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US20030003077A1 - Recombinant adenoviral vectors and their utility in the treatment of various types of fibrosis: hepatic, renal, pulmonary, as well as hypertrophic scars - Google Patents

Recombinant adenoviral vectors and their utility in the treatment of various types of fibrosis: hepatic, renal, pulmonary, as well as hypertrophic scars Download PDF

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US20030003077A1
US20030003077A1 US10/098,359 US9835902A US2003003077A1 US 20030003077 A1 US20030003077 A1 US 20030003077A1 US 9835902 A US9835902 A US 9835902A US 2003003077 A1 US2003003077 A1 US 2003003077A1
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mmp
fibrosis
gene
recombinant adenoviral
adenoviral vector
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Juan Armendariz Borunda
Estuardo Aguilar Cordova
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TGT Laboratories SA de CV
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Publication of US20030003077A1 publication Critical patent/US20030003077A1/en
Priority to US10/724,292 priority Critical patent/US8043855B2/en
Priority to US11/064,504 priority patent/US7858368B2/en
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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6489—Metalloendopeptidases (3.4.24)
    • C12N9/6491—Matrix metalloproteases [MMP's], e.g. interstitial collagenase (3.4.24.7); Stromelysins (3.4.24.17; 3.2.1.22); Matrilysin (3.4.24.23)
    • A—HUMAN NECESSITIES
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    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10211—Aviadenovirus, e.g. fowl adenovirus A
    • C12N2710/10241—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10243—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination

Definitions

  • the present invention relates to the creation of RECOMBINANT ADENOVIRAL vectors bearing exogenous genes that encode for therapeutic proteins useful in the treatment of HEPATIC cirrhosis and generalized FIBROSIS, such as renal FIBROSIS, pulmonary FIBROSIS, HYPERTROPHIC scars and keloid of the skin, and/or in other target organs susceptible to suffer from it. It also relates to a mechanism of tissue-specific recognition of the affected cells by means of delivery of therapeutic genes to cirrhotic organs.
  • the invention provides an effective way for the treatment of fibrosis through the employment of recombinant adenoviral vectors which are claimed here, as well as the process to prepare these vectors, the pharmaceutical composition that contains them, and their therapeutic uses in the treatment of several fibrosis, which has great commercial expectancy in the pharmaceutical industry and also presents an important alternative as gene therapy for the treatment of chronic-degenerative diseases characterized by fibrosis, with great therapeutic application in the field of Medicine.
  • Hepatic cirrhosis is a disease resulting from hepatic chronic damage. Damage might be toxic (chronic ingestion of alcohol), infectious (viral hepatitis, mainly by hepatitis B and/or C virus), immunological, (primary biliary cirrhosis), by biliary obstruction, (secondary biliary cirrhosis), metabolic (Wilson's disease).
  • cirrhosis has characteristics in common: synthesis and excessive deposition of proteins of extracellular matrix (ECM), mainly collagen I and to a lesser extent collagens IV and III), and consequently the formation of nodules of hepatocytes, abnormal vascularization and portal hypertension (Antoni P P, Ishak K G, Nayak Nc, Poulsen H E, Sheuer P J, Sobin L H. These physiopathological processes lead to an alteration in the blood supply and in consequence in the nutrition of hepatic cells. Regardless of the ethiological agent and morphologic differences, all forms of cirrhosis have as a common end, hepatic failure causing the patient's death.
  • ECM extracellular matrix
  • the liver is not able to maintain the physiologic concentration of solutes in the terminal hepatic vein, in other words, HEPATIC failure sets in.
  • This capillarization with the formation of the continuous endothelia (collagen of basement membrane) and the accumulation of other collagenic proteins, represents a barrier to the normal and bi-directional exchange of molecules between the plasma and hepatocytes, as can be appreciated in FIG. 1, where hepatic cirrhosis is characterized by the accumulation in the liver of type I collagen. With an excessive deposition of this protein, the free exchange of nutrients between blood and liver cells is impeded, the inactivation of toxic agents by this organ can not be carried out, becoming this the main cause of the pathophysiology of the disease. To date, no therapeutic agent that could revert and/or prevent with a 100% effectiveness the progressive accumulation of hepatic collagen has been described.
  • This technology can be implemented with viral or non-viral vectors.
  • Previous studies have been designed using plasmids and liposomes (DOTMA), cationic and anionic, etc.
  • DOTMA plasmids and liposomes
  • cationic and anionic etc.
  • the most commonly used include the use of retrovirus and adenovirus.
  • retroviral vectors have been used to introduce genes in hepatocytes (J T, and Curiel D T, Adenoviruses as Vectors for gene Therapy. Science and Medicine/1997 44-53).
  • precautions have to be taken since these vectors can generate potential replication-competent viruses.
  • 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.
  • no study has reported incidences of mutagenesis by insertion or activation of oncogenes by the incorporation of the replication-deficient retrovirus.
  • 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 Hepatic Growth Factors and through partial hepatectomy, surgical procedure by 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.
  • 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 “fibrogenic process”, 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.
  • U.S. Pat. No. 5,240,846 refers 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.
  • CFTR gene therapy gene therapy
  • 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.
  • 5,910,487 describes the use of plasmidic vectors for sending therapeutic molecules, but there is no association with the delivery of genes of metalloproteases MMP-8 latent and/or active, MMP-1, MMP-2, MMP-9, MMP-13; or “uPA (wild type uPA and/or its modified versions) or “Smad7” or the truncated receptors for transforming growth factor- ⁇ (TGF- ⁇ type II) 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.
  • 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. Also, 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 and 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 human metalloproteases MMP-8, latent and/or active, MMP-1, MMP-2, MMP-9 and MMP-13; human urokinase Plasminogen Activator (wild type and/or modified huPA), Smad7, and the truncated receptor for TGF- ⁇ type II, claimed herein. Other members of the family of genes represented are also included.
  • 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 cirrhotic livers or to fibrotic kidney or lungs.
  • U.S. Pat. No. 5,872,154 describes a method to reduce the immune response induced by an adenoviral recombinant 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.
  • 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 which contains the portion of an adenoviral vector in combination with an adeno-associated virus and a selected gene. Thorough it large quantities of recombinant vectors are produced, but they are not carrying cloned therapeutic genes as is described in this invention, in which specific therapeutic genes for the treatment of renal and hepatic fibrosis and hypertrophic scars are used.
  • U.S. Pat. No. 5,547,932 claims a compound of complexes of nucleic acids 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, is related to conjugated adenovirus bound through an antibody to a substance with affinity to nucleic acids.
  • U.S. Pat. No. 5,585,362 relates to an improved adenoviral vector and methods to obtain and use such vectors.
  • the use of adenoviral vectors is not mentioned in said patent.
  • the adenoviral vectors described in the present invention were used like vectors for sending therapeutic genes.
  • 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 in specific cells of the economy, such as hepatocytes.
  • This adenovirus can include a gene that codes for a therapeutic agent.
  • the outstanding technical difference of the instant invention compared to the state of the art is the specificity of the therapeutic agent as human metalloproteases MMP8 active and latent, MMP-1, MMP-2, MMP-9 and MMP-13; human uPA (urokinase Plasminogen Activator, wild type and/or modified), the truncated receptor for TGF- ⁇ type II and “Smad7” for the treatment of various fibrosis.
  • 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.
  • the vector or the virus to be assayed is the hepatitis B virus.
  • 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.
  • 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.
  • 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.
  • the technical characteristics of the present invention are focused on the specific use of therapeutic genes to treat fibrosis of different kinds: hepatic, renal and pulmonary fibrosis, as well as hypertrophic scars.
  • adenoviral vectors were determined 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).
  • 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 bloodstream. 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).
  • 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. Its union to cells occurs by means of the protein component of the capside and the virus enters into the cell by receptor-mediated endocytosis.
  • Ad2 type 2
  • Ad5 type 5
  • 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).
  • 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 right extreme of the adenoviral genome.
  • Vectors based on the insertion of the exogenous gen at the right extreme of the adenoviral genome or by the replacement of the E3 region maintain their replication capability.
  • An object of the present invention is to provide a procedure to prepare recombinant adenoviral vectors pAdGFP-MMP8, by means of the cloning of the reporter genes: lac-7 and GFP and the therapeutic gene of collagenase or metalloprotease MMP-8 in its latent and/or active forms.
  • Another object of the invention is to provide an adenoviral recombinant vector 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.
  • genes are, but are not limited to MMP-8 active and latent, MMP-1, MMP-2, MMP-9 and MMP-13; and uPA (wild type and/or modified).
  • compositions which contain the recombinant adenoviral vectors in quantities therapeutically effective of viral particles for the treatment of generalized fibrosis; as well as their uses and therapeutic applications in the treatment of fibrosis.
  • An advantage of greater importance in the treatment of the generalized fibrosis, particularly of hepatic cirrhosis, is that the delivery of therapeutic genes is carried out through tissue-specific recognition by the way of administration employed.
  • Another advantage of the therapeutic uses of the invention which is directed initially to revert hepatic cirrhosis, is the treatment of generalized fibrosis in other target organs susceptible to suffer from it, including, without limitation, the treatment of fibrosis in lung, heart, skin, kidney, among others, in mammalian animals, including human beings.
  • Another object is the design of a technology to send genes efficiently to livers of animals affected by cirrhosis that resemble two types of cirrhosis that usually affect human beings (Alcoholic cirrhosis and Primary Biliary Cirrhosis).
  • Another advantage resulting from the fibrosis treatment is that recombinant adenovirus does not induce lethal toxicity in none of the injected animals with the vectors.
  • Another objective of the invention allows us to discriminate the modification of the staining reaction with X-Gal between the endogenous tissue galactosidase activity and the bacterial -galactosidase induced by the infectious action of the adenoviral vector.
  • the use of the green fluorescent protein permits us to verify the in vivo transduction of different organs in rats to verify if the vector administration was appropriate, if the expression remains, and besides not killing the animals it is possible to conduct follow up observation after surgery.
  • a process of preparation is given, through which adenoviral recombinant vectors, pharmaceutical compounds and therapeutic uses for the fibrosis treatment, especially for the treatment of hepatic cirrhosis.
  • FIG. 1 shows the cellular physiopathology of hepatic cirrhosis
  • FIG. 2 shows the proof of concept on how gene therapy works by reverting the cirrhosis process
  • FIG. 3 is the schematic representation, which shows the cloning and production of the adenoviral vector Ad5 -gal;
  • FIG. 4 shows the schematic development of the AdEasy system to generate recombinant adenoviruses, specifically the pAdGFP-MMP-8;
  • FIG. 5 shows the analysis of the expression of -galactosidase in cultured cells.
  • FIG. 6 shows the expression determination of green fluorescent protein (GFP) expression in cultured cells
  • FIG. 7 shows the expression of -galactosidase in different organs after the infusion with Ad5 gal through the iliac vein.
  • FIG. 8 shows the analysis of the tropism of the vector Ad5-gal to different organs of cirrhotic experiment animals by chronic intoxication with CCl 4 , demonstrating that the main target organ is the liver;
  • FIG. 9 shows the analysis of the tropism of vector Ad5-gal to different organs of cirrhotic experiment animals. Cirrhosis was induced by bile duct ligation and it was demonstrated that the main target organ is the liver.
  • FIG. 10 shows histological sections of representative images of the in vivo efficiency transduction assays of the vector Ad5-gal in cirrhotic rats with chronic administration of CCl 4 ;
  • FIG. 11 shows histological sections of representative images of the in vivo efficiency transduction assays of the vector Ad5-gal in cirrhotic rats by common bile duct ligation;
  • FIG. 12 shows the in vivo determination of the expression of the green fluorescent protein
  • FIG. 13 shows the cloning strategy of the latent MMP8 and active MMP-8
  • FIG. 14 shows the mechanisms of complex formation with DNA of MMP-8s for in vitro transfection essays in cells of hepatic origin (HepG2);
  • FIG. 15 shows the verification through electrophoresis in agarose gels of the success of cloning of MMP8 cDNAs in the appropriate plasmids
  • FIG. 16 shows the transfection efficiency in HepG2 cells (Cells of hepatic origin) with the plasmids of -gal a ctosidase and cDNA-MMP-8;
  • FIG. 17 shows the analysis by polymerase chain reaction associated to reverse transcriptase (RT-PCR) of MMP-8 messenger RNAs
  • FIG. 18 shows analysis of the collagenolytic activity in the protein secreted to the culture medium by HepG2 cells after transfection with cDNAs for latent MMP8 and active MMP8;
  • FIG. 19 shows the hormonal regulation of the MMP-8 gene expression under the transcriptional control of the regulable promoter PEPCK and
  • FIG. 20 shows the dose-response assay of the different doses used to determine the best response of “in vivo” hepatic transduction with the -galactosidase reporter gene.
  • AdR recombinant adenoviral vectors
  • AdRs containing LacZ and GFP (green fluorescent protein) reporter genes are capable of transducing livers of cirrhotic rats even if the lobular architecture of the liver is distorted.
  • livers therapeutic genes such as human metalloproteases or collagenases human MMP-8 active and latent, MMP-1, MMP-2, MMP-9 and MMP-3; human Urokinase Plasminogen Activator (uPA wild type and/or modified); the truncated receptor for TGF- ⁇ type II and Smad 7, which degrade the excess of collagenic proteins deposited and/or prevent the exacerbated synthesis of collagenic proteins, as it is shown in FIGS. 2 and 18; and/or genes which encode for proteins stimulating hepatic regeneration such as uPA, in order to re-establish the normal functioning of the liver, as is shown in FIG. 2.
  • human metalloproteases or collagenases human MMP-8 active and latent, MMP-1, MMP-2, MMP-9 and MMP-3
  • human Urokinase Plasminogen Activator uPA wild type and/or modified
  • TGF- ⁇ type II and Smad 7 the truncated receptor for TGF- ⁇ type II and Smad
  • the current invention initiates a research line to carry out gene therapy as an alternative for the treatment of chronic degenerative disease, specifically of hepatic cirrhosis in human beings, through the establishment of an efficient vehicle to send genes to the liver which will produce therapeutic proteins to help re-establish the normal functions of the liver, see FIG. 2.
  • FIG. 2 shows how sending efficiently a therapeutic gene to the liver, in this case, a collagenase (metalloproteases of matrix, MMPs), it is possible to promote degradation of collagen through the over-expression of these metalloproteases.
  • a collagenase metaloproteases of matrix, MMPs
  • FIG. 3 the strategy for the cloning and production of an adenoviral vector is shown.
  • the plasmid pDeltaE1sp1B contains adenovirus Ad5 sequences, in which the bacterial gene Lac-z was inserted. This plasmid was recombined with the pBHG10 to obtain complete viral particles after co-transfection in the cell line 293 .
  • the vector pAdGFP was obtained as follows: the MMP-8 gene (coming from the plasmid PEPCK-MMP-8) was cloned in the vehicle vector, pAdTrack-CMV, the resultant plasmid is linearized with the restriction endonuclease Pme I, and is then transformed in E.
  • FIG. 5 the expression of -galactosidase in cultured cells is shown.
  • A), B) and C) correspond to HepG2 cells (320X); D,, E) and F), are mouse peritoneal macrophages (100 ⁇ ).
  • C) and F) the transduced cells are shown with 1 ⁇ 10 8 viral particles/ml from the Ad5-Gal vector.
  • Three techniques were conducted to compare the degree of incorporation of the reporter gene Lac-Z which was administered to each culture dish in the form of plasmidic DNA PGK-Gal, through precipitation with Ca ++ phosphate (Chen C, and Okayama H.
  • Gal staining was standardized using different pHs of the suspension with the reactive Xgal (Weiss D J, Ligitt D., and Clark J G. In situ photochemical detection of galactosidase activity in lung: assessment of Xgal reagent in distinguished Lac-Z gene expression and endogenous ga lactosidase activity. Human being therapy, Sep. 1, 1997, 8:1545-1554).
  • the models of experimental hepatic cirrhosis used are: a) Chronic intoxication caused by carbon tetrachloride (CCl 4 ), in which hepatic cirrhosis is established starting from the 8 th week of peritoneal administration (Mion F, Geloen A, Agosto E. and Minaire Y. Carbon tetrachloride induced cirrhosis in rats: influence of the acute effects of the toxin on glucose metabolism. Hepatology 1996, Vol. 23, No.
  • Ad5 gal was administered at the same time and from the same lot to control rats without cirrhosis.
  • Rats with 5 and 8 weeks of CCl 4 intoxication and rats with 2 and 4 weeks of bile duct ligation (BDL) were sacrificed 72 hrs after administration of recombinant adenovirus for the histological analysis and determination of the expression of the ga lactosidase protein (gal) encoded by the AdR.
  • liver, spleen, heart, lungs, kidneys and brain were extracted, tissue sections were cut in cube shapes of 5 to 6 mm., which were absorbed in freeze medium Tissue-Tek O.C.T., the tissues were frozen at ⁇ 30° C.
  • liver sections of cirrhotic rats were obtained and tissues absorbed in paraffin were cut and stained with Sirius red which specifically stains collagenic proteins (Armendariz-Borunda J., and Rojkind M., A simple quantitative method for collagen typing in tissue samples: Its application to Human liver with schistosomiasis. Collagen Rel. Res 1984, Vol. 4, 35-47).
  • Sirius red specifically stains collagenic proteins
  • FIG. 2 Said figure shows the role of pro-inflammatory and pro-fibrogenic cytokines produced in vivo by Kupffer cells which, in turn, activate the hepatic stellate cells (HSC) to have them produce excess collagens deposited in the subendothelial space, obstructing the exchange between hepatocytes and sinusoids (Armendariz-Borunda J., Katayama K., and Seyer J. M.: Transcriptional mechanisms of type I collagen gene expression are differentially regulated by IL-1beta, TNFalfa and TGF into cells. J. Biol. Chem.
  • FIG. 6 shows the expression of green fluorescent protein (GFP) in cultured cells.
  • FIG. 7 shows the expression of gal in different organs after infusion with Ad5 gal by iliac vein. Fixation, washing and Xgal solutions using different pHs were used to discriminate among the endogenous expression and the bacterial exogenous galactosidase.
  • a pH 7.0 was used and in Figure B the pH was 8.5.
  • the graphics show dearly that the main target organ is the liver, both in healthy rats as well as in rats with chronic administration of CCl 4 .
  • Spleen and lung present a degree of trasduction below 1%, and thus this is not evident from the graphs.
  • Rats received doses of 3 ⁇ 10 11 viral particles/ml of Ad5gal vector.
  • the healthy control rats presented a total of 70% of hepatocytes transduced, while spleen and lung showed less than 1% transduction. In the other organs no transduction was found.
  • Tissue sections were obtained from healthy rats as described before and compared with tissues from rats with 2 and 4 weeks of BDL.
  • FIG. 10 histological results are shown with the hepatic cirrhosis model induced by the chronic administration of CCl 4 , where A) represents a liver section of a normal rat, 72 hours after the administration of Ad5 gal, by iliac vein (one representative cut of the experiments of a total of 5 rats). More than 70% of the hepatocytes are positive to the expression of gal (200 ⁇ ); D) The same liver as in Figure A, but stained with Sirius Red to observe collagen synthesis and deposition (200 ⁇ ); B) liver with 5 weeks of chronic intoxication with CCl 4 . About 30-40% of the hepatocytes were successfully transduced; E).
  • FIG. 12 shows a laparotomy of a healthy Wistar rat that received pAdGFP-MMP-8 vector. The expression of the GFP is clearly seen in the liver and in insignificant amounts in the spleen. A very important fact is that the injection of adenoviral vectors did not induce lethal toxicity in experiment animals, both healthy and controls.
  • the preferred way to apply the present invention is through endovenous administration of the recombinant adenoviral vectors of this invention or the pharmaceutical compound which contains them, in which therapeutically effective amount is administered with an unitary dose regimen convenient to an individual with fibrosis.
  • This regimen can be adjusted according to the affliction degree.
  • unitary doses of about 10 7 to 10 14 viral particles for individual are employed.
  • a pharmaceutical compound including the adenoviral recombinant vectors of this invention can be conducted 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 limitation, starch, glucose, lactose, sacharose, gel, malt, rice, wheat flour, chalk, silica-gel, magnesium stearate, sodium stearate, powder of glyceril monostearate, 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.
  • HepG2 cells is a cell line of parenchymal origin derived from a human hepatoma, and were cultured in 60 mm culture dish, 37° C. in a wet atmosphere, with 95% air and CO 2 5% Sicile in Eagle's medium modified by Dulbecco (DMEM), supplemented with 10% fetal bovine serum, 2 mM L-Glutamax and antibiotics (100 U/ml penicillin and 100 g./ml. streptomycin).
  • DMEM Dulbecco
  • Two plasmids were used with 2 kinds of MMP-8 genes to transfect the hepatic cells:
  • the plasmid pcDNA-MMP-8 which contains the cDNA which encodes for latent MMP-8 (pro-MMP-8) together with the strong viral promoter of cytomegalovirus (CMV); and the plasmid pcDNA 3 MMP-8 containing the cDNA which encodes for the active MMP-8, together with the CMV promoter.
  • Said plasmid has the gene which encodes the enzyme -galactosidase inserted adjacent to the SV40 virus promoter.
  • each plasmid was introduced to E. coli DH5TM, (th is process is known as transformation), according to the instructions of the supplier. (Life Technologies, Gaithersburg, Md.): in a reaction tube 50 I of the competent strain DH5 were used and 2 l of plasmids (1-10 ng of DNA) were added. After mixing, it was incubated on ice during 30 minutes, a thermal shock (37° C. for 20 seconds) was applied and it was immediately chilled on ice for 2 minutes.
  • DNA transfection with calcium phosphate in which the exogenous DNA is precipitated as a fine complex on the cell surface, to be later incorporated by the cell and transiently integrated in the chromosomal DNA.
  • DNA is used in the form of complex with polylysine-lactose, because of hepatic cells have a specific receptor for Galactose in their cell membrane.
  • HepG2 cells were cultured at 70-80% confluence and then transfected with plasmids pcDNA-MMP-8, pcDNA 3 -MMP-8 and pSV 2 -galactosidase. Transfection was carried out by DNA precipitation with calcium phosphate (Graham, and Van derEb, 1973; Chen and Okayama 1988) and by complex formation with polylysine-lactose (Martinez-Fong et. al, 1994).
  • cultured cells were added with the newly formed precipitate, product of the addition to plasmidic DNA of a solution of DNA with CaCl 2 2M, in buffer solution HEPES pH 7.12 in case of the transfection with calcium phosphate or DNA complex with polylysine-lactose is added.
  • Cells are incubated from 4-16 hours to allow the precipitate to appear to the cell surface, and later the DNA can be endocyted and introduced transiently to the nucleus.
  • the culture medium is replaced for a fresh one, see FIG. 14, where HepG2 cells are cultured with DMEM medium with 10% bovine fetal serum.
  • MMP-8 gene was sent in different forms: naked, in complex with CaPO 4 or in complex with polylysine-lactose.
  • the polylysine-lactose complex is formed when 14.8 mg of poly-L-Lysine (0.1N) react with 200 l of -lactose 0.5 N (lactose-polylysine ratio: 1.0 N). Then, 20 mg of reducing agent sodium cyanoborohydride 3 M is added and it is incubated at 37° C. for 48 hours with constant stirring at 225 rpm. Then, the reaction goes through a desalting column (BioRad 10-DG) previously conditioned with phosphate buffer (PBS pH 7.2), which is eluted with the same buffer.
  • phosphate buffer PBS pH 7.2
  • Carbohydrate content is determined to the eluted fractions by the method of DuBois (1956) to analyze the degree of lactosylation of the complex and the contents of polylysine according the method of Shen et. al. (1984), which is considered as a base to evaluate the final concentration of the PL complex.
  • the fraction with a mayor concentration of PL is used for its further reaction with the DNA of the plasmid containing the gene of interest, as is shown in FIGS. 14 and 16.
  • This system determines the activity of the -galactosidase enzyme as a measure of the level of expression of the transfected gene of interest along with Lac Z gene which encodes for this enzyme.
  • the ga lactosidase is a bacterial enzyme which catalyzes the conversion of the uncolored substrate X-gal to a product of blue coloration. Because of this, the -galactosidase activity observed in eucaryotic cells subjected to transfection will indicate the successful incorporation of the gene of interest associated to the bacterial gene.
  • the assay of -gal for the stain of cells in culture dish consists in the fixation of cells at 4° C.
  • DEPC diethylpirocarbonate
  • cDNA complementary DNA
  • RNA samples were taken to a volume of 8 l with deionized, sterilized water and incubated at ⁇ 70° C. for 10 minutes. Then, the sample was stirred in iced water during 5 minutes and still in the ice, the following reagents were added: 4 l of 5 ⁇ buffer for the RT enzyme, 4 l dNTP's mix 2.5 mM, 1 l random primers (1 g/l), 1 l inhibitor of RNAase (one U/l) and finally 2 l of the Reverse Transcriptase enzyme (200 U/l). The reaction mix was incubated at room temperature for 10 minutes and then at 37° C. for one hour. At the end of this time, it was placed immediately in a temperature of 95° C. for 10 minutes, and then it was placed on iced water during 5 minutes with constant stirring and it was stored at ⁇ 70° C. until its further use.
  • a PCR reaction was set up using the primers or oligonucleotides specific for this gene according to the experimental conditions described hereinafter: in a reaction tube containing 2 l of cDNA 5 l of 2.5 mM MgCl 2 , 5 l 5 ⁇ buffer for the polimerase enzyme, from leukemia murine virus of Moloney (MMLV), l of 2.5 mM dNTPs, 5 l of the sense primer 3 ⁇ M, 5 l of the antisense primer 3 ⁇ M, 1 l of the polymerase enzyme (U/l) and it is taken to a final volume of 50 l with deionized water (Innis et al, 1990).
  • MMLV leukemia murine virus of Moloney
  • U/l polymerase enzyme
  • the oligonucleotide sense primer specific for MMP-8 is 5′-AGCTGTCAGAGGCTGGAGGTAGAAA-3′, and the antisense primer is 5′-CCTGAAAGCATAGTTGGGATACAT-3′ (Cole et al., 1996).
  • the mix was placed in a thermalcycler during 30 cycles according to the following program: denaturation (94° C., 5 min), annealing (60° C., 1 min.) and extension (72° C., 1.5 min). Then, PCR products are submitted to electrophoresis (60 mV, 1.5 h) in a 1.5% agarose gel.
  • cell supernatant containing 1-1.5 gr of protein were incubated at 27° C. during 18 hours with 5 g of native collagen type I and 60 l of the incubation buffer: 50 mM Tris-HCl, 5 mM CaCl 2 , 0.02% NaN 3 , 50 mM arginine, 1% Triton X-100 and in absence or presence of 1 mM APMA, pH 7.6.
  • 30 l of product of reaction were mixed with 30 l of sample buffer for proteins and electrophoresis in SDS-polyacrilamide gels (7.5%) was run to identify the degradation products 1 A and 2 A of collagen type 1.
  • FIG. 15 shows an electrophoresis of the DNA fragments released by cutting MMP-8 plasmids with restriction enzymes.
  • Lane A Marker of bp of 1 Kb DNA ladder (Gibco BRL); B).
  • the bands stained with ethydium bromide correspond to each of cDNA (between 506 and 560 base pairs) for mature and latent MMP-8 cDNA, respectively.
  • the co-transfection of this plasmid was realized along with the reporter gene of -galactosidase. In this way, cells observed in the microscope with blue staining, indicate indirectly that they have also incorporated to the plasmid of interest.
  • FIG. 16 shows the expression of -galactosidase in HepG2 cells, co-transfected with free plasmid, in form of complex with CaPO 4 , or in its form of complex with polylysine-lactose.
  • This figure shows that the DNA binding with polylysine-lactose was accomplished because the higher the polylysine concentration, the clearer the retardation of -gal plasmid.
  • the ratio selected to transfect the cells was the one that delayed 80% of plasmid migration.
  • FIG. 17 shows an analysis by RT-PCR of messenger RNA for MMP-8 and MMP-13.
  • This plasmid was used as a further positive control of transfection); in which a DNA electrophoresis of PCR amplified products, of the cDNA for MMP-8 delivered as a complex with CaPO 4 and polylysine-lactose, has been transcribed for both cases in transfected HepG2 cells. It is observed that product signal of PCR of MMP-8 (359 base pairs), was more intense when plasmid was delivered as a complex with polylysine-lactose.
  • FIG. 18 shows the enzymatic activity of type I collagen degradation of the protein secreted in the culture medium, which was observed in the transfected cells with the gene of latent MMP-8.
  • Negative controls type I collagen without addition of supernatants of cells (lane 1 ) and with addition of Trypsin (lane 3 ), collagen with supernatants of cells without transfection (lane 2 ).
  • Positive controls type I collagen with supernatant of human leukocytes (lane 3 ), type I collagen with addition of 0.015% bacterial collagenase (lane 4 ); and degradation products of native type I collagen, separated in a 6% polyacrylamide gel, after it was incubated with supernatant of transfected cells with latent and active MMP-8 genes. It was observed how in both cases the collagenolytic activity is clear in presence of APMA in the case of latent MMP-8, and its inhibition for EDTA for both latent and active MMP-8.
  • FIG. 19 shows evidence that activities of the enzymes that specifically degrade collagen can be controlled (turned off and/or turned on) through the cloning of its respective cDNAs that are themselves under the transcriptional control of promoters of regulable genes, such as the PEPCK (Phosphoenol-piruvate carboxikinase) gene.
  • PEPCK Phosphoenol-piruvate carboxikinase
  • GFP green fluorescent protein
  • the injection in portal vein could be more efficient to get to the target cells in the liver, providing them a favorable innoculum of viral particles to the entire liver before being diluted into the bloodstream.
  • This route is efficient, but it has the disadvantage that it requires a laparotomy.
  • peritoneal administration is a faster and simpler infusion, but it does not promote hepatocyte transduction.
  • the results of the present invention show that the injection of 3 ⁇ 10 11 viral particles by iliac vein in normal Wistar rats of approximately 200 g. produces a very high level of expression (70% of transduced hepatocytes).
  • hepatocytes which eventually could be transduced with therapeutic genes, such as metalloproteases (MMP-8) and/or genes which encode for stimulating proteins for hepatic regeneration such as uPA (Urokinase Plasminogen Activator) and Smad 7.
  • MMP-8 metalloproteases
  • uPA Urokinase Plasminogen Activator
  • FIG. 1 [0107]FIG. 1:
  • CEH stellate hepatic cell.
  • CES Endothelial sinusoidal cell.
  • CK Kupffer Cell.
  • ESET Subendothelial space.
  • HE Hepatocytes.
  • HIDC Liver with chronic damage.
  • SINU Seusoid
  • FIG. 2 [0116]
  • DCA Degradation of collagen
  • MMPs Metalloproteases
  • FIG. 3 [0121]
  • CT293 Co-transfection in cells 293
  • FIG. 4 [0124]
  • CTBK Co-transfection in bacteria and selection in Kanamicine.
  • GENADR Geneation of recombinant adenovirus.
  • FIG. 7 [0134]
  • X-GAL7 Reactive X-gal, pH 7.0
  • X-GAL 8.5 Reactive X-gal, pH 8.5
  • FIG. 8 [0146]
  • %CT % of transduced cells
  • FIG. 9 [0158]
  • FIG. 13 [0170]
  • PROT Protein
  • FIG. 14 [0173]
  • FIG. 16 [0181]
  • ADND Naked DNA
  • GELRADN-PL Retardation gel for polylysine
  • FIG. 18 [0184]FIG. 18:
  • CA With APMA.
  • CACE With APMA and EDTA.
  • CaPO4 Phosphate
  • COB Bacterial Collagenase
  • FIG. 20 [0196]
  • %CT % of transduced cells.
  • PV Viral particles

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US8236300B2 (en) 2002-12-02 2012-08-07 Biovec, Llc In vivo and ex vivo gene transfer into renal tissue using gutless adenovirus vectors
US8367056B2 (en) 2002-12-02 2013-02-05 Biovec, Llc In vivo and ex vivo gene transfer into renal tissue using gutless adenovirus vectors
US20050036988A1 (en) * 2003-05-28 2005-02-17 Ruian Xu Compositions and methods for preventing and treating liver cirrhosis
US20150368669A1 (en) * 2004-03-30 2015-12-24 Industry-Academic Cooperation Foundation, Yonsei University Gene delivery system containing relaxin gene and pharmaceutical composition using relaxin
US20120134964A1 (en) * 2006-04-10 2012-05-31 New York University Human matrix metalloproteinase-8 gene delivery enhances the oncolytic activity of a replicating adenovirus
WO2010085262A1 (en) * 2009-01-26 2010-07-29 Biovec, Llc In vivo and ex vivo gene transfer into renal tissue using gutless adenovirus vectors

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DE60017924T2 (de) 2006-03-30
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JP4173663B2 (ja) 2008-10-29
EP1221490A2 (de) 2002-07-10
CA2385538C (en) 2007-11-06
US20050201984A1 (en) 2005-09-15
US8043855B2 (en) 2011-10-25
US20040156827A1 (en) 2004-08-12
HK1049860B (en) 2006-02-24
JP2004500040A (ja) 2004-01-08
DE1221490T1 (de) 2003-05-28
AR025692A1 (es) 2002-12-11
ES2183752T1 (es) 2003-04-01
WO2001021761A3 (es) 2001-09-07
US7858368B2 (en) 2010-12-28
CO5420199A1 (es) 2004-07-30
AU7322600A (en) 2001-04-24
CA2385538A1 (en) 2001-03-29
PE20010610A1 (es) 2001-05-25
DE60017924D1 (de) 2005-03-10
WO2001021761A2 (es) 2001-03-29
ES2183752T3 (es) 2005-07-16

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