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WO1999023215A2 - Matieres et procedes permettant de prevenir les atteintes cellulaires chez l'homme et l'animal - Google Patents

Matieres et procedes permettant de prevenir les atteintes cellulaires chez l'homme et l'animal Download PDF

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Publication number
WO1999023215A2
WO1999023215A2 PCT/US1998/023270 US9823270W WO9923215A2 WO 1999023215 A2 WO1999023215 A2 WO 1999023215A2 US 9823270 W US9823270 W US 9823270W WO 9923215 A2 WO9923215 A2 WO 9923215A2
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Prior art keywords
polynucleotide
cell
cells
expression vector
tissue
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Ceased
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PCT/US1998/023270
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English (en)
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WO1999023215A3 (fr
WO1999023215A9 (fr
Inventor
Anupam Agarwal
Harry S. Nick
Gary A. Visner
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University of Florida
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University of Florida
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Priority to AU13745/99A priority Critical patent/AU1374599A/en
Publication of WO1999023215A2 publication Critical patent/WO1999023215A2/fr
Publication of WO1999023215A3 publication Critical patent/WO1999023215A3/fr
Publication of WO1999023215A9 publication Critical patent/WO1999023215A9/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C12N9/0083—Miscellaneous (1.14.99)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0089—Oxidoreductases (1.) acting on superoxide as acceptor (1.15)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/022—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from an adenovirus

Definitions

  • Heme oxygenase - l(HO-l) is a 32 kDa microsomal enzyme that is important in the degradation of heme.
  • HO-1 catalyzes the conversion of heme to biliverdin, releasing iron and carbon monoxide. Biliverdin is subsequently converted to bilirubin which is then excreted from the body.
  • HO-1 expression is inducible not only by the heme substrate, but also by a variety of agents that are associated with, or that cause oxidative stress.
  • a human HO-1 cDNA nucleotide sequence, and the amino acid sequence encoded thereby, has been determined (Yoshida, T., P. Biro, T. Cohen, R.M. Muller, and S. Shibahara (1988)
  • heme oxygenase- 1 has also recently been implicated in mouse cardiac xenograft survival in rats (Soares, M.P., Lin, Y., Anrather, J., Csizmdia, E., Takigami, K., Sato, K., Grey, S.T., Colvin, R.B., Choi, A.M., Poss, K.D., and Bach, F.H. (1998) "Expression of heme oxygenase- 1 can determine cardiac xenograft survival" Nature Medicine 4: 1073-1077).
  • Patients receiving medications for infection ⁇ e.g., gentamicin), cancer ⁇ e.g., cisplatin), immunosuppression ⁇ e.g., cyclosporin), or radiocontrast agents have a high risk for sustaining damage to cells and tissues, such as, for example, damage to kidneys which can result in acute renal failure.
  • Treatment for acute renal failure is expensive, necessitating dialysis and prolonged hospital stay. There is an increase in morbidity and mortality as well. Accordingly, there remains a need in the art for therapies that can prevent or ameliorate cell/tissue damage and/or failure associated with release of heme proteins.
  • the subject invention concerns methods for providing a person or animal with cytoprotection from heme protein-related cell damage and oxidative stress.
  • the subject method pertains to gene therapy using polynucleotide expression vectors or plasmids that include a nucleotide sequence encoding HO-1.
  • Cells or tissue transformed with the subject vectors or plasmids overexpress HO-1 enzyme which catalyzes degradation of heme proteins.
  • the subject invention also concerns novel polynucleotide expression vectors and plasmids comprising a polynucleotide sequence encoding HO-1.
  • an expression vector comprising a cytomegalovirus promoter/enhancer and the protein coding region of the HO- 1 gene (pcDNA3.1 /HO- 1) is exemplified.
  • vectors of the subject invention provide for overexpression of HO-1 in those cells.
  • FIG. 1A shows construction of a plasmid designated as pcDNA3.1/Zeo.
  • Figure IB shows a Northern blot of RNA from human renal tubule cells transfected with the pcDNA3.1/Zeo plasmid encoding the HO-1 enzyme.
  • Figure 1C shows a Western blot of proteins from human renal tubule cells transfected with the pcDNA3.1/Zeo plasmid encoding the HO-1 enzyme. The Western blot confirms the presence of a 32 kD protein.
  • Figure 2 shows cytoprotective effects of transient overexpression of the HO-1 gene in human proximal tubule cells exposed to cisplatin.
  • Figure 3 shows micrographs of human renal tubule epithelial cells that are either
  • Figure 4 A shows the amino acid sequence (in standard single letter code) of the human HO-1 enzyme.
  • Figure 4B shows a cDNA sequence encoding the HO-1 enzyme.
  • Figure 5 shows overexpression of HO-1 decreases cisplatin mediated toxicity as measured by LDH assay.
  • Figure 6 shows cytoprotective effect of hemin pretreatment in proximal tubule cells exposed to cisplatin.
  • Figure 7 shows the effect of hemin pretreatment on cisplatin-induced apoptosis in human proximal tubule cells.
  • Figure 8 shows the effects of overexpression of HO-1 in HEK293 cells exposed to cisplatin as measured by mitochondrial viability by the MTT assay.
  • a method of the subject invention comprises gene therapy of targeted cells or tissues to introduce polynucleotide sequences which when expressed result in overexpression of an enzyme having HO-1 activity.
  • Cells and tissues contemplated for treatment by the subject methods include, for example, kidney, spinal cord, brain, liver, lung, intestine and skin.
  • the subject method comprises utilizing gene therapy techniques to introduce polynucleotide sequences into targeted cells or tissues which when expressed in those cells or tissues result in overexpression of an HO-1 enzyme, or a biologically active fragment or variant thereof.
  • the present invention concerns a method for providing overexpression of HO-1 protein to provide cytoprotection in human renal proximal tubule kidney cells.
  • Cells in the proximal tubule of the nephron are most susceptible to a wide variety of injurious agents, e.g., drugs (gentamicin, cisplatin, cyclosporine), contrast agents used in radiologic procedures, environmental toxins (cadmium) and ischemia-reperfusion renal injury.
  • One embodiment of the subject method provides a polynucleotide expression vector comprising at least that region of an HO-1 gene that encodes an HO-1 enzyme, or a biologically active fragment or variant thereof, to transform kidney cells to express HO-1 in a patient in need of such treatment.
  • a polynucleotide vector of the present invention Once the cells have been transformed with a polynucleotide vector of the present invention, the HO-1 gene is overexpressed in those cells.
  • the expression vector is integrated into the genome of the target cell to provide stable overexpression of an HO-1 enzyme in those cells.
  • Polynucleotide expression vectors of the present invention can be introduced into cells or tissues by in vivo or ex vivo means.
  • the polynucleotide vectors are introduced in vivo by a variety of viral and non- viral means.
  • Suitable nonviral means include, for example, polynucleotide complexed with cationic lipids, and encapsulation of the polynuceotides in liposome vesicles.
  • Suitable viral vectors can be prepared from retroviruses, such as murine leukemia viruses and HTV-derived vectors , adeno-associated viruses, and adeno virus. Other suitable viral vectors are known in the art and are contemplated in the present invention.
  • Polynucleotide vectors of the present invention for HO-1 gene therapy can be administered to a person in need of such treatment according to standard procedures and methods known in the art.
  • the polynucleotide vectors are administered in a biologically compatible solution by direct injection or contact with the target cells and/or tissue of the patient.
  • the subject vectors can be administered to protect kidney cells from damage from cisplatin and other agents, by injecting in vivo a polynucleotide expression vector directly in kidney tissue or by contacting kidney tissue in vivo with a polynucleotide vector or plasmid of the invention.
  • the polynucleotide vector can be delivered to the kidney via catheter inserted into the proximal tubule. The amount of vector to be administered can be readily determined by a person of ordinary skill in the art.
  • HO-1 polynucleotide sequences included within the scope of the present invention include those sequences that encode HO-1 polypeptide that are derived from animals, including mammals.
  • the sequence is a human sequence of HO-1.
  • HO-1 polynucleotide sequences contemplated in the subject invention also include HO-1 genes having the natural sequence, as well as allelic variants and degenerate variants that encode an enzyme having HO-1 activity.
  • the subject invention also concerns polynucleotide vectors and plasmids comprising polynucleotide sequences that encode a fragment or variant of an active HO-1 protein as long as that fragment or variant has substantially the same biological activity as the natural full length protein. Such fragments and variants can be readily prepared using standard methods.
  • contemplated in all aspects of the invention are HO-1 polypeptides, and the polynucleotide sequences that encode them, as well as biologically active fragments and variants.
  • the vectors of the subject invention can also be designed for targeted delivery and/or expression of the HO-1 gene in kidney cells. Targeted expression can be achieved by using suitable polynucleotide regulatory control elements to control expression of vector linked genes in particular cells or types of cells.
  • the subject invention also concerns polynucleotide expression vectors and plasmids comprising a polynucleotide sequence that encodes a heme oxygenase- 1 enzyme, or a biologically active fragment or variant thereof.
  • An exemplified polynucleotide plasmid of the subject invention has been designated pcDNA3.1/HO-l ( Figure 1A). This plasmid was prepared by inserting an EcoRI/Xbal fragment that includes the HO-1 protein coding region into the pcDNA3.1/zeo plasmid.
  • This vector can be prepared by using standard procedures well known in the art. It comprises a cytomegalovirus promoter/enhancer and the protein coding region of the HO-1 gene.
  • the polynucleotide vectors contain regulatory elements that provide for high levels of expression of an HO-1 encoding polynucleotide in the cells. More preferably, the polynucleotide vectors include promoter and/or enhancer elements selected for high levels of expression of any operably linked genes, such as HO-1, in mammalian cells. In an exemplified embodiment, the polynucleotide vector comprises cytomegalovirus promoter and/or enhancer sequences operably linked to that region of the HO-1 gene that encodes an active form of the enzyme.
  • the polynucleotide vector also comprises a polynucleotide encoding selectable marker, such as Zeocin, neoR, thymidine kinase, beta-galactosidase, chloroamphenicol acetyl transferase (CAT), dihydrofolate reductase (DHFR) and the like, which allows one to select for those cells that are stably transformed by the polynucleotide vector.
  • the vector can also comprise a reporter gene for detection of HO- 1 expression.
  • the polynucleotide sequences, including polynucleotide vectors and plasmids, of the subject invention can be composed of either DNA or RNA. Nucleotide analogs that can replace the normal nucleotides found in DNA and RNA can also be used in the subject polynucleotides, vectors, and plasmids.
  • the subject invention also concerns cells and tissues transformed with a polynucleotide expression vector comprising a polynucleotide sequence which encodes a heme oxygenase- 1 enzyme. Transformed cells in tissues contemplated within the scope of the invention include kidney, spinal cord, brain, liver, lung, intestine and skin. Kidney cells that can be transformed according to the subject invention include, for example, human proximal tubule cells.
  • the subject invention also concerns polynucleotides comprising a first polynucleotide sequence that encodes an HO-1 protein, or a biologically active fragment or variant thereof, in combination with a second polynucleotide sequence that encodes a protein having anti-oxidant activity.
  • anti-oxidant proteins that can be encoded by the second polynucleotide and that are contemplated within the scope of the invention include superoxide dismutase (SOD) polypeptides.
  • SOD superoxide dismutase
  • the second polynucleotide sequence encodes a mutant manganese SOD protein that lacks or that has decreased product inhibition as compared to wild type SOD.
  • the polynucleotide encoding the SOD is operatively linked to a polynucleotide leader sequence that targets the polynucleotide to mitochondria.
  • the second polynucleotide sequence encodes a mutant manganese SOD protein that lacks or that has decreased product inhibition as compared to wild type SOD and is operatively linked to a polynucleotide leader sequence that targets the polynucleotide to mitochondria
  • the first and second polynucleotides can be operatively linked on a single vector or they can be present on separate vectors.
  • the first and second polynucleotides are operatively linked on a single vector.
  • first and second polynucleotides on the vector are unimportant as long as proper protein expression from each of the protein encoding polynucleotides can be achieved once the vector is delivered to a target cell.
  • the first and second polynucleotides are operatively linked on a single vector wherein an internal ribosome entry site (IRES) (Rees, S., Coote, J., Stables, S., Harris, S.
  • IRS internal ribosome entry site
  • polynucleotide vectors of the invention comprising first and/or second polynucleotides can further comprise regulatory sequences that enhance or promote expression of the polynucleotide sequences encoding the polypeptides.
  • the regulatory sequences are sequences that can be induced to upregulate expression of the encoded proteins in the target cell.
  • the regulatory sequences upregulate expression of the encoded proteins in the target cell in response to a physiological phenomena, such as, for example, an inflammatory or immune response in the host animal.
  • Suitable regulatory sequences include promoter sequences, enhancer sequences, and intron sequences.
  • the present invention also concerns methods for regulating or inhibiting cellular apoptosis.
  • apoptosis is regulated or inhibited in a target cell by expression of a polynucleotide sequence that encodes an HO-1 polypeptide.
  • a polynucleotide encoding an HO-1 polypeptide is delivered to and expressed in a target cell.
  • the expression of an endogenous polynucleotide encoding an HO-1 polypeptide in a target cell is induced by administering an agent capable of inducing endogenous HO-1 expression.
  • transplantation success of a xenograft or allograft in a host animal is enhanced by expression of a polynucleotide sequence that encodes an HO-1 polypeptide.
  • a polynucleotide encoding an HO-1 polypeptide is delivered to and expressed in a target cell of the transplanted tissue or organ.
  • an endogenous polynucleotide encoding an HO-1 polypeptide in a target cell of the transplanted tissue or organ is induced by administering an agent capable of inducing endogenous HO-1 expression.
  • Tissue and organs contemplated for transplantation include, but are not limited to, heart, lung, liver, kidney, skin, spinal cord and fetal tissues.
  • the present invention also concerns methods for treating or preventing conditions such as atherosclerosis, acute respiratory distress conditions, ischemia-reperfusion injury, radiation-induced injury and other conditions that involve oxidative stress.
  • the subject invention also concerns methods for treating cadmium exposure which causes nephrotoxicity.
  • a target cell is treated or induced to express a polynucleotide sequence that encodes an HO-1 polypeptide.
  • a polynucleotide encoding an HO-1 polypeptide is delivered to and expressed in a target cell.
  • the expression of an endogenous polynucleotide encoding an HO-1 polypeptide in a target cell is induced by administering an agent capable of inducing endogenous HO-1 expression.
  • the subject invention also pertains to polynucleotides having sequences that are antisense to polynucleotides that encode HO-1, or a fragment or variant thereof.
  • the antisense polynucleotides include those that are antisense to both DNA encoding HO-1, as well as RNA transcribed from that DNA.
  • the antisense sequences of the subject invention can be delivered to and expressed in target cells, such as cancer cells, for which one would like to decrease or prevent expression of HO-1.
  • target cells such as cancer cells, for which one would like to decrease or prevent expression of HO-1.
  • the subject invention also concerns methods for cancer therapy by inhibiting expression of HO-1 in targeted cancer cells using the antisense sequences of the invention.
  • LDH assay Transfected HEK 293 cells were plated in 24 well plates and incubated at 37 °C for 24 hours. The cells were exposed to CP(lOO ⁇ M). Sixteen hours after exposure, lOO ⁇ l of supernatant was transferred in 96 wll plates and 200 ⁇ l of working solution containing diaphorase, NAD + , iodo-tetrazolium chloride and sodium lactate were added.
  • the enzyme reaction was stopped by the addition of 50 ⁇ l of IN HC1.
  • the absorbance of samples at 492 nm and 620 nm was measured.
  • the percent specific LDH release was calculated by the absorbance of treated samples over control and Triton-X treated samples.
  • Figure IB shows a Northern blot, using the pcDNA3.1/Zeo plasmid as a probe, of RNA from both untreated human renal tubule cells (lane 1) and human renal tubule cells transformed with an expression vector having the shorter HO-1 cDNA according to the subject invention (lane 2).
  • the untreated cells express only endogenous HO-1 mRNA.
  • Lane 2 of Figure IB shows that the transformed cells express the endogenous HO-1 mRNA and overexpress the shorter HO-1 mRNA transcribed from the subject vector.
  • LDH Lactate dehydrogenase
  • FIG. 2 shows that cells expressing the HO-1 gene exhibited an approximately 30% decrease in LDH release at both the 50 ⁇ M and 100 ⁇ M concentrations of cisplatin.
  • overexpression of HO-1 in these cells provided a cytoprotective effect against damage from cisplatin as measured by LDH release from the cells.
  • Figure 3 shows the morphology of renal tubule cells either untreated (Panel A) or treated with cisplatin (Panel B) or pretreated with hemin (an inducer of HO-1 expression) followed by cisplatin exposure (Panel C).
  • cisplatin an inducer of HO-1 expression
  • Figure 3 shows the morphology of renal tubule cells either untreated (Panel A) or treated with cisplatin (Panel B) or pretreated with hemin (an inducer of HO-1 expression) followed by cisplatin exposure (Panel C).
  • hemin an inducer of HO-1 expression
  • Hemin+CP 17.6%, p ⁇ 0.05).
  • HEK293 cells were transfected with a vector containing the entire protein coding region of the human HO-1 gene (HHO) under the promoter/enhancer of cytomegalovirus (pcDNA3.1/HHO). The vector alone was used as a transfection control. Overexpression was confirmed by northern and immunoblot analyses which revealed a ⁇ 40 fold and -10 fold increase in mRNA and protein, respectively, over control cells ( Figure IB and 1C).
  • This assay requires the presence of active mitochondrial dehydrogenases to reduce the dye MTT to formazan, a purple colored dye.

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Abstract

Cette invention concerne un nouveau procédé permettant de traiter un homme ou un animal pour empêcher les lésions des cellules ou des tissus lorsque l'homme ou l'animal présente un risque d'atteinte associé aux protéines hèmes, tel que l'insuffisance rénale aiguë qui est associée au traitement à la cisplatine des patients atteints de cancer. Le procédé comprend l'utilisation d'un nouveau vecteur qui, via un transfert de gène in vivo, produit la surexpression du gène HO-1 dans le rein. Cette invention concerne également des vecteurs polynucléotidiques et des plasmides comprenant les séquences polynucléotidiques qui codent une enzyme HO-1. Ces vecteurs peuvent être utilisés pour transformer des cellules de rein pour qu'elles expriment le gène HO-1.
PCT/US1998/023270 1997-10-31 1998-10-30 Matieres et procedes permettant de prevenir les atteintes cellulaires chez l'homme et l'animal Ceased WO1999023215A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU13745/99A AU1374599A (en) 1997-10-31 1998-10-30 Materials and methods for preventing cellular injury in humans and animals

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US6386397P 1997-10-31 1997-10-31
US60/063,863 1997-10-31
US10540098P 1998-10-23 1998-10-23
US60/105,400 1998-10-23

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WO1999023215A2 true WO1999023215A2 (fr) 1999-05-14
WO1999023215A3 WO1999023215A3 (fr) 1999-07-15
WO1999023215A9 WO1999023215A9 (fr) 1999-08-19

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000036113A3 (fr) * 1998-12-17 2000-08-31 Sangstat Medical Corp Methodes permettant d'ameliorer les chances de survie d'une greffe par modulation de l'activite heme oxygenase

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992007935A1 (fr) * 1990-11-01 1992-05-14 The Scripps Research Institute Proteines de fusion ciblees par clycosaminoglycane, leurs conception, construction et compositions

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000036113A3 (fr) * 1998-12-17 2000-08-31 Sangstat Medical Corp Methodes permettant d'ameliorer les chances de survie d'une greffe par modulation de l'activite heme oxygenase
US7151090B2 (en) 1998-12-17 2006-12-19 Sangstat Medical Corporation Methods for enhancing graft survival by modulating heme oxygenase activity

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AU1374599A (en) 1999-05-24
WO1999023215A3 (fr) 1999-07-15
WO1999023215A9 (fr) 1999-08-19

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