ataaacctag aagaagagga cgatgacaac gaagacgaag tagacgagca  19320 

agctgagcag caaaaaactc acgtatttgg gcaggcgcct tattctggta taaatattac  19380 

aaaggagggt attcaaatag gtgtcgaagg tcaaacacct aaatatgccg ataaaacatt  19440 

tcaacctgaa cctcaaatag gagaatctca gtggtacgaa actgaaatta atcatgcagc  19500 

tgggagagtc cttaaaaaga ctaccccaat gaaaccatgt tacggttcat atgcaaaacc  19560 

cacaaatgaa aatggagggc aaggcattct tgtaaagcaa caaaatggaa agctagaaag  19620 

tcaagtggaa atgcaatttt tctcaactac tgaggcgacc gcaggcaatg gtgataactt  19680 

gactcctaaa gtggtattgt acagtgaaga tgtagatata gaaaccccag acactcatat  19740 

ttcttacatg cccactatta aggaaggtaa ctcacgagaa ctaatgggcc aacaatctat  19800 

gcccaacagg cctaattaca ttgcttttag ggacaatttt attggtctaa tgtattacaa  19860 

cagcacgggt aatatgggtg ttctggcggg ccaagcatcg cagttgaatg ctgttgtaga  19920 

tttgcaagac agaaacacag agctttcata ccagcttttg cttgattcca ttggtgatag  19980 

aaccaggtac ttttctatgt ggaatcaggc tgttgacagc tatgatccag atgttagaat  20040 

tattgaaaat catggaactg aagatgaact tccaaattac tgctttccac tgggaggtgt  20100 

gattaataca gagactctta ccaaggtaaa acctaaaaca ggtcaggaaa atggatggga  20160 

aaaagatgct acagaatttt cagataaaaa tgaaataaga gttggaaata attttgccat  20220 

ggaaatcaat ctaaatgcca acctgtggag aaatttcctg tactccaaca tagcgctgta  20280 

tttgcccgac aagctaaagt acagtccttc caacgtaaaa atttctgata acccaaacac  20340 

ctacgactac atgaacaagc gagtggtggc tcccgggtta gtggactgct acattaacct  20400 

tggagcacgc tggtcccttg actatatgga caacgtcaac ccatttaacc accaccgcaa  20460 

tgctggcctg cgctaccgct caatgttgct gggcaatggt cgctatgtgc ccttccacat  20520 

ccaggtgcct cagaagttct ttgccattaa aaacctcctt ctcctgccgg gctcatacac  20580 

ctacgagtgg aacttcagga aggatgttaa catggttctg cagagctccc taggaaatga  20640 

cctaagggtt gacggagcca gcattaagtt tgatagcatt tgcctttacg ccaccttctt  20700 

ccccatggcc cacaacaccg cctccacgct tgaggccatg cttagaaacg acaccaacga  20760 

ccagtccttt aacgactatc tctccgccgc caacatgctc taccctatac ccgccaacgc  20820 

taccaacgtg cccatatcca tcccctcccg caactgggcg gctttccgcg gctgggcctt  20880 

cacgcgcctt aagactaagg aaaccccatc actgggctcg ggctacgacc cttattacac  20940 

ctactctggc tctataccct acctagatgg aaccttttac ctcaaccaca cctttaagaa  21000 

ggtggccatt acctttgact cttctgtcag ctggcctggc aatgaccgcc tgcttacccc  21060 

caacgagttt gaaattaagc gctcagttga cggggagggt tacaacgttg cccagtgtaa  21120 

catgaccaaa gactggttcc tggtacaaat gctagctaac tacaacattg gctaccaggg  21180 

cttctatatc ccagagagct acaaggaccg catgtactcc ttctttagaa acttccagcc  21240 

catgagccgt caggtggtgg atgatactaa atacaaggac taccaacagg tgggcatcct  21300 

acaccaacac aacaactctg gatttgttgg ctaccttgcc cccaccatgc gcgaaggaca  21360 

ggcctaccct gctaacttcc cctatccgct tataggcaag accgcagttg acagcattac  21420 

ccagaaaaag tttctttgcg atcgcaccct ttggcgcatc ccattctcca gtaactttat  21480 

gtccatgggc gcactcacag acctgggcca aaaccttctc tacgccaact ccgcccacgc  21540 

gctagacatg acttttgagg tggatcccat ggacgagccc acccttcttt atgttttgtt  21600 

tgaagtcttt gacgtggtcc gtgtgcaccg gccgcaccgc ggcgtcatcg aaaccgtgta  21660 

cctgcgcacg cccttctcgg ccggcaacgc cacaacataa agaagcaagc aacatcaaca  21720 

acagctgccg ccatgggctc cagtgagcag gaactgaaag ccattgtcaa agatcttggt  21780 

tgtgggccat attttttggg cacctatgac aagcgctttc caggctttgt ttctccacac  21840 

aagctcgcct gcgccatagt caatacggcc ggtcgcgaga ctgggggcgt acactggatg  21900 

gcctttgcct ggaacccgca ctcaaaaaca tgctacctct ttgagccctt tggcttttct  21960 

gaccagcgac tcaagcaggt ttaccagttt gagtacgagt cactcctgcg ccgtagcgcc  22020 

attgcttctt cccccgaccg ctgtataacg ctggaaaagt ccacccaaag cgtacagggg  22080 

cccaactcgg ccgcctgtgg actattctgc tgcatgtttc tccacgcctt tgccaactgg  22140 

ccccaaactc ccatggatca caaccccacc atgaacctta ttaccggggt acccaactcc  22200 

atgctcaaca gtccccaggt acagcccacc ctgcgtcgca accaggaaca gctctacagc  22260 

ttcctggagc gccactcgcc ctacttccgc agccacagtg cgcagattag gagcgccact  22320 

tctttttgtc acttgaaaaa catgtaaaaa taatgtacta gagacacttt caataaaggc  22380 

aaatgctttt atttgtacac tctcgggtga ttatttaccc ccacccttgc cgtctgcgcc  22440 

gtttaaaaat caaaggggtt ctgccgcgca tcgctatgcg ccactggcag ggacacgttg  22500 

cgatactggt gtttagtgct ccacttaaac tcaggcacaa ccatccgcgg cagctcggtg  22560 

aagttttcac tccacaggct gcgcaccatc accaacgcgt ttagcaggtc gggcgccgat  22620 

atcttgaagt cgcagttggg gcctccgccc tgcgcgcgcg agttgcgata cacagggttg  22680 

cagcactgga acactatcag cgccgggtgg tgcacgctgg ccagcacgct cttgtcggag  22740 

atcagatccg cgtccaggtc ctccgcgttg ctcagggcga acggagtcaa ctttggtagc  22800 

tgccttccca aaaagggcgc gtgcccaggc tttgagttgc actcgcaccg tagtggcatc  22860 

aaaaggtgac cgtgcccggt ctgggcgtta ggatacagcg cctgcataaa agccttgatc  22920 

tgcttaaaag ccacctgagc ctttgcgcct tcagagaaga acatgccgca agacttgccg  22980 

gaaaactgat tggccggaca ggccgcgtcg tgcacgcagc accttgcgtc ggtgttggag  23040 

atctgcacca catttcggcc ccaccggttc ttcacgatct tggccttgct agactgctcc  23100 

ttcagcgcgc gctgcccgtt ttcgctcgtc acatccattt caatcacgtg ctccttattt  23160 

atcataatgc ttccgtgtag acacttaagc tcgccttcga tctcagcgca gcggtgcagc  23220 

cacaacgcgc agcccgtggg ctcgtgatgc ttgtaggtca cctctgcaaa cgactgcagg  23280 

tacgcctgca ggaatcgccc catcatcgtc acaaaggtct tgttgctggt gaaggtcagc  23340 

tgcaacccgc ggtgctcctc gttcagccag gtcttgcata cggccgccag agcttccact  23400 

tggtcaggca gtagtttgaa gttcgccttt agatcgttat ccacgtggta cttgtccatc  23460 

agcgcgcgcg cagcctccat gcccttctcc cacgcagaca cgatcggcac actcagcggg  23520 

ttcatcaccg taatttcact ttccgcttcg ctgggctctt cctcttcctc ttgcgtccgc  23580 

ataccacgcg ccactgggtc gtcttcattc agccgccgca ctgtgcgctt acctcctttg  23640 

ccatgcttga ttagcaccgg tgggttgctg aaacccacca tttgtagcgc cacatcttct  23700 

ctttcttcct cgctgtccac gattacctct ggtgatggcg ggcgctcggg cttgggagaa  23760 

gggcgcttct ttttcttctt gggcgcaatg gccaaatccg ccgccgaggt cgatggccgc  23820 

gggctgggtg tgcgcggcac cagcgcgtct tgtgatgagt cttcctcgtc ctcggactcg  23880 

atacgccgcc tcatccgctt ttttgggggc gcccggggag gcggcggcga cggggacggg  23940 

gacgacacgt cctccatggt tgggggacgt cgcgccgcac cgcgtccgcg ctcgggggtg  24000 

gtttcgcgct gctcctcttc ccgactggcc atttccttct cctataggca gaaaaagatc  24060 

atggagtcag tcgagaagaa ggacagccta accgccccct ctgagttcgc caccaccgcc  24120 

tccaccgatg ccgccaacgc gcctaccacc ttccccgtcg aggcaccccc gcttgaggag  24180 

gaggaagtga ttatcgagca ggacccaggt tttgtaagcg aagacgacga ggaccgctca  24240 

gtaccaacag aggataaaaa gcaagaccag gacaacgcag aggcaaacga ggaacaagtc  24300 

gggcgggggg acgaaaggca tggcgactac ctagatgtgg gagacgacgt gctgttgaag  24360 

catctgcagc gccagtgcgc cattatctgc gacgcgttgc aagagcgcag cgatgtgccc  24420 

ctcgccatag cggatgtcag ccttgcctac gaacgccacc tattctcacc gcgcgtaccc  24480 

cccaaacgcc aagaaaacgg cacatgcgag cccaacccgc gcctcaactt ctaccccgta  24540 

tttgccgtgc cagaggtgct tgccacctat cacatctttt tccaaaactg caagataccc  24600 

ctatcctgcc gtgccaaccg cagccgagcg gacaagcagc tggccttgcg gcagggcgct  24660 

gtcatacctg atatcgcctc gctcaacgaa gtgccaaaaa tctttgaggg tcttggacgc  24720 

gacgagaagc gcgcggcaaa cgctctgcaa caggaaaaca gcgaaaatga aagtcactct  24780 

ggagtgttgg tggaactcga gggtgacaac gcgcgcctag ccgtactaaa acgcagcatc  24840 

gaggtcaccc actttgccta cccggcactt aacctacccc ccaaggtcat gagcacagtc  24900 

atgagtgagc tgatcgtgcg ccgtgcgcag cccctggaga gggatgcaaa tttgcaagaa  24960 

caaacagagg agggcctacc cgcagttggc gacgagcagc tagcgcgctg gcttcaaacg  25020 

cgcgagcctg ccgacttgga ggagcgacgc aaactaatga tggccgcagt gctcgttacc  25080 

gtggagcttg agtgcatgca gcggttcttt gctgacccgg agatgcagcg caagctagag  25140 

gaaacattgc actacacctt tcgacagggc tacgtacgcc aggcctgcaa gatctccaac  25200 

gtggagctct gcaacctggt ctcctacctt ggaattttgc acgaaaaccg ccttgggcaa  25260 

aacgtgcttc attccacgct caagggcgag gcgcgccgcg actacgtccg cgactgcgtt  25320 

tacttatttc tatgctacac ctggcagacg gccatgggcg tttggcagca gtgcttggag  25380 

gagtgcaacc tcaaggagct gcagaaactg ctaaagcaaa acttgaagga cctatggacg  25440 

gccttcaacg agcgctccgt ggccgcgcac ctggcggaca tcattttccc cgaacgcctg  25500 

cttaaaaccc tgcaacaggg tctgccagac ttcaccagtc aaagcatgtt gcagaacttt  25560 

aggaacttta tcctagagcg ctcaggaatc ttgcccgcca cctgctgtgc acttcctagc  25620 

gactttgtgc ccattaagta ccgcgaatgc cctccgccgc tttggggcca ctgctacctt  25680 

ctgcagctag ccaactacct tgcctaccac tctgacataa tggaagacgt gagcggtgac  25740 

ggtctactgg agtgtcactg tcgctgcaac ctatgcaccc cgcaccgctc cctggtttgc  25800 

aattcgcagc tgcttaacga aagtcaaatt atcggtacct ttgagctgca gggtccctcg  25860 

cctgacgaaa agtccgcggc tccggggttg aaactcactc cggggctgtg gacgtcggct  25920 

taccttcgca aatttgtacc tgaggactac cacgcccacg agattaggtt ctacgaagac  25980 

caatcccgcc cgccaaatgc ggagcttacc gcctgcgtca ttacccaggg ccacattctt  26040 

ggccaattgc aagccatcaa caaagcccgc caagagtttc tgctacgaaa gggacggggg  26100 

gtttacttgg acccccagtc cggcgaggag ctcaacccaa tccccccgcc gccgcagccc  26160 

tatcagcagc agccgcgggc ccttgcttcc caggatggca cccaaaaaga agctgcagct  26220 

gccgccgcca cccacggacg aggaggaata ctgggacagt caggcagagg aggttttgga  26280 

cgaggaggag gaggacatga tggaagactg ggagagccta gacgaggaag cttccgaggt  26340 

cgaagaggtg tcagacgaaa caccgtcacc ctcggtcgca ttcccctcgc cggcgcccca  26400 

gaaatcggca accggttcca gcatggctac aacctccgct cctcaggcgc cgccggcact  26460 

gcccgttcgc cgacccaacc gtagatggga caccactgga accagggccg gtaagtccaa  26520 

gcagccgccg ccgttagccc aagagcaaca acagcgccaa ggctaccgct catggcgcgg  26580 

gcacaagaac gccatagttg cttgcttgca agactgtggg ggcaacatct ccttcgcccg  26640 

ccgctttctt ctctaccatc acggcgtggc cttcccccgt aacatcctgc attactaccg  26700 

tcatctctac agcccatact gcaccggcgg cagcggcagc ggcagcaaca gcagcggcca  26760 

cacagaagca aaggcgaccg gatagcaaga ctctgacaaa gcccaagaaa tccacagcgg  26820 

cggcagcagc aggaggagga gcgctgcgtc tggcgcccaa cgaacccgta tcgacccgcg  26880 

agcttagaaa caggattttt cccactctgt atgctatatt tcaacagagc aggggccaag  26940 

aacaagagct gaaaataaaa aacaggtctc tgcgatccct cacccgcagc tgcctgtatc  27000 

acaaaagcga agatcagctt cggcgcacgc tggaagacgc ggaggctctc ttcagtaaat  27060 

actgcgcgct gactcttaag gactagtttc gcgccctttc tcaaatttaa gcgcgaaaac  27120 

tacgtcatct ccagcggcca cacccggcgc cagcacctgt cgtcagcgcc attatgagca  27180 

aggaaattcc cacgccctac atgtggagtt accagccaca aatgggactt gcggctggag  27240 

ctgcccaaga ctactcaacc cgaataaact acatgagcgc gggaccccac atgatatccc  27300 

gggtcaacgg aatccgcgcc caccgaaacc gaattctctt ggaacaggcg gctattacca  27360 

ccacacctcg taataacctt aatccccgta gttggcccgc tgccctggtg taccaggaaa  27420 

gtcccgctcc caccactgtg gtacttccca gagacgccca ggccgaagtt cagatgacta  27480 

actcaggggc gcagcttgcg ggcggctttc gtcacagggt gcggtcgccc gggcagggta  27540 

taactcacct gacaatcaga gggcgaggta ttcagctcaa cgacgagtcg gtgagctcct  27600 

cgcttggtct ccgtccggac gggacatttc agatcggcgg cgccggccgt ccttcattca  27660 

cgcctcgtca ggcaatccta actctgcaga cctcgtcctc tgagccgcgc tctggaggca  27720 

ttggaactct gcaatttatt gaggagtttg tgccatcggt ctactttaac cccttctcgg  27780 

gacctcccgg ccactatccg gatcaattta ttcctaactt tgacgcggta aaggactcgg  27840 

cggacggcta cgactgaatg ttaagtggag aggcagagca actgcgcctg aaacacctgg  27900 

tccactgtcg ccgccacaag tgctttgccc gcgactccgg tgagttttgc tactttgaat  27960 

tgcccgagga tcatatcgag ggcccggcgc acggcgtccg gcttaccgcc cagggagagc  28020 

ttgcccgtag cctgattcgg gagtttaccc agcgccccct gctagttgag cgggacaggg  28080 

gaccctgtgt tctcactgtg atttgcaact gtcctaacct tggattacat caagatcttt  28140 

gttgccatct ctgtgctgag tataataaat acagaaatta aaatatactg gggctcctat  28200 

cgccatcctg taaacgccac cgtcttcacc cgcccaagca aaccaaggcg aaccttacct  28260 

ggtactttta acatctctcc ctctgtgatt tacaacagtt tcaacccaga cggagtgagt  28320 

ctacgagaga acctctccga gctcagctac tccatcagaa aaaacaccac cctccttacc  28380 

tgccgggaac gtacgagtgc gtcaccggcc gctgcaccac acctaccgcc tgaccgtaaa  28440 

ccagactttt tccggacaga cctcaataac tctgtttacc agaacaggag gtgagcttag  28500 

aaaaccctta gggtattagg ccaaaggcgc agctactgtg gggtttatga acaattcaag  28560 

caactctacg ggctattcta attcaggttt ctctagaatc ggggttgggg ttattctctg  28620 

tcttgtgatt ctctttattc ttatactaac gcttctctgc ctaaggctcg ccgcctgctg  28680 

tgtgcacatt tgcatttatt gtcagctttt taaacgctgg ggtcgccacc caagatgatt  28740 

aggtacataa tcctaggttt actcaccctt gcgtcagccc acggtaccac ccaaaaggtg  28800 

gattttaagg agccagcctg taatgttaca ttcgcagctg aagctaatga gtgcaccact  28860 

cttataaaat gcaccacaga acatgaaaag ctgcttattc gccacaaaaa caaaattggc  28920 

aagtatgctg tttatgctat ttggcagcca ggtgacacta cagagtataa tgttacagtt  28980 

ttccagggta aaagtcataa aacttttatg tatacttttc cattttatga aatgtgcgac  29040 

attaccatgt acatgagcaa acagtataag ttgtggcccc cacaaaattg tgtggaaaac  29100 

actggcactt tctgctgcac tgctatgcta attacagtgc tcgctttggt ctgtacccta  29160 

ctctatatta aatacaaaag cagacgcagc tttattgagg aaaagaaaat gccttaattt  29220 

actaagttac aaagctaatg tcaccactaa ctgctttact cgctgcttgc aaaacaaatt  29280 

caaaaagtta gcattataat tagaatagga tttaaacccc ccggtcattt cctgctcaat  29340 

accattcccc tgaacaattg actctatgtg ggatatgctc cagcgctaca accttgaagt  29400 

caggcttcct ggatgtcagc atctgacttt ggccagcacc tgtcccgcgg atttgttcca  29460 

gtccaactac agcgacccac cctaacagag atgaccaaca caaccaacgc ggccgccgct  29520 

accggactta catctaccac aaatacaccc caagtttctg cctttgtcaa taactgggat  29580 

aacttgggca tgtggtggtt ctccatagcg cttatgtttg tatgccttat tattatgtgg  29640 

ctcatctgct gcctaaagcg caaacgcgcc cgaccaccca tctatagtcc catcattgtg  29700 

ctacacccaa acaatgatgg aatccataga ttggacggac tgaaacacat gttcttttct  29760 

cttacagtat gattaaatga gacatgattc ctcgagtttt tatattactg acccttgttg  29820 

cgcttttttg tgcgtgctcc acattggctg cggtttctca catcgaagta gactgcattc  29880 

cagccttcac agtctatttg ctttacggat ttgtcaccct cacgctcatc tgcagcctca  29940 

tcactgtggt catcgccttt atccagtgca ttgactgggt ctgtgtgcgc tttgcatatc  30000 

tcagacacca tccccagtac agggacagga ctatagctga gcttcttaga attctttaat  30060 

tatgaaattt actgtgactt ttctgctgat tatttgcacc ctatctgcgt tttgttcccc  30120 

gacctccaag cctcaaagac atatatcatg cagattcact cgtatatgga atattccaag  30180 

ttgctacaat gaaaaaagcg atctttccga agcctggtta tatgcaatca tctctgttat  30240 

ggtgttctgc agtaccatct tagccctagc tatatatccc taccttgaca ttggctggaa  30300 

acgaatagat gccatgaacc acccaacttt ccccgcgccc gctatgcttc cactgcaaca  30360 

agttgttgcc ggcggctttg tcccagccaa tcagcctcgc cccacttctc ccacccccac  30420 

tgaaatcagc tactttaatc taacaggagg agatgactga caccctagat ctagaaatgg  30480 

acggaattat tacagagcag cgcctgctag aaagacgcag ggcagcggcc gagcaacagc  30540 

gcatgaatca agagctccaa gacatggtta acttgcacca gtgcaaaagg ggtatctttt  30600 

gtctggtaaa gcaggccaaa gtcacctacg acagtaatac caccggacac cgccttagct  30660 

acaagttgcc aaccaagcgt cagaaattgg tggtcatggt gggagaaaag cccattacca  30720 

taactcagca ctcggtagaa accgaaggct gcattcactc accttgtcaa ggacctgagg  30780 

atctctgcac ccttattaag accctgtgcg gtctcaaaga tcttattccc tttaactaat  30840 

aaaaaaaaat aataaagcat cacttactta aaatcagtta gcaaatttct gtccagttta  30900 

ttcagcagca cctccttgcc ctcctcccag ctctggtatt gcagcttcct cctggctgca  30960 

aactttctcc acaatctaaa tggaatgtca gtttcctcct gttcctgtcc atccgcaccc  31020 

actatcttca tgttgttgca gatgaagcgc gcaagaccgt ctgaagatac cttcaacccc  31080 

gtgtatccat atgacacgga aaccggtcct ccaactgtgc cttttcttac tcctcccttt  31140 

gtatccccca atgggtttca agagagtccc cctggggtac tctctttgcg cctatccgaa  31200 

cctctagtta cctccaatgg catgcttgcg ctcaaaatgg gcaacggcct ctctctggac  31260 

gaggccggca accttacctc ccaaaatgta accactgtga gcccacctct caaaaaaacc  31320 

aagtcaaaca taaacctgga aatatctgca cccctcacag ttacctcaga agccctaact  31380 

gtggctgccg ccgcacctct aatggtcgcg ggcaacacac tcaccatgca atcacaggcc  31440 

ccgctaaccg tgcacgactc caaacttagc attgccaccc aaggacccct cacagtgtca  31500 

gaaggaaagc tagccctgca aacatcaggc cccctcacca ccaccgatag cagtaccctt  31560 

actatcactg cctcaccccc tctaactact gccactggta gcttgggcat tgacttgaaa  31620 

gagcccattt atacacaaaa tggaaaacta ggactaaagt acggggctcc tttgcatgta  31680 

acagacgacc taaacacttt gaccgtagca actggtccag gtgtgactat taataatact  31740 

tccttgcaaa ctaaagttac tggagccttg ggttttgatt cacaaggcaa tatgcaactt  31800 

aatgtagcag gaggactaag gattgattct caaaacagac gccttatact tgatgttagt  31860 

tatccgtttg atgctcaaaa ccaactaaat ctaagactag gacagggccc tctttttata  31920 

aactcagccc acaacttgga tattaactac aacaaaggcc tttacttgtt tacagcttca  31980 

aacaattcca aaaagcttga ggttaaccta agcactgcca aggggttgat gtttgacgct  32040 

acagccatag ccattaatgc aggagatggg cttgaatttg gttcacctaa tgcaccaaac  32100 

acaaatcccc tcaaaacaaa aattggccat ggcctagaat ttgattcaaa caaggctatg  32160 

gttcctaaac taggaactgg ccttagtttt gacagcacag gtgccattac agtaggaaac  32220 

aaaaataatg ataagctaac tttgtggacc acaccagctc catctcctaa ctgtagacta  32280 

aatgcagaga aagatgctaa actcactttg gtcttaacaa aatgtggcag tcaaatactt  32340 

gctacagttt cagttttggc tgttaaaggc agtttggctc caatatctgg aacagttcaa  32400 

agtgctcatc ttattataag atttgacgaa aatggagtgc tactaaacaa ttccttcctg  32460 

gacccagaat attggaactt tagaaatgga gatcttactg aaggcacagc ctatacaaac  32520 

gctgttggat ttatgcctaa cctatcagct tatccaaaat ctcacggtaa aactgccaaa  32580 

agtaacattg tcagtcaagt ttacttaaac ggagacaaaa ctaaacctgt aacactaacc  32640 

attacactaa acggtacaca ggaaacagga gacacaactc caagtgcata ctctatgtca  32700 

ttttcatggg actggtctgg ccacaactac attaatgaaa tatttgccac atcctcttac  32760 

actttttcat acattgccca agaataaaga atcgtttgtg ttatgtttca acgtgtttat  32820 

ttttcaattg cagaaaattt caagtcattt ttcattcagt agtatagccc caccaccaca  32880 

tagcttatac agatcaccgt accttaatca aactcacaga accctagtat tcaacctgcc  32940 

acctccctcc caacacacag agtacacagt cctttctccc cggctggcct taaaaagcat  33000 

catatcatgg gtaacagaca tattcttagg tgttatattc cacacggttt cctgtcgagc  33060 

caaacgctca tcagtgatat taataaactc cccgggcagc tcacttaagt tcatgtcgct  33120 

gtccagctgc tgagccacag gctgctgtcc aacttgcggt tgcttaacgg gcggcgaagg  33180 

agaagtccac gcctacatgg gggtagagtc ataatcgtgc atcaggatag ggcggtggtg  33240 

ctgcagcagc gcgcgaataa actgctgccg ccgccgctcc gtcctgcagg aatacaacat  33300 

ggcagtggtc tcctcagcga tgattcgcac cgcccgcagc ataaggcgcc ttgtcctccg  33360 

ggcacagcag cgcaccctga tctcacttaa atcagcacag taactgcagc acagcaccac  33420 

aatattgttc aaaatcccac agtgcaaggc gctgtatcca aagctcatgg cggggaccac  33480 

agaacccacg tggccatcat accacaagcg caggtagatt aagtggcgac ccctcataaa  33540 

cacgctggac ataaacatta cctcttttgg catgttgtaa ttcaccacct cccggtacca  33600 

tataaacctc tgattaaaca tggcgccatc caccaccatc ctaaaccagc tggccaaaac  33660 

ctgcccgccg gctatacact gcagggaacc gggactggaa caatgacagt ggagagccca  33720 

ggactcgtaa ccatggatca tcatgctcgt catgatatca atgttggcac aacacaggca  33780 

cacgtgcata cacttcctca ggattacaag ctcctcccgc gttagaacca tatcccaggg  33840 

aacaacccat tcctgaatca gcgtaaatcc cacactgcag ggaagacctc gcacgtaact  33900 

cacgttgtgc attgtcaaag tgttacattc gggcagcagc ggatgatcct ccagtatggt  33960 

agcgcgggtt tctgtctcaa aaggaggtag acgatcccta ctgtacggag tgcgccgaga  34020 

caaccgagat cgtgttggtc gtagtgtcat gccaaatgga acgccggacg tagtcatatt  34080 

tcctgaagca aaaccaggtg cgggcgtgac aaacagatct gcgtctccgg tctcgccgct  34140 

tagatcgctc tgtgtagtag ttgtagtata tccactctct caaagcatcc aggcgccccc  34200 

tggcttcggg ttctatgtaa actccttcat gcgccgctgc cctgataaca tccaccaccg  34260 

cagaataagc cacacccagc caacctacac attcgttctg cgagtcacac acgggaggag  34320 

cgggaagagc tggaagaacc atgttttttt ttttattcca aaagattatc caaaacctca  34380 

aaatgaagat ctattaagtg aacgcgctcc cctccggtgg cgtggtcaaa ctctacagcc  34440 

aaagaacaga taatggcatt tgtaagatgt tgcacaatgg cttccaaaag gcaaacggcc  34500 

ctcacgtcca agtggacgta aaggctaaac ccttcagggt gaatctcctc tataaacatt  34560 

ccagcacctt caaccatgcc caaataattc tcatctcgcc accttctcaa tatatctcta  34620 

agcaaatccc gaatattaag tccggccatt gtaaaaatct gctccagagc gccctccacc  34680 

ttcagcctca agcagcgaat catgattgca aaaattcagg ttcctcacag acctgtataa  34740 

gattcaaaag cggaacatta acaaaaatac cgcgatcccg taggtccctt cgcagggcca  34800 

gctgaacata atcgtgcagg tctgcacgga ccagcgcggc cacttccccg ccaggaacct  34860 

tgacaaaaga acccacactg attatgacac gcatactcgg agctatgcta accagcgtag  34920 

ccccgatgta agctttgttg catgggcggc gatataaaat gcaaggtgct gctcaaaaaa  34980 

tcaggcaaag cctcgcgcaa aaaagaaagc acatcgtagt catgctcatg cagataaagg  35040 

caggtaagct ccggaaccac cacagaaaaa gacaccattt ttctctcaaa catgtctgcg  35100 

ggtttctgca taaacacaaa ataaaataac aaaaaaacat ttaaacatta gaagcctgtc  35160 

ttacaacagg aaaaacaacc cttataagca taagacggac tacggccatg ccggcgtgac  35220 

cgtaaaaaaa ctggtcaccg tgattaaaaa gcaccaccga cagctcctcg gtcatgtccg  35280 

gagtcataat gtaagactcg gtaaacacat caggttgatt catcggtcag tgctaaaaag  35340 

cgaccgaaat agcccggggg aatacatacc cgcaggcgta gagacaacat tacagccccc  35400 

ataggaggta taacaaaatt aataggagag aaaaacacat aaacacctga aaaaccctcc  35460 

tgcctaggca aaatagcacc ctcccgctcc agaacaacat acagcgcttc acagcggcag  35520 

cctaacagtc agccttacca gtaaaaaaga aaacctatta aaaaaacacc actcgacacg  35580 

gcaccagctc aatcagtcac agtgtaaaaa agggccaagt gcagagcgag tatatatagg  35640 

actaaaaaat gacgtaacgg ttaaagtcca caaaaaacac ccagaaaacc gcacgcgaac  35700 

ctacgcccag aaacgaaagc caaaaaaccc acaacttcct caaatcgtca cttccgtttt  35760 

cccacgttac gtaacttccc attttaagaa aactacaatt cccaacacat acaagttact  35820 

ccgccctaaa acctacgtca cccgccccgt tcccacgccc cgcgccacgt cacaaactcc  35880 

accccctcat tatcatattg gcttcaatcc aaaataaggt atattattga tgatg       35935 

 
           
             2  
             1964  
             DNA  
             Homo sapiens  
             
               CDS  
               (100)...(1395)  
             
           
            2 

aagcttcggg ccagggtcca cctgtccccg cagcgccgtc gcgccctcct gccgcaggcc     60 

accgaggccg ccgccgtcta gcgccccgac ctcgccacc atg aga gcc ctg ctg       114 
                                           Met Arg Ala Leu Leu 
                                            1               5 

gcg cgc ctg ctt ctc tgc gtc ctg gtc gtg agc gac tcc aaa ggc agc      162 
Ala Arg Leu Leu Leu Cys Val Leu Val Val Ser Asp Ser Lys Gly Ser 
                 10                  15                  20 

aat gaa ctt cat caa gtt cca tcg aac tgt gac tgt cta aat gga gga      210 
Asn Glu Leu His Gln Val Pro Ser Asn Cys Asp Cys Leu Asn Gly Gly 
             25                  30                  35 

aca tgt gtg tcc aac aag tac ttc tcc aac att cac tgg tgc aac tgc      258 
Thr Cys Val Ser Asn Lys Tyr Phe Ser Asn Ile His Trp Cys Asn Cys 
         40                  45                  50 

cca aag aaa ttc gga ggg cag cac tgt gaa ata gat aag tca aaa acc      306 
Pro Lys Lys Phe Gly Gly Gln His Cys Glu Ile Asp Lys Ser Lys Thr 
     55                  60                  65 

tgc tat gag ggg aat ggt cac ttt tac cga gga aag gcc agc act gac      354 
Cys Tyr Glu Gly Asn Gly His Phe Tyr Arg Gly Lys Ala Ser Thr Asp 
 70                  75                  80                  85 

acc atg ggc cgg ccc tgc ctg ccc tgg aac tct gcc act gtc ctt cag      402 
Thr Met Gly Arg Pro Cys Leu Pro Trp Asn Ser Ala Thr Val Leu Gln 
                 90                  95                 100 

caa acg tac cat gcc cac aga tct gat gct ctt cag ctg ggc ctg ggg      450 
Gln Thr Tyr His Ala His Arg Ser Asp Ala Leu Gln Leu Gly Leu Gly 
            105                 110                 115 

aaa cat aat tac tgc agg aac cca gac aac cgg agg cga ccc tgg tgc      498 
Lys His Asn Tyr Cys Arg Asn Pro Asp Asn Arg Arg Arg Pro Trp Cys 
        120                 125                 130 

tat gtg cag gtg ggc cta aag ccg ctt gtc caa gag tgc atg gtg cat      546 
Tyr Val Gln Val Gly Leu Lys Pro Leu Val Gln Glu Cys Met Val His 
    135                 140                 145 

gac tgc gca gat gga aaa aag ccc tcc tct cct cca gaa gaa tta aaa      594 
Asp Cys Ala Asp Gly Lys Lys Pro Ser Ser Pro Pro Glu Glu Leu Lys 
150                 155                 160                 165 

ttt cag tgt ggc caa aag act ctg agg ccc cgc ttt aag att att ggg      642 
Phe Gln Cys Gly Gln Lys Thr Leu Arg Pro Arg Phe Lys Ile Ile Gly 
                170                 175                 180 

gga gaa ttc acc acc atc gag aac cag ccc tgg ttt gcg gcc atc tac      690 
Gly Glu Phe Thr Thr Ile Glu Asn Gln Pro Trp Phe Ala Ala Ile Tyr 
            185                 190                 195 

agg agg cac cgg ggg ggc tct gtc acc tac gtg tgt gga ggc agc ctc      738 
Arg Arg His Arg Gly Gly Ser Val Thr Tyr Val Cys Gly Gly Ser Leu 
        200                 205                 210 

atc agc cct tgc tgg gtg atc agc gcc aca cac tgc ttc att gat tac      786 
Ile Ser Pro Cys Trp Val Ile Ser Ala Thr His Cys Phe Ile Asp Tyr 
    215                 220                 225 

cca aag aag gag gac tac atc gtc tac ctg ggt cgc tca agg ctt aac      834 
Pro Lys Lys Glu Asp Tyr Ile Val Tyr Leu Gly Arg Ser Arg Leu Asn 
230                 235                 240                 245 

tcc aac acg caa ggg gag atg aag ttt gag gtg gaa aac ctc atc cta      882 
Ser Asn Thr Gln Gly Glu Met Lys Phe Glu Val Glu Asn Leu Ile Leu 
                250                 255                 260 

cac aag gac tac agc gct gac acg ctt gct cac cac aat gac att gcc      930 
His Lys Asp Tyr Ser Ala Asp Thr Leu Ala His His Asn Asp Ile Ala 
            265                 270                 275 

ttg ctg aag atc cgt tcc aag gag ggc agg tgt gcg cag cca tcc cgg      978 
Leu Leu Lys Ile Arg Ser Lys Glu Gly Arg Cys Ala Gln Pro Ser Arg 
        280                 285                 290 

act ata cag acc atc tgc ctg ccc tcg atg tat aac gat ccc cag ttt     1026 
Thr Ile Gln Thr Ile Cys Leu Pro Ser Met Tyr Asn Asp Pro Gln Phe 
    295                 300                 305 

ggc aca agc tgt gag atc act ggc ttt gga aaa gag aat tct acc gac     1074 
Gly Thr Ser Cys Glu Ile Thr Gly Phe Gly Lys Glu Asn Ser Thr Asp 
310                 315                 320                 325 

tat ctc tat ccg gag cag ctg aaa atg act gtt gtg aag ctg att tcc     1122 
Tyr Leu Tyr Pro Glu Gln Leu Lys Met Thr Val Val Lys Leu Ile Ser 
                330                 335                 340 

cac cgg gag tgt cag cag ccc cac tac tac ggc tct gaa gtc acc acc     1170 
His Arg Glu Cys Gln Gln Pro His Tyr Tyr Gly Ser Glu Val Thr Thr 
            345                 350                 355 

aaa atg ctg tgt gct gct gac cca cag tgg aaa aca gat tcc tgc cag     1218 
Lys Met Leu Cys Ala Ala Asp Pro Gln Trp Lys Thr Asp Ser Cys Gln 
        360                 365                 370 

gga gac tca ggg gga ccc ctc gtc tgt tcc ctc caa ggc cgc atg act     1266 
Gly Asp Ser Gly Gly Pro Leu Val Cys Ser Leu Gln Gly Arg Met Thr 
    375                 380                 385 

ttg act gga att gtg agc tgg ggc cgt gga tgt gcc ctg aag gac aag     1314 
Leu Thr Gly Ile Val Ser Trp Gly Arg Gly Cys Ala Leu Lys Asp Lys 
390                 395                 400                 405 

cca ggc gtc tac acg aga gtc tca cac ttc tta ccc tgg atc cgc agt     1362 
Pro Gly Val Tyr Thr Arg Val Ser His Phe Leu Pro Trp Ile Arg Ser 
                410                 415                 420 

cac acc aag gaa gag aat ggc ctg gcc ctc tga gggtccccag ggaggaaacg   1415 
His Thr Lys Glu Glu Asn Gly Leu Ala Leu  * 
            425                 430 

ggcaccaccc gctttcttgc tggttgtcat ttttgcagta gagtcatctc catcagaagc   1475 

ttttggggag cagagacact aacgacttca gggcagggct ctgatattcc atgaatgtat   1535 

caggaaatat atatgtgtgt gtatgtttgc acacttgttg tgtgggctgt gagtgtaagt   1595 

gtgagtaaga gctggtgtct gattgttaag tctaaatatt tccttaaact gtgtggactg   1655 

tgatgccaca cagagtggtc tttctggaga ggttataggt cactcctggg gcctcttggg   1715 

tcccccacgt gacagtgcct gggaatgtac ttattctgca gcatgacctg tgaccagcac   1775 

tgtctcagtt tcactttcac atagatgtcc ctttcttggc cagttatccc ttccttttag   1835 

cctagttcat ccaatcctca ctgggtgggg tgaggaccac tccttacact gaatatttat   1895 

atttcactat ttttatttat atttttgtaa ttttaaataa aagtgatcaa taaaatgtga   1955 

tttttctga                                                           1964 

 
           
             3  
             1359  
             DNA  
             Homo sapiens  
           
            3 

ggctctagat cgccaccatg cacaggagga gaagcaggag catgcatcgt cgccgcagtc     60 

gaagctgtcg ggaagaccaa aaacctgttt atcaacagcc atcgaaccca tcgaactgtg    120 

actgtctaaa tggaggaaca tgtgtgtcca acaagtactt ctccaacatt cactggtgca    180 

actgcccaaa gaaattcgga gggcagcact gtgaaataga taagtcaaaa acctgctatg    240 

aggggaatgg tcacttttac cgaggaaagg ccagcactga caccatgggc cggccctgcc    300 

tgccctggaa ctctgccact gtccttcagc aaacgtacca tgcccacaga tctgatgctc    360 

ttcagctggg cctggggaaa cataattact gcaggaaccc agacaaccgg aggcgaccct    420 

ggtgctatgt gcaggtgggc ctaaagccgc ttgtccaaga gtgcatggtg catgactgcg    480 

cagatggaaa aaagccctcc tctcctccag aagaattaaa atttcagtgt ggccaaaaga    540 

ctctgaggcc ccgctttaag attattgggg gagaattcac caccatcgag aaccagccct    600 

ggtttgcggc catctacagg aggcaccggg ggggctctgt cacctacgtg tgtggaggca    660 

gcctcatcag cccttgctgg gtgatcagcg ccacacactg cttcattgat tacccaaaga    720 

aggaggacta catcgtctac ctgggtcgct caaggcttaa ctccaacacg caaggggaga    780 

tgaagtttga ggtggaaaac ctcatcctac acaaggacta cagcgctgac acgcttgctc    840 

accacaatga cattgccttg ctgaagatcc gttccaagga gggcaggtgt gcgcagccat    900 

cccggactat acagaccatc tgcctgccct cgatgtataa cgatccccag tttggcacaa    960 

gctgtgagat cactggcttt ggaaaagaga attctaccga ctatctctat ccggagcagc   1020 

tgaaaatgac tgttgtgaag ctgatttccc accgggagtg tcagcagccc cactactacg   1080 

gctctgaagt caccaccaaa atgctgtgtg ctgctgaccc acagtggaaa acagattcct   1140 

gccagggaga ctcaggggga cccctcgtct gttccctcca aggccgcatg actttgactg   1200 

gaattgtgag ctggggccgt ggatgtgccc tgaaggacaa gccaggcgtc tacacgagag   1260 

tctcacactt cttaccctgg atccgcagtc acaccaagga agagaatggc ctggccctcg   1320 

aagaagatac cagtgaaaaa gacgagctct gagggtacc                          1359 

 
           
             4  
             2094  
             DNA  
             Homo sapiens  
             
               CDS  
               (339)...(2042)  
             
           
            4 

gttggcgagg agtttcctgt ttcccccgca gcgctgagtt gaagttgagt gagtcactcg     60 

cgcgcacgga gcgacgacac ccccgcgcgt gcacccgctc gggacaggag ccggactcct    120 

gtgcagcttc cctcggccgc cgggggcctc cccgcgcctc gccggcctcc aggccccctc    180 

ctggctggcg agcgggcgcc acatctggcc cgcacatctg cgctgccggc ccggcgcggg    240 

gtccggagag ggcgcggcgc ggaggcgcag ccaggggtcc gggaaggcgc cgtccgctgc    300 

gctgggggct cggtctatga cgagcagcgg ggtctgcc atg ggt cgg ggg ctg ctc    356 
                                          Met Gly Arg Gly Leu Leu 
                                           1               5 

agg ggc ctg tgg ccg ctg cac atc gtc ctg tgg acg cgt atc gcc agc      404 
Arg Gly Leu Trp Pro Leu His Ile Val Leu Trp Thr Arg Ile Ala Ser 
             10                  15                  20 

acg atc cca ccg cac gtt cag aag tcg gtt aat aac gac atg ata gtc      452 
Thr Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val 
         25                  30                  35 

act gac aac aac ggt gca gtc aag ttt cca caa ctg tgt aaa ttt tgt      500 
Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys 
     40                  45                  50 

gat gtg aga ttt tcc acc tgt gac aac cag aaa tcc tgc atg agc aac      548 
Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn 
 55                  60                  65                  70 

tgc agc atc acc tcc atc tgt gag aag cca cag gaa gtc tgt gtg gct      596 
Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala 
                 75                  80                  85 

gta tgg aga aag aat gac gag aac ata aca cta gag aca gtt tgc cat      644 
Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His 
             90                  95                 100 

gac ccc aag ctc ccc tac cat gac ttt att ctg gaa gat gct gct tct      692 
Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser 
        105                 110                 115 

cca aag tgc att atg aag gaa aaa aaa aag cct ggt gag act ttc ttc      740 
Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe 
    120                 125                 130 

atg tgt tcc tgt agc tct gat gag tgc aat gac aac atc atc ttc tca      788 
Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser 
135                 140                 145                 150 

gaa gaa tat aac acc agc aat cct gac ttg ttg cta gtc ata ttt caa      836 
Glu Glu Tyr Asn Thr Ser Asn Pro Asp Leu Leu Leu Val Ile Phe Gln 
                155                 160                 165 

gtg aca ggc atc agc ctc ctg cca cca ctg gga gtt gcc ata tct gtc      884 
Val Thr Gly Ile Ser Leu Leu Pro Pro Leu Gly Val Ala Ile Ser Val 
            170                 175                 180 

atc atc atc ttc tac tgc tac cgc gtt aac cgg cag cag aag ctg agt      932 
Ile Ile Ile Phe Tyr Cys Tyr Arg Val Asn Arg Gln Gln Lys Leu Ser 
        185                 190                 195 

tca acc tgg gaa acc ggc aag acg cgg aag ctc atg gag ttc agc gag      980 
Ser Thr Trp Glu Thr Gly Lys Thr Arg Lys Leu Met Glu Phe Ser Glu 
    200                 205                 210 

cac tgt gcc atc atc ctg gaa gat gac cgc tct gac atc agc tcc acg     1028 
His Cys Ala Ile Ile Leu Glu Asp Asp Arg Ser Asp Ile Ser Ser Thr 
215                 220                 225                 230 

tgt gcc aac aac atc aac cac aac aca gag ctg ctg ccc att gag ctg     1076 
Cys Ala Asn Asn Ile Asn His Asn Thr Glu Leu Leu Pro Ile Glu Leu 
                235                 240                 245 

gac acc ctg gtg ggg aaa ggt cgc ttt gct gag gtc tat aag gcc aag     1124 
Asp Thr Leu Val Gly Lys Gly Arg Phe Ala Glu Val Tyr Lys Ala Lys 
            250                 255                 260 

ctg aag cag aac act tca gag cag ttt gag aca gtg gca gtc aag atc     1172 
Leu Lys Gln Asn Thr Ser Glu Gln Phe Glu Thr Val Ala Val Lys Ile 
        265                 270                 275 

ttt ccc tat gag gag tat gcc tct tgg aag aca gag aag gac atc ttc     1220 
Phe Pro Tyr Glu Glu Tyr Ala Ser Trp Lys Thr Glu Lys Asp Ile Phe 
    280                 285                 290 

tca gac atc aat ctg aag cat gag aac ata ctc cag ttc ctg acg gct     1268 
Ser Asp Ile Asn Leu Lys His Glu Asn Ile Leu Gln Phe Leu Thr Ala 
295                 300                 305                 310 

gag gag cgg aag acg gag ttg ggg aaa caa tac tgg ctg atc acc gcc     1316 
Glu Glu Arg Lys Thr Glu Leu Gly Lys Gln Tyr Trp Leu Ile Thr Ala 
                315                 320                 325 

ttc cac gcc aag ggc aac cta cag gag tac ctg acg cgg cat gtc atc     1364 
Phe His Ala Lys Gly Asn Leu Gln Glu Tyr Leu Thr Arg His Val Ile 
            330                 335                 340 

agc tgg gag gac ctg cgc aag ctg ggc agc tcc ctc gcc cgg ggg att     1412 
Ser Trp Glu Asp Leu Arg Lys Leu Gly Ser Ser Leu Ala Arg Gly Ile 
        345                 350                 355 

gct cac ctc cac agt gat cac act cca tgt ggg agg ccc aag atg ccc     1460 
Ala His Leu His Ser Asp His Thr Pro Cys Gly Arg Pro Lys Met Pro 
    360                 365                 370 

atc gtg cac agg gac ctc aag agc tcc aat atc ctc gtg aag aac gac     1508 
Ile Val His Arg Asp Leu Lys Ser Ser Asn Ile Leu Val Lys Asn Asp 
375                 380                 385                 390 

cta acc tgc tgc ctg tgt gac ttt ggg ctt tcc ctg cgt ctg gac cct     1556 
Leu Thr Cys Cys Leu Cys Asp Phe Gly Leu Ser Leu Arg Leu Asp Pro 
                395                 400                 405 

act ctg tct gtg gat gac ctg gct aac agt ggg cag gtg gga act gca     1604 
Thr Leu Ser Val Asp Asp Leu Ala Asn Ser Gly Gln Val Gly Thr Ala 
            410                 415                 420 

aga tac atg gct cca gaa gtc cta gaa tcc agg atg aat ttg gag aat     1652 
Arg Tyr Met Ala Pro Glu Val Leu Glu Ser Arg Met Asn Leu Glu Asn 
        425                 430                 435 

gtt gag tcc ttc aag cag acc gat gtc tac tcc atg gct ctg gtg ctc     1700 
Val Glu Ser Phe Lys Gln Thr Asp Val Tyr Ser Met Ala Leu Val Leu 
    440                 445                 450 

tgg gaa atg aca tct cgc tgt aat gca gtg gga gaa gta aaa gat tat     1748 
Trp Glu Met Thr Ser Arg Cys Asn Ala Val Gly Glu Val Lys Asp Tyr 
455                 460                 465                 470 

gag cct cca ttt ggt tcc aag gtg cgg gag cac ccc tgt gtc gaa agc     1796 
Glu Pro Pro Phe Gly Ser Lys Val Arg Glu His Pro Cys Val Glu Ser 
                475                 480                 485 

atg aag gac aac gtg ttg aga gat cga ggg cga cca gaa att ccc agc     1844 
Met Lys Asp Asn Val Leu Arg Asp Arg Gly Arg Pro Glu Ile Pro Ser 
            490                 495                 500 

ttc tgg ctc aac cac cag ggc atc cag atg gtg tgt gag acg ttg act     1892 
Phe Trp Leu Asn His Gln Gly Ile Gln Met Val Cys Glu Thr Leu Thr 
        505                 510                 515 

gag tgc tgg gac cac gac cca gag gcc cgt ctc aca gcc cag tgt gtg     1940 
Glu Cys Trp Asp His Asp Pro Glu Ala Arg Leu Thr Ala Gln Cys Val 
    520                 525                 530 

gca gaa cgc ttc agt gag ctg gag cat ctg gac agg ctc tcg ggg agg     1988 
Ala Glu Arg Phe Ser Glu Leu Glu His Leu Asp Arg Leu Ser Gly Arg 
535                 540                 545                 550 

agc tgc tcg gag gag aag att cct gaa gac ggc tcc cta aac act acc     2036 
Ser Cys Ser Glu Glu Lys Ile Pro Glu Asp Gly Ser Leu Asn Thr Thr 
                555                 560                 565 

aaa tag ctcttctggg gcaggctggg ccatgtccaa agaggctgcc cctctcacca      2092 
Lys  * 

aa                                                                  2094 

 
           
             5  
             2393  
             DNA  
             Homo sapiens  
             
               CDS  
               (105)...(2291)  
             
           
            5 

ggggggctca gagccgactg gctcttttag gcactgactc cgaacaggat tctttcaccc     60 

aggcatctcc tccagaggga tccgccagcc cgtccagcag cacc atg tgg gtg acc     116 
                                                 Met Trp Val Thr 
                                                  1 

aaa ctc ctg cca gcc ctg ctg ctg cag cat gtc ctc ctg cat ctc ctc      164 
Lys Leu Leu Pro Ala Leu Leu Leu Gln His Val Leu Leu His Leu Leu 
 5                   10                  15                  20 

ctg ctc ccc atc gcc atc ccc tat gca gag gga caa agg aaa aga aga      212 
Leu Leu Pro Ile Ala Ile Pro Tyr Ala Glu Gly Gln Arg Lys Arg Arg 
                 25                  30                  35 

aat aca att cat gaa ttc aaa aaa tca gca aag act acc cta atc aaa      260 
Asn Thr Ile His Glu Phe Lys Lys Ser Ala Lys Thr Thr Leu Ile Lys 
             40                  45                  50 

ata gat cca gca ctg aag ata aaa acc aaa aaa gtg aat act gca gac      308 
Ile Asp Pro Ala Leu Lys Ile Lys Thr Lys Lys Val Asn Thr Ala Asp 
         55                  60                  65 

caa tgt gct aat aga tgt act agg aat aaa gga ctt cca ttc act tgc      356 
Gln Cys Ala Asn Arg Cys Thr Arg Asn Lys Gly Leu Pro Phe Thr Cys 
     70                  75                  80 

aag gct ttt gtt ttt gat aaa gca aga aaa caa tgc ctc tgg ttc ccc      404 
Lys Ala Phe Val Phe Asp Lys Ala Arg Lys Gln Cys Leu Trp Phe Pro 
 85                  90                  95                 100 

ttc aat agc atg tca agt gga gtg aaa aaa gaa ttt ggc cat gaa ttt      452 
Phe Asn Ser Met Ser Ser Gly Val Lys Lys Glu Phe Gly His Glu Phe 
                105                 110                 115 

gac ctc tat gaa aac aaa gac tac att aga aac tgc atc att ggt aaa      500 
Asp Leu Tyr Glu Asn Lys Asp Tyr Ile Arg Asn Cys Ile Ile Gly Lys 
            120                 125                 130 

gga cgc agc tac aag gga aca gta tct atc act aag agt ggc atc aaa      548 
Gly Arg Ser Tyr Lys Gly Thr Val Ser Ile Thr Lys Ser Gly Ile Lys 
        135                 140                 145 

tgt cag ccc tgg agt tcc atg ata cca cac gaa cac agc ttt ttg cct      596 
Cys Gln Pro Trp Ser Ser Met Ile Pro His Glu His Ser Phe Leu Pro 
    150                 155                 160 

tcg agc tat cgg ggt aaa gac cta cag gaa aac tac tgt cga aat cct      644 
Ser Ser Tyr Arg Gly Lys Asp Leu Gln Glu Asn Tyr Cys Arg Asn Pro 
165                 170                 175                 180 

cga ggg gaa gaa ggg gga ccc tgg tgt ttc aca agc aat cca gag gta      692 
Arg Gly Glu Glu Gly Gly Pro Trp Cys Phe Thr Ser Asn Pro Glu Val 
                185                 190                 195 

cgc tac gaa gtc tgt gac att cct cag tgt tca gaa gtt gaa tgc atg      740 
Arg Tyr Glu Val Cys Asp Ile Pro Gln Cys Ser Glu Val Glu Cys Met 
            200                 205                 210 

acc tgc aat ggg gag agt tat cga ggt ctc atg gat cat aca gaa tca      788 
Thr Cys Asn Gly Glu Ser Tyr Arg Gly Leu Met Asp His Thr Glu Ser 
        215                 220                 225 

ggc aag att tgt cag cgc tgg gat cat cag aca cca cac cgg cac aaa      836 
Gly Lys Ile Cys Gln Arg Trp Asp His Gln Thr Pro His Arg His Lys 
    230                 235                 240 

ttc ttg cct gaa aga tat ccc gac aag ggc ttt gat gat aat tat tgc      884 
Phe Leu Pro Glu Arg Tyr Pro Asp Lys Gly Phe Asp Asp Asn Tyr Cys 
245                 250                 255                 260 

cgc aat ccc gat ggc cag ccg agg cca tgg tgc tat act ctt gac cct      932 
Arg Asn Pro Asp Gly Gln Pro Arg Pro Trp Cys Tyr Thr Leu Asp Pro 
                265                 270                 275 

cac acc cgc tgg gag tac tgt gca att aaa aca tgc gct gac aat act      980 
His Thr Arg Trp Glu Tyr Cys Ala Ile Lys Thr Cys Ala Asp Asn Thr 
            280                 285                 290 

atg aat gac act gat gtt cct ttg gaa aca act gaa tgc atc caa ggt     1028 
Met Asn Asp Thr Asp Val Pro Leu Glu Thr Thr Glu Cys Ile Gln Gly 
        295                 300                 305 

caa gga gaa ggc tac agg ggc act gtc aat acc att tgg aat gga att     1076 
Gln Gly Glu Gly Tyr Arg Gly Thr Val Asn Thr Ile Trp Asn Gly Ile 
    310                 315                 320 

cca tgt cag cgt tgg gat tct cag tat cct cac gag cat gac atg act     1124 
Pro Cys Gln Arg Trp Asp Ser Gln Tyr Pro His Glu His Asp Met Thr 
325                 330                 335                 340 

cct gaa aat ttc aag tgc aag gac cta cga gaa aat tac tgc cga aat     1172 
Pro Glu Asn Phe Lys Cys Lys Asp Leu Arg Glu Asn Tyr Cys Arg Asn 
                345                 350                 355 

cca gat ggg tct gaa tca ccc tgg tgt ttt acc act gat cca aac atc     1220 
Pro Asp Gly Ser Glu Ser Pro Trp Cys Phe Thr Thr Asp Pro Asn Ile 
            360                 365                 370 

cga gtt ggc tac tgc tcc caa att cca aac tgt gat atg tca cat gga     1268 
Arg Val Gly Tyr Cys Ser Gln Ile Pro Asn Cys Asp Met Ser His Gly 
        375                 380                 385 

caa gat tgt tat cgt ggg aat ggc aaa aat tat atg ggc aac tta tcc     1316 
Gln Asp Cys Tyr Arg Gly Asn Gly Lys Asn Tyr Met Gly Asn Leu Ser 
    390                 395                 400 

caa aca aga tct gga cta aca tgt tca atg tgg gac aag aac atg gaa     1364 
Gln Thr Arg Ser Gly Leu Thr Cys Ser Met Trp Asp Lys Asn Met Glu 
405                 410                 415                 420 

gac tta cat cgt cat atc ttc tgg gaa cca gat gca agt aag ctg aat     1412 
Asp Leu His Arg His Ile Phe Trp Glu Pro Asp Ala Ser Lys Leu Asn 
                425                 430                 435 

gag aat tac tgc cga aat cca gat gat gat gct cat gga ccc tgg tgc     1460 
Glu Asn Tyr Cys Arg Asn Pro Asp Asp Asp Ala His Gly Pro Trp Cys 
            440                 445                 450 

tac acg gga aat cca ctc att cct tgg gat tat tgc cct att tct cgt     1508 
Tyr Thr Gly Asn Pro Leu Ile Pro Trp Asp Tyr Cys Pro Ile Ser Arg 
        455                 460                 465 

tgt gaa ggt gat acc aca cct aca ata gtc aat tta gac cat ccc gta     1556 
Cys Glu Gly Asp Thr Thr Pro Thr Ile Val Asn Leu Asp His Pro Val 
    470                 475                 480 

ata tct tgt gcc aaa acg aaa caa ttg cga gtt gta aat ggg att cca     1604 
Ile Ser Cys Ala Lys Thr Lys Gln Leu Arg Val Val Asn Gly Ile Pro 
485                 490                 495                 500 

aca cga aca aac ata gga tgg atg gtt agt ttg aga tac aga aat aaa     1652 
Thr Arg Thr Asn Ile Gly Trp Met Val Ser Leu Arg Tyr Arg Asn Lys 
                505                 510                 515 

cat atc tgc gga gga tca ttg ata aag gag agt tgg gtt ctt act gca     1700 
His Ile Cys Gly Gly Ser Leu Ile Lys Glu Ser Trp Val Leu Thr Ala 
            520                 525                 530 

cga cag tgt ttc cct tct cga gac ttg aaa gat tat gaa gct tgg ctt     1748 
Arg Gln Cys Phe Pro Ser Arg Asp Leu Lys Asp Tyr Glu Ala Trp Leu 
        535                 540                 545 

gga att cat gat gtc cac gga aga gga gat gag aaa tgc aaa cag gtt     1796 
Gly Ile His Asp Val His Gly Arg Gly Asp Glu Lys Cys Lys Gln Val 
    550                 555                 560 

ctc aat gtt tcc cag ctg gta tat ggc cct gaa gga tca gat ctg gtt     1844 
Leu Asn Val Ser Gln Leu Val Tyr Gly Pro Glu Gly Ser Asp Leu Val 
565                 570                 575                 580 

tta atg aag ctt gcc agg cct gct gtc ctg gat gat ttt gtt agt acg     1892 
Leu Met Lys Leu Ala Arg Pro Ala Val Leu Asp Asp Phe Val Ser Thr 
                585                 590                 595 

att gat tta cct aat tat gga tgc aca att cct gaa aag acc agt tgc     1940 
Ile Asp Leu Pro Asn Tyr Gly Cys Thr Ile Pro Glu Lys Thr Ser Cys 
            600                 605                 610 

agt gtt tat ggc tgg ggc tac act gga ttg atc aac tat gat ggc cta     1988 
Ser Val Tyr Gly Trp Gly Tyr Thr Gly Leu Ile Asn Tyr Asp Gly Leu 
        615                 620                 625 

tta cga gtg gca cat ctc tat ata atg gga aat gag aaa tgc agc cag     2036 
Leu Arg Val Ala His Leu Tyr Ile Met Gly Asn Glu Lys Cys Ser Gln 
    630                 635                 640 

cat cat cga ggg aag gtg act ctg aat gag tct gaa ata tgt gct ggg     2084 
His His Arg Gly Lys Val Thr Leu Asn Glu Ser Glu Ile Cys Ala Gly 
645                 650                 655                 660 

gct gaa aag att gga tca gga cca tgt gag ggg gat tat ggt ggc cca     2132 
Ala Glu Lys Ile Gly Ser Gly Pro Cys Glu Gly Asp Tyr Gly Gly Pro 
                665                 670                 675 

ctt gtt tgt gag caa cat aaa atg aga atg gtt ctt ggt gtc att gtt     2180 
Leu Val Cys Glu Gln His Lys Met Arg Met Val Leu Gly Val Ile Val 
            680                 685                 690 

cct ggt cgt gga tgt gcc att cca aat cgt cct ggt att ttt gtc cga     2228 
Pro Gly Arg Gly Cys Ala Ile Pro Asn Arg Pro Gly Ile Phe Val Arg 
        695                 700                 705 

gta gca tat tat gca aaa tgg ata cac aaa att att tta aca tat aag     2276 
Val Ala Tyr Tyr Ala Lys Trp Ile His Lys Ile Ile Leu Thr Tyr Lys 
    710                 715                 720 

gta cca cag tca tag ctgaagtaag tgtgtctgaa gcacccacca atacaactgt     2331 
Val Pro Gln Ser  * 
725 

cttttacatg aagatttcag agaatgtgga atttaaaatg tcacttacaa caatcctaag   2391 

ac                                                                  2393 

 
           
             6  
             19  
             DNA  
             Artificial Sequence  
             
               Primer(sense) for amplify HGF gene  
             
           
            6 

atgctcattg gaccctggt                                                  19 

 
           
             7  
             18  
             DNA  
             Artificial Sequence  
             
               Primer(antisense) for amplify HGF gene  
             
           
            7 

gcctggcaag cttcatta                                                   18 

 
           
             8  
             23  
             DNA  
             Artificial Sequence  
             
               Primer(sense) for amplify c-met gene  
             
           
            8 

cagtgatgat ctcaatgggc aat                                             23 

 
           
             9  
             22  
             DNA  
             Artificial Sequence  
             
               Primer(antisense) for amplify c-met gene  
             
           
            9 

aatgccctct tcctatgact tc                                              22 

 
           
             10  
             21  
             DNA  
             Artificial Sequence  
             
               Primer(sense) for amplify collagen I gene  
             
           
            10 

caagaatggc gaccgtggtg a                                               21 

 
           
             11  
             21  
             DNA  
             Artificial Sequence  
             
               Primer(antisense) for amplify collagen I gene  
             
           
            11 

ggtgtgactc gtgcagccat c                                               21 

 
           
             12  
             20  
             DNA  
             Artificial Sequence  
             
               Primer(sense) for amplify collagen III gene  
             
           
            12 

agatggatca agtggacatc                                                 20 

 
           
             13  
             20  
             DNA  
             Artificial Sequence  
             
               Primer(antisense) for amplify collagen III gene  
             
           
            13 

catgtttctc cggtttccat                                                 20