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WO1997030167A1 - Procede de traitement des troubles du foie - Google Patents

Procede de traitement des troubles du foie Download PDF

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WO1997030167A1
WO1997030167A1 PCT/US1997/001564 US9701564W WO9730167A1 WO 1997030167 A1 WO1997030167 A1 WO 1997030167A1 US 9701564 W US9701564 W US 9701564W WO 9730167 A1 WO9730167 A1 WO 9730167A1
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otc
virus
liver
recombinant
gene
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PCT/US1997/001564
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James M. Wilson
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The Trustees Of The University Of Pennsylvania
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1003—Transferases (2.) transferring one-carbon groups (2.1)
    • C12N9/1018—Carboxy- and carbamoyl transferases (2.1.3)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
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    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/001—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination
    • C12N2830/002—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
    • C—CHEMISTRY; METALLURGY
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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
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/80—Vector systems having a special element relevant for transcription from vertebrates
    • C12N2830/85—Vector systems having a special element relevant for transcription from vertebrates mammalian

Definitions

  • the present invention relates generally to the treatment of inherited or acquired liver failure due to compromised ureagenesis; and specifically, to the treatment of the liver with recombinant viruses.
  • Ureagenesis is the process by which ammonia (produced in the metabolism and from deamination of amino acids in the liver and kidney) is removed from the body by conversion to urea.
  • the urea cycle involves the combination of ammonia, carbon dioxide and ATP, which in the presence of the enzyme carbamyl phosphate synthetase forms carbamyl phosphate. This compound reacts with ornithine in the presence of the mitochondrial enzyme ornithine transcarbamylase (OTC) to form citrulline.
  • OTC mitochondrial enzyme ornithine transcarbamylase
  • Arginine is produced from citrulline by a two-step process involving aspartic acid, ATP, and the enzymes argininosuccinate synthetase and arginino-succinate lyase . Finally arginine is hydrolyzed in the presence of the enzyme arginase to yield urea and ornithine. Urea is transported to the kidneys and excreted in the urine and ornithine is recycled back into the urea cycle. Specific inborn genetic errors of enzymes of the urea cycle have been observed in humans. Such defects lead to a syndrome of early onset hyperammonemia, which can cause severe disability or death.
  • a deficiency of the hepatic enzyme OTC is associated with compromised ureagenesis and derangements in nitrogen metabolism leading to intolerance to protein.
  • liver function deteriorates so does the capability of the patient to breakdown protein, leading to accumulations of ammonia in the blood, which is a marker for metabolic consequences of life-threatening hyperammonemic encephalopathy in humans.
  • the development of encephalopathy can lead to coma and eventually death.
  • This X-linked recessive disorder is the most common inborn error of urea synthesis, with an estimated prevalence of 1:40,000 to 1:80,000 births [Nagata, N. et al, Amer. J. Med. Genet.. 39: 228-229 (1991)], i.e., affecting about 850 newborns per year. See, also, Batshaw, M. L. et al, New Engl. J. Med.. 306: 1387-1392
  • Urea cycle disorders are extreme examples of the results of nonspecific liver damage and compromise of ureagenesis due to fallout of hepatocytes.
  • Acquired diseases of the liver such as cirrhosis or cancers, can cause inefficient urea cycling, due to a deficiency in the number of properly functioning hepatocytes.
  • Such inefficiency in the urea cycle can result in intolerance to protein ingestion, mental deficiency, retarded development and function of the nervous system and excessive amounts of free ammonia in the blood.
  • urea cycle disorders such as OTC deficiency.
  • Metabolic derangements in ureagenesis were corrected in several severely afflicted hemizygotes following orthotopic liver transplantation [Largilliere, C. et al, J. Pediat.. 115: 415-417 (1989) cited above; Broelsch, C.E. et al, Ann. Surg.. 212: 368-377 (1990); and Todo, S. et al, Heoatology. 15: 419-422 (1992)].
  • a therapeutic strategy such as organ replacement has problems due to the intrusiveness of the method, as well as the critical lack of organ donors.
  • liver enzyme deficiencies, as well as other urea cycle disorders continue to be devastating illnesses.
  • Recombinant adenoviruses have been evaluated as vectors for liver-directed gene therapy [Herz, J. , and Gerard, R. D., Proc. Natl. Acad. Sci., U.S.A.. 90: 2812-2816 (1993); Kozarsky, K. et al, J. Biol. Chem.. 269: 13695-13702 (1994); Stratford-Perricaudet, L. D. et al, Hum. Gene Ther.. 1 : 241-256 (1990); Morsy, M. et al, J. Clin. Invest.. 92: 1580-1586 (1993)].
  • Adenovirus is rendered defective for use as a vector by deleting the immediate early genes Ela and Elb and incorporating a minigene expressing the therapeutic protein.
  • Adenovirus is efficiently targeted to hepatocytes in vivo following intravenous infusion. High level transgene expression can be achieved in virtually 100% of hepatocytes, most of which are fully differentiated and not dividing.
  • the first use of El deleted viruses for gene therapy was in newborn spf** 11 mice [Stratford-Perricaudet, cited above]. Infusion of a vector containing a rat OTC CDNA into the newborn animals led to an increase in hepatic OTC actively in 4/15 mice which persisted for 1-2 months and was associated with decreased urinary orotic acid excretion.
  • the present invention provides a method of enhancing ureagenesis in a subject in need thereof comprising administering to said subject a recombinant virus capable of delivering a therapeutic transgene which expresses at least one urea cycle enzyme.
  • the recombinant virus can express more than a single urea cycle enzyme.
  • the invention provides a method of enhancing ureagenesis in a subject in need thereof comprising administering to said subject a combination of recombinant viruses, each virus capable of delivering a therapeutic transgene which expresses a different urea cycle enzyme.
  • the invention provides a method of preventing or treating hepatic encephalopathy due to hyperammonemia by enhancing ureagenesis by the methods of the invention.
  • the methods described above include a step of administering to said subject an effective amount of an immune modulator, said modulator substantially inhibiting the formation of neutralizing antibodies directed against the virus or of substantially reducing CTL elimination of virally-infected cells.
  • the invention provides a recombinant adenovirus capable of delivering a therapeutic transgene which expresses at least one urea cycle enzyme.
  • Fig. 1 is a diagrammatic map of recombinant adenoviruses. See, Example 2.
  • Fig. 2A is a graph illustrating the evaluation by Knodell score of periportal and bridging necrosis vs days post infusion of the recipient mouse liver receiving infusion of 5xl ⁇ 10 particles/mouse of H5.OlOCBhOTC (1st gen. hOTC) or H5.110CBhOTC (2nd gen. hOTC) recombinant adenovirus.
  • Fig. 2B is a graph illustrating the pathological response of Fig. 2A in mice receiving infusion of 1X10 11 particles of H5.OlOCMVmOTC (1st gen. mOTC) or H5.110CMVmOTC (2nd gen. OTC) recombinant adenovirus.
  • Fig. 2C is a graph illustrating the evaluation of intraglobular degeneration and focal necrosis in livers of mice treated as in Fig. 2A.
  • Fig. 2D is a graph illustrating the evaluation of intraglobular degeneration and focal necrosis in livers of mice treated as in Fig. 2B.
  • Fig. 2E is a graph illustrating the evaluation of portal inflammation response of the recipient mouse liver treated as in Fig. 2A.
  • Fig. 2F is a graph illustrating the evaluation of portal inflammation response of the recipient mouse liver treated as in Fig. 2B.
  • Fig. 3A is a graph plotting urinary orotate excretion vs. days post infusion for spf/Y mice infused with 5X10 11 particles of H5.OlOCBhOTC or H5.010CMVlacZ.
  • Fig. 3B is a graph plotting urinary orotate excretion vs. days post infusion for spf/Y mice infused with SxlO 11 particles of H ⁇ .llOCBhOTC or H5.HOCBlacZ.
  • Fig. 3C is a graph plotting plasma glutamine levels vs. days post infusion for spf/Y mice infused with ⁇ xlO 11 particles of H5.110CBOTC or H5.HOCBlacZ.
  • Fig. 4 is a bar graph illustrating the liver OTC activity in spf mice infused with recombinant adenoviruses: H5.OlOCBhOTC, H5.OlOCMVhOTC, H5.OlOCMVmOTC, or H5.OlOCMVlacZ. Untreated spf mice and untreated C3H mice are controls. Data are presented as OTC activity ( ⁇ mol citrulline/mg protein/hr) .
  • Fig. 5A is a graph plotting urinary orotate excretion level vs days post infusion in spf mice infused with 2x10" particles of H5.OlOCMVmOTC, H5.llOCMVmOTC or H5.110CMVlacZ.
  • Urinary orotic acid levels were measured in duplicate for each sample.
  • Urinary orotate/mg creatinine are presented as a % of pretreatment levels and are the mean + SEM of at least 4 determinations.
  • Fig. 5B is a graph plotting plasma glutamine levels vs days post infusion in spf mice infused with the recombinant adenoviruses as described in Fig. 5A. Plasma glutamine are presented as a % of pretreatment levels and are the mean ⁇ SEM of between 4 to 10 determinations.
  • Fig. 5C is a graph plotting urinary orotate excretion level vs days post infusion in spf* ⁇ mice infused with 2x10" particles of H5.llOCMVmOTC. Urinary orotic acid levels were measured as described in Fig. 5A.
  • Fig. 5D is a graph plotting plasma glutamine levels vs days post infusion in spf" 1 - mice infused with 2xlO n particles of H5.llOCMVmOTC. Plasma glutamine levels are presented as described in Fig. 5C.
  • Fig. 6 is a graph illustrating the dose response to nitrogen challenge (NH 4 C1 in mmol/kg) of spf mice (closed circle) or C3H mice (open circle) . Average scores for each group were indicated. Scores for spf mice are noted by asterisk.
  • Fig. 7 is a graph illustrating the clinical response to nitrogen challenge of spf mice treated with gene therapy measured in days post viral infusion.
  • Untreated control C3H mice open triangles
  • untreated control spf mice closed triangles
  • spf mice treated with OTC virus open circles
  • lacZ virus closed circles
  • Average scores for each group are indicated. Scores for C3H control mice and spf mice treated with OTC virus were noted by asterisk.
  • the present invention provides a novel method for treating or preventing hepatic encephalopathy caused by compromised ureagenesis in mammals, regardless of the cause of improper functioning of the urea cycle. This method is useful in treating inefficient ureagenesis due to inherited genetic defects in urea cycle enzymes. This method is equally useful in treating compromised ureagenesis which results from non-specific acquired liver failure.
  • a urea cycle enzyme gene is delivered to the liver cells by administering a recombinant virus, preferably an adenovirus, capable of expressing the gene in vivo .
  • a recombinant virus preferably an adenovirus
  • Expression of a deficient urea cycle enzyme in vivo improves the efficiency of the processing of ammonia to urea in the subject's liver. This improvement can be obtained even in a liver in which the deficiency in the enzyme function is due to malfunctioning liver cells or an abnormally low number of properly functioning liver cells, e.g., caused by liver damage due to cirrhosis, rather than to a particular genetic deficiency.
  • One or more of the missing or deficiently-expressed urea cycle enzymes may be supplied by one or more recombinant viral vectors, administered together or sequentially.
  • somatic gene transfer can prevent the development of encephalopathy due to hyperammonemia.
  • the rate-limiting step of an enzymatic pathway can change when the concentration of reactants, intermediates, and/or products is altered (by enzyme or gene deficiencies and tissue disorders) .
  • a vector carrying a gene capable of expressing a single urea cycle enzyme will be sufficient to improve the condition of hepatic encephalopathy in the context of liver failure.
  • the methods of this invention also encompass the delivery of more than one gene therapy vector, each of which may express at least one of the five above-mentioned urea cycle genes, or a single vector capable of expressing more than one urea cycle enzyme.
  • the methods of this invention preferably target gene therapy vectors to hepatocytes to correct underlying metabolic derangements.
  • the present invention thus corrects urea cycle metabolic derangements by hepatocyte-directed gene transfer rather than by complete organ replacement.
  • the methods of this invention preferably use recombinant adenoviruses carrying a urea cycle enzyme gene for the treatment of liver metabolic disease, i.e., hepatic encephalopathy.
  • liver metabolic disease i.e., hepatic encephalopathy.
  • Hemizygous male or homozygous female OTC-deficient mice are recognized as authentic animal models of OTC deficiency, useful for the study of human hyperammonemia (see Example 1) .
  • These OTC- deficient mice develop hyperammonemia, seizures and coma when challenged with ammonia, thus simulating episodes that characterize the human disease. These life threatening episodes of hyperammonemia are used to demonstrate the efficacy of recombinant adenoviruses for correcting the metabolic defect in liver.
  • Recombinant Viruses carrying a urea cycle enzyme gene for the treatment of liver metabolic disease, i.e., hepatic encephalopathy.
  • Recombinant viruses that are capable of delivering and stably integrating a functional, normal urea cycle enzyme gene to hepatocytes are used in this method.
  • the urea cycle enzyme gene will be referred to as the OTC gene.
  • any of the other four liver enzyme genes carbamyl phosphate synthetase (CPS) , arginino-succinate lysase (AL) , arginase (ARG) , and argininosuccinate synthetase (AS) , may be used according to the same techniques.
  • recombinant viruses for use in the present invention are desirably deleted in one or more viral genes, and contain a "minigene" containing the liver enzyme gene (e.g., OTC) under the control of regulatory sequences.
  • a "minigene” containing the liver enzyme gene e.g., OTC
  • Optional helper viruses and/or packaging cell lines supply to the recombinant viruses any gene products necessary for the deleted viral genes to ultimately be replicated and/or expressed.
  • Suitable viruses useful in gene therapy are well known, including retroviruses, vaccinia viruses, poxviruses, adenoviruses and adeno-associated viruses, among others.
  • viruses for use in the methods of the invention are adenoviruses [see, e.g., M. S. Horwitz et al, "Adenoviridae and Their Replication", Virology. second edition, pp. 1712, ed. B. N. Fields et al, Raven Press Ltd., New York (1990); M. Rosenfeld et al, Cell. 6_8:143-155 (1992); J. F. Engelhardt et al, Human Genet. Ther.. 1:759-769 (1993); Yang IV; J. Wilson, Nature. 3_65:691-692 (Oct. 1993); B. J.
  • Adenoviruses can be purified in large quantities and highly concentrated, and the virus can transduce genes into non-dividing cells.
  • the adenovirus sequences may be obtained from any known adenovirus type, including the presently identified 41 human types [Horwitz et al, Virology. 2d ed., B. N. Fields, Raven Press, Ltd., New York (1990)].
  • the DNA sequences of a number of adenovirus types are available from Genbank, including type Ad5 [Genbank Accession No. M73260].
  • a variety of adenovirus strains are available from the American Type Culture Collection, Rockville, Maryland, or available by request from a variety of commercial and institutional sources. Particularly desirable are human type C adenoviruses (Ad), including serotypes Ad2 and Ad5, which are not associated with human malignancies.
  • the adenovirus is preferably rendered replication defective by deleting the early gene locus that encodes Ela and Elb [K. F. Kozarsky and J. M. Wilson, Curr. Opin. Genet. Dev.. 2:499-503 (1993)].
  • Recombinant, defective adenoviruses optionally bearing other mutations, e.g., temperature sensitive mutations, deletions and hybrid vectors formed by adenovirus/adeno- associated virus sequences may also be used in this invention [see, for example, the viruses described in Kozarsky, cited above, and other references cited herein, which are incorporated by reference] .
  • the methods employed for the selection of viral sequences useful in a recombinant virus, the cloning and construction of the liver enzyme "minigene" and its insertion into a desired virus and the production of a infectious recombinant virus by use of helper viruses and the like are within the skill in the art given the teachings provided herein.
  • Useful recombinant adenoviruses for delivery of the liver enzyme gene (e.g., OTC) to the liver can contain adenovirus nucleic acid sequences ranging from a minimum sequence amount (a virus containing only the adenovirus cis-elements necessary for replication and virion encapsidation, but otherwise deleted of all adenovirus genes) to viruses characterized by deletions of only selected adenovirus genes. In either case, deleted gene products can be supplied in the recombinant virus production process by a packaging or helper cell line. Desirable "minimal" recombinant adenoviruses (Ad) vectors useful in the present invention are described in detail in co-owned International Patent Application
  • Recombinant, replication-deficient adenoviruses useful for the methods of this invention alternatively contain more than the minimal adenovirus sequences.
  • These other Ad vectors may be characterized by deletions of various portions of gene regions of the virus, and infectious virus particles formed by the optional use of helper viruses and/or packaging cell lines. See, e.g.,
  • first generation recombinant viruses are formed by deleting all or a sufficient portion of the adenoviral early immediate early gene Ela (which spans mu 1.3 to 4.5) and delayed early gene Elb (which spans mu 4.6 to 11.2) so as to eliminate their normal biological functions.
  • These replication-defective El-deleted viruses are capable of replicating and producing infectious virus when grown on an adenovirus- transformed, complementation human embryonic kidney cell line containing functional adenovirus Ela and Elb genes which provide the corresponding gene products in trans, the 293 cell [ATCC CRL1573].
  • the resulting virus is capable of infecting many cell types and can express a transgene but cannot replicate in most cells that do not carry the El region DNA unless the cell is infected at a very high multiplicity of infection.
  • Another recombinant adenovirus, a "second generation" virus is characterized by the above described deletion in the Ela and Elb genes, as well as additional deletions and mutations of the adenovirus genome in genes E2a and E3 as follows.
  • Second generation recombinant adenoviruses contain a mutation which produces temperature-sensitive (ts) virus, i.e., incorporation of the missense temperature-sensitive mutation in the DNA binding protein (DBP) E2a region found in the Ad5 H5tsl25 strain [P.
  • a "second generation" adenovirus is characterized by deletion of all or a portion of the adenovirus delayed early gene E3 (which spans mu 76.6 to 86.2) .
  • the function of E3 is irrelevant to the function and production of the recombinant virus particle.
  • recombinant viruses are constructed with a therapeutic minigene inserted into the El-deleted region of the known mutant Ad5 sub360 backbone [J. Logan et al, Proc. Natl. Acad. Sci. USA. 8_1:3655-3659 (1984)]; or the Ad5 mutant dl7001 backbone [Dr. William Wold, Washington University, St. Louis; see, e.g., J. E.
  • Both mutant viruses also contain a deletion in the E3 region of the adenoviral genome; in sub360, at 78.5 to 84.3 mu, and in dl7001, at 78.4 to 86 mu.
  • the life cycle of both sub360 and dl7001 display wild type characteristics.
  • Recombinant adenoviruses useful in this invention may also be constructed having a deletion of the El gene, at least a portion of the E3 region, and an additional deletion or mutation within adenovirus genes other than El and E3 to accommodate the liver enzyme minigene and/or other mutations which result in reduced expression of adenoviral protein and/or reduced viral replication.
  • all or a portion of the adenovirus delayed early genes E2a (which spans mu 67.9 to 61.5), E2b (which spans mu 29 to 14.2) and E4 (which spans mu 96.8 to 91.3) may be eliminated or mutated in the recombinant adenovirus.
  • liver enzyme minigene may be inserted into any deleted region of the selected Ad virus.
  • minigene is meant the combination of the liver enzyme gene and the other regulatory elements described below, which are necessary to transcribe the gene and express the gene product in vivo .
  • the therapeutic gene contained within the recombinant virus is one or more urea cycle enzymes, as described above and known in the art.
  • the human gene sequence for OTC is known [see, e.g., Jones, S. N. et al, J. Biol. Chem.. 265: 14684-14690 (1990) and references cited therein], as are the sequences for the other liver enzymes.
  • For the sequences of CPS see Y. Haraguchi et al, Gene. 107:335-340 (1991); for the sequences of AL, see W. E. O'Brien et al, Proc. Natl. Acad. Sci. USA. 8_3_:7211-7215 (1986); for the sequences of ARG, see Y. Haraguchi et al, Proc. Natl. Acad. Sci. USA f 84 .
  • liver enzyme gene is operatively linked to regulatory components in a manner which permits its transcription.
  • Such components include conventional regulatory elements necessary to drive expression of the liver enzyme transgene in a cell transfected with the recombinant virus.
  • the minigene also contains a selected promoter which is linked to the transgene and located, with other regulatory elements, within the selected viral sequences of the recombinant virus.
  • promoter is a routine matter within the skill of the art and is not a limitation of this invention.
  • Useful promoters may be constitutive promoters or regulated (inducible) promoters, which will enable control of the amount of the transgene to be expressed.
  • a desirable promoter is that of the cytomegalovirus (CMV) immediate early promoter/enhancer [see, e.g., Boshart et al, Cell. 41:521-530 (1985)].
  • CMV cytomegalovirus
  • Another desirable promoter/enhancer sequence is the chicken cytoplasmic ⁇ -actin promoter [T. A. Kost et al, Nucl. Acids Res.. ⁇ (23):8287 (1983)].
  • the minigene may also desirably contain nucleic acid sequences heterologous to the virus sequences including sequences providing signals required for efficient polyadenylation of the transcript (poly-A or Pa) and introns with functional splice donor and acceptor sites.
  • a common poly-A sequence which is employed in the exemplary vectors of this invention is that derived from the papovavirus SV-40.
  • the poly-A sequence generally is inserted in the minigene following the transgene sequences and before the viral sequences.
  • a common intron sequence is also derived from SV-40, and is referred to as the SV-40 T intron sequence.
  • a minigene of the present invention may also contain such an intron, desirably located between the promoter/enhancer sequence and the transgene.
  • Ad viruses containing an OTC minigene used to demonstrate this invention are described in detail in Example 1 below.
  • Other exemplary Ad viruses including Ad viruses containing an AS minigene (Example 6) , Ad viruses containing an ARG minigene (Example 7) Ad viruses containing a CPS minigene (Example 8) , and Ad viruses containing an AL minigene (Example 9) , are also described in detail. Assembly of the selected DNA sequences of the adenovirus, the reporter genes or therapeutic genes and other elements into the recombinant adenovirus and the use of the helper viruses to produce an infectious virus is performed using conventional techniques.
  • Such techniques include conventional cloning techniques of cDNA such as those described in texts [Sambrook et al, cited above], use of overlapping oligonucleotide sequences, polymerase chain reaction, and any suitable method which provides the desired nudeotide sequence.
  • Standard transfection and co-transfection techniques are employed, e.g., CaP0 4 transfection techniques using the human embryonic kidney 293 cell line, which provides Ela function to El-deleted adenovirus.
  • Other conventional methods employed include homologous recombination of the viral genomes, plaguing of viruses in agar overlay, methods of measuring signals generated by the reporter gene (e.g., enzymatic, colorimetic, etc.) and the like.
  • the vector is infected in vitro in the presence of an optional helper virus and/or a packaging cell line. Homologous recombination occurs between the helper and the vector, which permits the adenovirus-transgene sequences in the vector to be replicated and packaged into virion capsids, resulting in recombinant viral particles.
  • the current method for producing such virus particles is transfection-based. Briefly, helper virus is used to infect cells, which are then subsequently transfected with an adenovirus plasmid vector containing an OTC (or other liver enzyme) transgene by conventional methods. About 30 or more hours post-transfection, the cells are harvested, an extract prepared and the recombinant virus vector containing the OTC (or other liver enzyme) transgene is purified by buoyant density ultracentrifugation in a CsCl gradient.
  • the resulting recombinant adenovirus containing the liver enzyme minigene provides an efficient gene transfer vehicle for delivery of the liver enzyme gene to a patient in vivo or ex vivo and provide for introduction of the gene into a liver cell, alternatively, for the production of a medicament useful in treatment of a non- inherited liver disorder.
  • These recombinant viruses can also be employed to produce the selected liver enzyme in vitro , if desired.
  • the recombinant virus may be administered to a patient, preferably suspended in a biologically compatible solution or pharmaceutically acceptable delivery vehicle.
  • a suitable vehicle includes sterile saline.
  • aqueous and non-aqueous isotonic sterile injection solutions and aqueous and non-aqueous sterile suspensions known to be pharmaceutically acceptable carriers and well known to those of skill in the art may be employed for this purpose.
  • the medicament or virus is administered in sufficient amounts to transfect the hepatocytes and provide sufficient levels of transduction and expression of the liver enzyme gene to improve the functioning of the urea cycle, without undue adverse effects or with medically acceptable physiological effects which can be determined by those skilled in the medical arts.
  • Conventional and pharmaceutically acceptable routes of administration include direct delivery to the liver, intranasal, intravenous, intramuscular, subcutaneous, intradermal, oral and other parental routes of administration. Routes of administration may be combined, if desired.
  • a therapeutically effective human dosage of the recombinant viruses is generally in the range of from about 0.1 to about 100 ml, and more preferably from about 0.1 to about 20 ml, of saline solution containing concentrations of from about 1 x IO 7 to 5 x l ⁇ n pfu/ l viruses, and preferably about 1 x 10" pfu/ml.
  • a preferred adult human dosage is about 1 ml saline solution at the above concentrations.
  • the dosage will be adjusted to balance the therapeutic benefit against any adverse effects.
  • the levels of expression of the gene encoding the selected urea cycle enzyme can be monitored to determine the selection, adjustment or frequency of dosage administration.
  • each enzyme gene will be present in a separate recombinant adenovirus, and that such viruses will be administered at approximately the same time and in approximately equivalent dosages.
  • two or more urea cycle enzyme genes may be expressed from the presence of two minigenes in the same recombinant virus.
  • adenovirus- ediated gene therapy Current limitations of adenovirus- ediated gene therapy include the potential of cytotoxic T lymphocyte (CTL) responses to residually-expressed viral protein and possibly to OTC (or other liver enyzme) in patients with null mutations.
  • CTL cytotoxic T lymphocyte
  • OTC or other liver enyzme
  • previous studies have described the development of neutralizing antibodies to input viral proteins that block gene transfer upon second administration [Kozarsky et al, cited above] .
  • Improved vectors deleted for adenoviral genes may diminish CTL responses to viral protein, but will have no effect on neutralizing antibody to viral capsid protein or CTL responses to OTC.
  • Activation of CD4 T helper cells to input viral proteins is necessary for both B cell and CTL activation.
  • Transient blockade of the initial CD4 T cell activation at the time of virus instillation has been shown to both prolong transgene expression and prevent formation of neutralizing antibody [Yang, Y. et al, Nature Med.. 1, 890-893 (1995) (Yang VI) ] .
  • the method according to this invention for preventing or treating hepatic encephalopathy due to hyperammonemia and/or compromised ureagenesis in inherited or acquired liver disorders involves the coadministration of an immune modulator together with the recombinant adenovirus carrying an OTC gene.
  • Other preferred embodiments involve preventing or treating hepatic encephalopathy by coadministration of an immune modulator together with a recombinant adenovirus carrying an ARG, CPS, AS, or AL genes.
  • combinations of these recombinant adenoviruses may be utilized.
  • a suitable amount of a preferably short-acting, immune modulator may be administered either concurrently with, or before or after administration of the recombinant adenovirus.
  • the selected immune modulator is defined herein as an agent capable of inhibiting the formation of neutralizing antibodies directed against the recombinant virus of this invention or capable of inhibiting cytolytic T lymphocyte (CTL) elimination of the virus.
  • the immune modulator may interfere with the interactions between the T helper subsets (T H1 or T m ) and B cells to inhibit neutralizing antibody formation.
  • the immune modulator may inhibit the interaction between T H1 cells and CTLs to reduce the occurrence of CTL elimination of the virus. More specifically, the immune modulator transiently interferes with, or blocks, the function of the CD4 T cells.
  • the method of selection of immune modulators is disclosed in detail in co-pending United States patent application No. 08/394,032, which is incorporated herein by reference.
  • selection of the immune modulator may be based upon the mechanism sought to be interrupted or blocked. See, e.g., Y. Yang et al, Nat. Medic.. l(9):890-893 (1995), incorporated by reference herein.
  • the method of inhibiting an adverse immune response to the gene therapy vector involves nonspecific inactivation of CD4 cells with monoclonal antibody, preferably, "humanized” antibodies which prevent the recipient from mounting an immune response to the blocking antibody.
  • Such "humanization” may be accomplished by methods known to the art. See, for example, G.E. Mark and E. A.
  • the immune modulator can be a cytokine, such as interleukin-12 [see, e.g., Y. Yang et al, Nat. Med.. 1(9):1-4 (Sept., 1995)] or gamma interferon [S. C. Morris et al, J. Immunol.. 152:1047-
  • cytokine such as interleukin-12 [see, e.g., Y. Yang et al, Nat. Med.. 1(9):1-4 (Sept., 1995)] or gamma interferon [S. C. Morris et al, J. Immunol.. 152:1047-
  • Another desirable immune modulator for use in this method which selectively inhibits the CD4+ T cell subset T H1 function at the time of primary administration of the viral vector includes interleukin-4 [see, e.g., Yokota et al, Proc. Natl. Acad. Sci.. USA. 83_:5894-5898 (1986); United States Patent No. 5,017,691].
  • Such cytokines for use in this method are preferably in protein form, e.g., recombinantly produced using known techniques, or obtained commercially.
  • cytokine gene may be engineered into a recombinant virus and expressed in a target cell in vivo or ex vivo . It is also anticipated that active peptides, fragments, subunits or analogs of these cytokines which share the function of these proteins, will also be useful in this method.
  • the method preferably involves administration of more than one cytokine, specific dosing regimens and/or co ⁇ administration of an additional immune regulator, such as an antibody.
  • an additional immune regulator such as an antibody.
  • cytokines are advantageous because cytokines are natural products, and thus not likely to generate any adverse immune responses in the patient to which they are administered.
  • Still other immune modulators useful in this method are agents that specifically inhibit or deplete CD4+ cells, for example, by antibody to the CD4 protein.
  • agents include anti-T cell antibodies, such as anti-OKT 3+ [see, e.g., US Patent No. 4,658,019; European Patent Application No. 501,233, published September 2, 1992, among others; and commercially available antibody GK1.5 (ATCC Accession No. TIB207) ] . Depletion of CD4+ cells is shown to inhibit the CTL elimination of the virus.
  • any agent that interferes with, or blocks the interactions necessary for, the activation of B cells by T H cells, and thus the production of neutralizing antibodies is useful in the methods of this invention.
  • an antibody to CD40 ligand (anti-CD40L) [available from Bristol-Myers Squibb Co; see, e.g., European patent application 555,880, published August 18, 1993] or a soluble CD40 molecule can be a selected immune modulator in this method.
  • a soluble form of B7 or an antibody to CD28 or CTLA4, e.g., CTLA4-Ig [available from Bristol-Myers Squibb Co; see, e.g., European patent application 606,217, published July 20, 1994] can also be the selected immune modulator in this method.
  • immune modulators or agents that non- specifically inhibit immune function i.e., cyclosporin A or cyclophosphamide
  • cyclosporin A or cyclophosphamide may also be useful in this method.
  • the subject may be undergoing long term treatment with a non-specific immunosuppressant. If not, however, a short course of treatment with cyclosporin A or cyclophosphamide is preferred where these agents are utilized.
  • a suitable amount or dosage of the immune modulator will depend primarily on the identity of the modulator, the amount of the recombinant virus bearing the OTC transgene which is initially administered to the patient, and the method of delivery of the virus. Other secondary factors such as those mentioned above, may also be considered by a physician in determining the dosage of immune modulator to be delivered to the patient.
  • a therapeutically effective human dosage of a protein immune modulator e.g., IL-12 or ⁇ -IFN
  • IL-12 or ⁇ -IFN is generally in the range of from about 0.5 ⁇ g to about 5 mg per about 1 x 10 7 pfu/ml recombinant adenovirus.
  • Various dosages may be determined by one of skill in the art to balance the therapeutic benefit against any side effects.
  • the co-administration of recombinant virus and immune modulator occur within a close time proximity to each other.
  • the immune modulator is administered simultaneously with the recombinant virus expressing the urea cycle enzyme gene.
  • the immune modulator is administered prior to or subsequent to administration of the virus. It is presently preferred to administer the modulator concurrently with or no longer than one to three days prior to the administration of the virus.
  • the immune modulator may be administered separately from the recombinant virus, or, if desired, it may be administered in admixture with the recombinant virus.
  • the immune modulator may be administered in a pharmaceutically acceptable carrier or diluent, such as saline.
  • the immune modulator when formulated separately from the virus, is desirably suspended in saline solution.
  • a solution may contain conventional components, e.g. pH adjusters, preservatives and the like.
  • pH adjusters e.g. pH adjusters, preservatives and the like.
  • the immune modulator may be itself administered as DNA, either separately from the vector or admixed with the recombinant virus bearing the transgene.
  • Methods exist in the art for the pharmaceutical preparation of the modulator as protein or as DNA See, e.g., J. Cohen, Science. 259:1691-1692 (1993) regarding DNA vaccines] .
  • the immune modulator is administered by the same route as the recombinant virus.
  • the immune modulator may be formulated directly into the composition containing the virus administered to the patient.
  • the immune modulator may be administered separately, preferably shortly before or after administration of the virus.
  • a composition containing one immune modulator may be administered separately from a composition containing a second immune modulator, and so on, depending on the number of immune modulators administered. These administrations may independently be before, simultaneously with, or after administration of the recombinant virus.
  • the administration of the selected immune modulator may be repeated during the treatment with the recombinant adenovirus carrying the transgene, during the period of time that the transgene is expressed, as monitored by assays suitable to evaluating the functioning of the urea cycle or with every booster of the recombinant virus.
  • each reinjection of the same recombinant virus may employ a different immune modulator.
  • Another aspect of the present invention includes the determination of which of the five urea cycle enzymes is the rate-limiting enzyme involved in any particular liver dysfunction characterized by compromised ureagenesis.
  • ureagenesis is compromised by partial liver resection in an accepted animal model of the indicated liver dysfunction.
  • Recombinant viruses containing different urea cycle enzyme genes of this invention are administered singularly. After each virus and individual urea cycle enzyme gene is administered, ureagenesis is evaluated by conventional methods known to the art. Based on the observation of the functioning of ureagenesis following administration of each vector, one compares the results of administration of each enzyme gene and thereby determines which enzyme is necessary to improve ureagenesis.
  • a combination of urea cycle enzymes in one or more recombinant adenoviruses are administered and their effects studied in the same manner to determine what combination of enzymes is necessary to improve ureagenesis.
  • a toxic drug is administered to bring the animal subject to the point of hyperammonemia. Then the recombinant viruses are administered singularly or in combination, as described above, to determine the best treatment for the indicated liver dysfunction.
  • Examples 1 and 2 discuss the animal model and OTC-expressing recombinant adenoviruses used in these experiments.
  • Example 3 discusses the assay methods used to assess efficacy of treatment of an OTC deficiency.
  • Example 4 demonstrates the utility of adenovirus-mediated gene therapy in preventing the biochemical and clinical sequelae of nitrogen challenge in OTC deficiency. Briefly described, adenovirus vectors of the present invention that express a normal murine OTC gene are infused into the blood of OTC deficient animals. Adenoviral vectors administered by this route target to liver and high level OTC expression is detected in virtually all hepatocytes. The animals are subsequently challenged with ammonia and evaluated for metabolic and clinical consequences.
  • Example 5 sets out the experimental protocols and results of treating the OTC-deficient mouse models with first and second generation adenoviruses carrying minigenes which differed in promoters and origin of the OTC gene.
  • the experimental protocols systematically evaluated the impact of both the transgene and virus on the safety and efficiency of adenovirus-mediated gene transfer to liver.
  • Recombinant "first generation" adenoviruses deleted in El contain either human OTC CDNA driven by a CMV-enhanced, ⁇ -actin promoter (H5.OlOCBhOTC) or mouse OTC CDNA driven by a strong CMV promoter (H5.OlOCMVmOTC) . Both viruses achieved high level gene transfer in vivo.
  • Example 6 describes construction of exemplary adenoviruses carrying AS.
  • Example 7 describes construction of exemplary adenoviruses carrying ARG.
  • Example 8 describes construction of exemplary adenoviruses carrying CPS.
  • Example 9 describes construction of an exemplary adenovirus carrying AL.
  • Example 10 describes use of the exemplary adenoviruses of the invention in the treatment of hyperammonemia.
  • Example 1 Murine Models
  • the best characterized murine model of OTC deficiency is the sparse fur (spf) mouse, in which a missense mutation in codon 117 of the OTC gene leads to a catalytically defective enzyme with hepatic OTC activity reduced to approximately 5-20% of wild-type levels at physiologic Ph [Veres, G. et al. Science. 237: 415-417 (1987) and Qureshi, I. A. et al, Pediat. Pes.. 13: 807-811 (1979)].
  • the 85% reduction in liver OTC activity in this mouse leads to an activation of de novo pyrimidine synthesis as evidenced by a 13-fold increase in excretion of orotic acid in the urine.
  • the other accepted murine model is the spf* 1 * (abnormal skin and hair) mutant mouse, in which a point mutation in the final base pair of exon 4 of the OTC gene leads to aberrant splicing with markedly reduced levels of OTC mRNA and only 5% of normal OTC activity
  • mice are characterized by dysfunctional or reduced (10-30%) residual OTC enzyme activity in liver, resulting in metabolic and clinical abnormalities consistent with a partial OTC deficiency in humans.
  • Hemizygous male and homozygous female pups have a mild phenotype that resembles human males and symptomatic females with partial OTC activity (i.e., late onset hemizygotes and symptomatic heterozygotes). For example, these mice are runted and have wrinkled skin with little to no fur early in development. The mice are asymptomatic on a normal diet with baseline abnormalities in plasma glutamine, citrulline, and arginine.
  • mice The mouse strains spf, s ⁇ f" h and C3HeB/J used in the Examples below were purchased from Jackson Laboratory (Bar Harbor, Maine) and maintained in the Wistar animal facility.
  • Female heterozygous spf/X or spfVx mice were bred with normal male (X/Y) C3HeB/J mice to generate experimental spf/Y or spf" h /Y mice. All animals used in the experiments described below were between 6 to 10 weeks of age.
  • Example 2 First and the Second Generation Recombinant Adenoviruses
  • OlOCBhOTC This El-deleted first generation recombinant adenovirus was prepared as follows. The coding sequence for human OTC was removed from plasmid pHO-731 on a 1.0 kb Hinfl fragment [Horwich, A. L. et al, Science. 224: 1068-1074 (1984)], blunted with Klenow, ligated with Bell linkers, and cloned in direct orientation into the BamHI site of the retroviral vector pgagBA [Grossman, M. et al, Hum. Gene Ther. f 3_: 501-510 (1992)].
  • the resulting plasmid contains adenovirus type 5 map units 0-1, a minigene capable of directing the expression of human OTC cDNA from a CMV-enhanced, chicken ⁇ -actin promoter, a polyadenylation signal, and adenovirus map units 9.7-16 in a plasmid backbone.
  • This plasmid is deleted of adenoviral sequences spanning 1 to 9.6 map units.
  • pAd.CBhOTC was linearized with Nhe I and transfected into 293 cells [ATCC CRL1573] with Clal/Xbal restricted sub360 genomic adenovirus type 5 DNA, which contains a small deletion in the E3 gene between m.u. 78.5-84.3 [Logan, J. , and Shenk, T., Proc. Natl. Acad. Sci.. U.S.A.. 81: 3655-3659 (1984)].
  • the resulting recombinant virus, designated H5.OlOCBhOTC was grown and purified through three rounds of plaque isolations.
  • H5.110CBhOTC This second generation recombinant virus differs from H5.OlOCBhOTC only by a single base pair substitution in the E2a gene which generates a temperature sensitive viral DNA binding protein (DBP) capable of growth at 32°C but not 39°C.
  • DBP temperature sensitive viral DNA binding protein
  • This virus was deleted in El (i.e., no Ad m.u. 1-9.2), defective in E2a due to the tsl25 mutation, and contains the above-described human OTC cDNA minigene.
  • the E2a ts mutation contained within this recombinant adenovirus was generated from the wild type Ad5 mutant strain H5.tsl25 [Ensinger, M. J. , and Ginsberg, H. S., J. Virol.. 10: 328-339 (1972)].
  • This temperature sensitive mutation inactivates essential gene product of E2a.
  • This second generation virus has shown improved efficacy in gene transfer to mouse liver, and mouse, rat, and primate lung, using lacZ containing constructs in place of the human OTC gene [Yang II; Engelhardt I; Engelhardt, J. et al, Hum. Gene Thera. f 5_: 1217-1229 (1994) (Engelhardt II); Goldman, M. J.
  • first and second generation recombinant viruses differ from the above-described viruses in that they contain a strong constitutive CMV viral enhancer/promoter and carry mouse OTC cDNA rather than human.
  • these adenoviral vectors deliver a species-homologous OTC gene to the experimental animal. It is possible that the murine OTC transgene product interacts with regulatory factors in the cell involved in ammonia homeostasis.
  • Mouse OTC cDNA was generated by RT-PCR, cloned into pGEM-T vector (Promega, Madison, Wl) and restricted with Spe I and Sac II. A 1.5kb fragment containing mouse cDNA was isolated, blunted and cloned into EcoRV site of an adenoviral vector pAd.CMV-link (a plasmid containing the adenoviral sequences 0 to 16 map units deleted of Ela and Elb as described in the other adenovirus vectors into which a CMV promoter-polylinker cassette was cloned) .
  • pAd.CMV-link a plasmid containing the adenoviral sequences 0 to 16 map units deleted of Ela and Elb as described in the other adenovirus vectors into which a CMV promoter-polylinker cassette was cloned
  • the new plasmid designated pAd.CMVmOTC, was linearized with EcoRI and cotransfected into 293 cells with Clal/Xbal restricted Ad5 viral DNA containing the sub360 mutation in E3 as described above to generate the first generation recombinant virus, H5.OlOCMVmOTC.
  • pAd.CMVmOTC was linearized with EcoRI and cotransfected into 293 cells with Clal/Xbal restricted Ad5 viral DNA containing the tsl25 mutation in E2a as described above and the sub360 mutation in E3, resulting in H5.llOCMVmOTC. (For nomenclature of recombinant adenoviruses see Engelhardt II) .
  • Both viruses were purified through three rounds of plaque isolation.
  • the mouse cDNA part of the recombinant viruses was sequenced in both directions.
  • H5. llOCMVlacZ This second generation lacZ control virus was constructed as described in Engelhardt I, and purified through three rounds of plaque isolation.
  • H5. OlOCMVhOTC For use in studying the influence of the promoter, this first generation recombinant virus was prepared similarly to H5.OlOCMVmOTC, but the human OTC gene above was used to replace the mouse OTC gene described above.
  • Suitable adenoviral vectors capable of expressing the other liver enzyme genes may be readily prepared using methods similar to those described for the above vectors, and substituting the desired liver enzyme gene.
  • Cells were harvested by scraping into mitochondria lysis buffer (0.5% Triton, 10 mM Hepes, pH 7.4, 2 mM DTT) , and total protein was extracted by three freeze-thaw cycles. Liver tissue was homogenized in mitochondria lysis buffer with a Polytron homogenizer. The homogenate was centrifuged in a microfuge at the maximum speed for 5 minutes; the supernatant was transferred to a new tube; and OTC enzyme activity was measured according to the assay described by Lee, J. T., and Nussbaum, R. L. , J. Clin. Invest., 8_4, 1762-1766 (1989) , with the following modifications.
  • reaction mixture 5 mM ornithine, 15 mM carbamyl phosphate, and 270 mM triethanolamine, pH 7.7
  • reaction mixture 5 mM ornithine, 15 mM carbamyl phosphate, and 270 mM triethanolamine, pH 7.7
  • liver slides were then embedded in OCT and sectioned for histochemical staining as described by Mizutani, A., J. Histochem. Cvtochem.. 16: 172-180 (1968) .
  • reaction medium which contained 12 mg carbamyl phosphate, Li salt; 20 mg L-ornithine dihydrochloride, 3.2 g sucrose; 16 ml 0.05M triethanolamine buffer, pH 7.2; 20 ml distilled water, and 4 ml 1% lead nitrate.
  • the lead nitrate solution was added dropwise with continuous stirring and the solution was readjusted to pH 7.2 with 1 N NaOH. The slightly turbid substrate mixture was filtered and used immediately.
  • Sections were incubated for 30 minutes in reaction medium at room temperature, washed with distilled water three times, immersed in 0.37% ammonium sulfide for 1 minute, rinsed with distilled water again and mounted for light microscopic observations. Dark brown deposits of lead sulfide indicated the sites of OTC activity.
  • Urinary orotic acid levels were measured in duplicate for each sample as described by Brusilow, S. W. , and Hauser, E. , J. Chromatograph.. 493: 388-391 (1989).
  • Plasma amino acids were analyzed by precolumn derivitization with o-phthaldialdehyde as previously described by Robinson et al, J. Neurochemi.. £1: 2099 2103 (1993)]. After centrifugation of heparinized blood, an aliquot of plasma was immediately precipitated with an equal volume of 0.8 N perchloric acid which contained the internal standards L-a-aminoadipate and L-a-amino-n- butyric acid. After centrifugation, an aliquot of the supematants was neutralized with 2 M KHC0 3 . These samples were derivitized using an autosampler. External standards were injected after every fifth specimen.
  • RNA hybridization analysis Total cellular RNA was isolated, fractionated on formaldehyde gel, and transferred onto Hybond-N nylon filters (Amersham, Arlington Heights, IL) . DNA fragments used as probes in RNA hybridizations were gel-purified and labeled with [ ⁇ - 32 P]dCTP by random priming.
  • Immunofluorescence staining of adenoviral late gene products was performed as described by Kozarsky et al., cited above.
  • the primary antibody was a polyclonal rabbit antibody specific to Ad5 late gene products (produced in Dr. Wilson's laboratory).
  • the secondary antibody was a fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit IgG (Chemicon, Temecula, CA) .
  • Protocol A Individual cohorts of male C3HeB/J and spf/Y mice (6-10 weeks old) were weighed and injected intraperitoneally with a single dose (ranging from 4 to 10 mmol/kg) of a 0.64M solution of NH 4 C1 (i.e., nitrogen challenge) . At 15-20 minutes after injection, blood was collected from the retro-orbital plexus of the mice and analyzed via HPLC for plasma amino acids and the mice were evaluated and scored by two observers using the scoring system described below.
  • Protocol B Blood samples were collected by retro-orbital bleeding three days before the experiment (day -3). On day 0, Spf/Y and C3H male mice at 6-10 weeks of age were injected with lxlO 11 particles of recombinant second generation adenovirus, either
  • H5.llOCMVmOTC or H5.HOCMVlacZ in 0.1 ml of phosphate buffered saline (PBS) via the tail vein.
  • PBS phosphate buffered saline
  • Control mice were injected with PBS only.
  • the animals were then injected with NH t Cl (10 mmol/kg) on days 1, 2, 7, 14, or 28 post virus infusion.
  • Blood was collected from the retro-orbital plexus of the mice and analyzed via HPLC for plasma amino acids and the mice were evaluated and scored by two observers using the scoring system described below.
  • the clinical scoring system was developed to quantify the clinical manifestations of hyperammonemia. After nitrogen challenge, clinical manifestations of hyperammonemia were scored using a system based on the appearance of ataxia (A) , seizures (S) and response to sound (R) . Ataxia is determined by pulling on the mouse's tail and observing gait. The response is scored as follows: 2, if the mouse is able to ambulate normally; 1, if the mouse staggers away; 0, inability to walk. Seizure intensities are scored as follows: 2, no seizure; 1, spontaneous myoclonus with spontaneous jerking movements and 0, tonic-clonic seizures characterized by rigid extension of all limbs.
  • hyper- responsiveness to sound is determined by ringing a bell with sound frequency of 100 db 5 to 6 times in series.
  • mice Under this scoring system, a normal mouse is expected to receive an additive score of seven (A2S2R3) . Severely affected mice would receive a score of one (A0S1R0) . Any mice that die during the challenge would automatically receive a score of 0. Mice that exhibited tonic-clonic seizures always died after the seizures. The observers did not know the identity of animals in terms of treatment group during the period of observation.
  • Plasma amino acid data and the clinical scores of responsiveness to nitrogen challenge from mice that received different treatments were analyzed by the single factor analysis of variance (ANOVA) .
  • Fig. 6 presents the relationship between dose of NH 4 C1 and clinical seguelae in the animals treated under Protocol A.
  • Acute hyperammonemia similar to that observed in patients, was simulated in spf mice following i.p. injection of NH 4 C1 (i.e., nitrogen challenge).
  • NH 4 C1 i.e., nitrogen challenge
  • There was a direct correlation in spf animals between dose of NH 4 C1 and severity of symptoms ranging from essentially no symptoms at 4 mmol/kg to virtually complete mortality at 10 mmol/kg. Animals developed ataxia, seizures, and frequently coma and death within 20 minutes of nitrogen challenge concurrent with the accumulation of plasma aspartate, alanine, and gluta ate.
  • Table 1 summarizes the impact of OTC gene delivery using adenoviral gene therapy vectors on plasma amino acids following nitrogen challenge when evaluated 1, 2, 7, 14, and 28 days after gene therapy.
  • Rows 1 and 6 of Table 1 represent a baseline of plasma amino acids in both C3H and asymptomatic spf/Y animals maintained on a normal chow diet (see, rows 1 and 6 of Table 1) .
  • the urea cycle intermediates citrulline and arginine were decreased in spf animals to 25% (p ⁇ 0.005) and 70% (p ⁇ 0.005) of levels founds in C3H mice providing evidence for defective ureagenesis.
  • Plasma glutamine and urine orotate were elevated 2-fold (p ⁇ 0.005) and 10-fold (data not shown) , respectively, in spf mice relative to C3H animals consistent with systemic accumulation of nitrogen in the setting of partial OTC deficiency.
  • Amino acids were measured as described in Example 3 in blood harvested prior to, and 20 minutes following, challenge with NH 4 C1. No significant differences in plasma amino acids consistent with systemic nitrogen accumulation were noted in C3H animals following nitrogen challenge. This was not the case in spf animals who realized 3-fold increase in alanine (p ⁇ 0.005), 8-fold increase in aspartate (p ⁇ 0.005), and 2-fold increase in glutamate (p ⁇ 0.005) after nitrogen challenge.
  • the C3H and spf animals were treated with equivalent doses of lacZ virus to evaluate nonspecific effects of gene therapy on nitrogen metabolism and baseline ureagenesis. This is of concern because adenoviral vectors cause some degree of liver inflammation in mice at the doses used in this experiment (see below) .
  • serum glutamine (p ⁇ 0.005) and alanine (p ⁇ 0.005) were mildly and transiently increased in C3H mice following gene transfer.
  • One group of Spf/Y mice were infused via the tail vein on day 0 with 5x10" particles of first generation H5.OlOCBhOTC or second generation H5.110CBhOTC human OTC-based recombinant adenovirus suspended in 0.1 ml of phosphate-buffered saline (PBS) . Animals were sacrificed at day 4, 7, and 14 post infusion.
  • PBS phosphate-buffered saline
  • Uninfected C3HeB/J, heterozygous spf/+, and hemizygous spf/Y mice were used as controls. Liver tissues from each animal were prepared for histochemical, biochemical, and molecular biological analysis.
  • One group of Spf/Y mice were infused via the tail vein on day 0 with 5xl0 10 particles/mouse of first generation H5.OlOCBhOTC or second generation H5.110CBhOTC human OTC-based recombinant adenovirus suspended in 0.1 ml of phosphate-buffered saline (PBS). Animals were sacrificed at day 7, 14, 21 and 28 post infusion.
  • PBS phosphate-buffered saline
  • a second group of Spf/Y mice were infused with lxlO 11 particles of first generation H5.OlOCMVmOTC or second generation H5.llOCMVmOTC mouse OTC-based recombinant adenovirus following the same protocol and these animals were sacrificed at day 7, 14, 21 and 28.
  • Spf/Y mice at 6-8 weeks of age were infused with 5xlO u particles of first generation viruses (H5.OlOCBhOTC or H5.010CMVlacZ) or second generation viruses (H ⁇ .llOCBhOTC or H5.HOCBlacZ) through tail vein.
  • Urine and plasma samples were collected the day before the virus infusion, and at day 4, 7, and 14 post-infusion.
  • Liver tissues of spf/Y mice infused with 2xlO ⁇ particles of recombinant virus as described in Protocol D were harvested 4 days later. Fresh frozen sections (6 ⁇ m) were fixed in 100% methanol for 10 minutes, and analyzed for adenoviral late gene expression by immunofluorescence using an antibody specific to hexon.
  • Spf/Y mice were infused with 2xlO ⁇ particles of one of the following recombinant adenoviruses: H5.OlOCBhOTC, H5.OlOCMVhOTC, H5.OlOCMVmOTC, or H5.010CMVlacZ. Untreated spf mice and untreated C3H mice were used as controls. Liver tissue was harvested 3 days post infusion and analyzed for OTC activity by lysate enzyme analysis.
  • Spf/Y mice at 6-8 weeks of age were infused with 2xl0 11 particle of first generation (H5.OlOCMVmOTC) or second generation virus (H5.llOCMVmOTC or H ⁇ .HOCMVlacZ) through tail vein.
  • Spf ⁇ /Y mice of similar age were infused with 2xl0 n particle of the second generation virus (H5.llOCMVmOTC) .
  • Urine and plasma samples were collected at day -3, day -l, and at weekly intervals after virus infusion. H.
  • Heterozygotes showed two populations of OTC expressing cells consistent with lyonization of the X chromosome.
  • Histochemical analysis of spf liver removed 4 days after infusion of 5 x l ⁇ " particles of first generation human OTC vector as described in
  • Experimental Protocol A revealed low level expression in most cells, diminishing to baseline by days 14 - 28, concurrent with the development of substantial but self-limited hepatitis (Fig. 2A) .
  • Liver tissues were harvested from the animals of Experimental Protocols A and B at indicated time points in Figs. 2A through 2F following infusion and evaluated for evidence of histopathology by light microscopic inspection of paraffin sections stained with hematoxylin and erosin. Histopathology was evaluated using the criteria developed by Knodell R. et al, Hepatol.. 1: 431-435 (1981)]. The three criteria scored were: I, periportal & bridging necrosis; II, intralobular degeneration & focal necrosis; and III, portal inflammation. Analyses were performed on three to four animals per time point. The histogram shown in Figs. 2A through 2F is the average of at least three independent observations with SEM shown as error bars.
  • OTC enzyme as measured by the histochemical stain was higher and prolonged in the second generation mOTC virus than in the first generation viruses or the hOTC containing viruses. Inflammation was reduced in the second generation mOTC virus than in the first generation viruses or the hOTC containing viruses (Fig. 2B) .
  • Figs. 3A and 3B illustrate urinary orotate excretion in spf mice infused with recombinant adenoviruses carrying human OTC cDNA, as described in Experimental Protocol C.
  • Urinary orotate/mg creatinine are presented as a % of pretreatment levels and are the mean + SEM of at least 6 determinations. There was no significant change in urinary orotate (Fig. 3A) in spf mice after infusion of 5 x 10 ⁇ particles of first generation human OTC vector H5.OlOCBhOTC, when compared to animals that received identical doses of H5.010CMVlacZ virus.
  • Urinary orotate nonspecifically decreased to approximately 50% of pretreatment levels with both viruses H5.OlOCBhOTC and H5.010CMVlacZ possibly due to the associated hepatitis.
  • Fig. 3C illustrates plasma glutamine levels in spf mice of Experimental Protocol C infused with second generation recombinant adenoviruses carrying human OTC CDNA. The levels are presented as a % of pretreatment levels and are the mean + SEM of between 4 to 10 determinations. There was no significant change in serum glutamine in spf mice after infusion of 5 x 10 U particles of first generation human OTC vector
  • H5.OlOCBhOTC when compared to animals that received identical doses of H5.OlOCMVlacZ virus.
  • RNA blot analysis of liver tissues from spf mice infused with recombinant adenoviruses according to Experimental Protocol D demonstrated a large blot for the hexon RNA in liver RNA from spf/Y mouse that received first generation human OTC virus and first generation mouse OTC virus.
  • Liver RNA from spf/Y mouse that received first and second generation human OTC virus and first and second generation mouse OTC virus revealed large blots for DBP.
  • RNA from untreated spf liver showed neither the hexon nor DBP blots.
  • the intensity of ribosomal RNA (18s and 28s) was similar in each lane, indicating equivalent quantities of electrophoresed RNA. Expression of late viral genes at the level of RNA was reduced in the second generation MOTC virus than in the first generation viruses or the HOTC containing viruses.
  • the OTC lysate assay performed on liver tissue from Experimental Protocol F produced the results illustrated in Fig. 4. Consistent with the above results, the highest OTC activity was detected in the spf mice treated with H5.OlOCMVmOTC. The OTC activity detected in the spf mice treated with H5.OlOCMVhOTC was equivalent to the OTC activity in the control C3H mice.
  • CMV/ ⁇ -actin promoter produced little enzyme activity above background in mouse spf liver.
  • a three-fold increase in activity was achieved when the CMV promoter/enhancer was used to express the human OTC CDNA.
  • An additional two to three-fold increase was achieved when the human OTC CDNA was replaced with the murine homolog in the CMV based vector.
  • the results from Protocol F demonstrated that normal OTC enzymatic activity was achieved with 2 to 5-fold less mouse OTC virus than the maximally tolerated dose of human OTC virus, which only partially corrected mouse OTC deficiency. This further supports the observation that the human OTC CDNA product functions inefficiently in restoring OTC levels in this mouse model.
  • Urinary orotic acid levels and plasma glutamate levels were measured as described above in Example 3 for the animals of Protocol G. The results are shown in Figs. 5A through 5D. Urinary orotate of animals treated with first generation mouse OTC virus decreased to 10% of pretreatment levels by day 7 and gradually returned to baseline by day 42 (Fig. 5A) . Correction of urinary orotate was significantly greater in animals treated with first generation MOTC virus than the nonspecific reduction seen with the lacZ virus, although it was not complete.
  • the suboptimal performance of the first generation El deleted vector carrying either MOTC or HOTC is attributed, in part, to the inherent immunogenicity of first generation constructs.
  • El deleted viruses express viral genes whose proteins are targets for destructive cellular immune responses.
  • the levels of viral late gene RNA and protein in the second generation viruses are diminished over those observed with the first generation viruses; associated hepatitis is also decreased.
  • mice infected with human OTC adenovirus detected cytotoxic T cells but not antibodies to human OTC protein. While primary antigen specific cellular immune responses may decrease stability of transgene expression at day 7 and beyond, they cannot limit efficiency at early time points such as day 3.
  • Previous studies have clearly implicated cellular immunity in the loss of transgene expression and associated inflammation that has characterized El deleted adenoviruses [Yang I, II, and III, cited above; Yang, Y. et al, Proc. Natl. Acad. Sci..
  • Transgene-derived MOTC differs by only one amino acid from the spf protein and is identical to the product of the spf Mh allele.
  • Transgene expression with the mouse OTC CDNA vectors persisted longer than what has been consistently observed with vectors expressing non-self transgenes such as ⁇ -galactosidase. Incorporating the tsl25 mutation into this vector diminished late viral gene expression and further prolonged transgene expression. The expression of transgene eventually diminished to undetectable levels within 3-4 months of gene transfer.
  • Example 6 Recombinant Adenovirus Containing Human Argininosuccinate Synthetase (AS) CDNA
  • a plasmid pAS4/l/9 containing human AS cDNA was obtained from the ATCC [catalog #57074], An 1.5 kb PstI fragment containing AS cDNA was isolated, blunted and cloned into EcoRV site of an adenoviral vector pAd.CMV- linkl [X. Ye et al, J. Biol. Chem.. 221:3639-3646 (1996)].
  • the new plasmid, designated pAd.CMVhAS was linearized with Nhel and cotransfected into 293 cells with Clal/Xbal restricted sub360 [J. Logan et al, Proc. Natl. Acad. Sci. USA.
  • This vector is useful in liver-directed gene therapy as described above with respect to the OTC- containing vectors.
  • Human ARG cDNA was obtained from human liver tissue by RT-PCR using primers generated using the published sequence [Y. Haraguchi, Proc. Natl. Acad. Sci. USA, 84.:412-415 (1987)].
  • the primer sequences are: 5 » primer: 5'-AGCTCAAGTGCAGCAAAGAG-3 • [SEQ ID NO:l] 3' primer: 5'-TGACATGGACACATAGTACCT-3' [SEQ ID NO:2].
  • the ARG cDNA generated was cloned into pGEM-T vector (Promega, Madison, Wl) and restricted with NotI and Sphl.
  • a second arginase-expressing vector was constructed using the methods (including the same restriction enzyme site) described above for H5.110CMVhArg, except that the viral backbone utilized was dl7001.
  • dl7001 is a type 5 adenovirus which contains a deletion in the E3 region between map units 78.4 through 86 and, more particularly, at nucleotides 594 and 3662 where nudeotide 1 corresponds to the EcoRI site of Ad5 at 27,331 bp. All E3 region open reading frames are deleted.
  • the resulting vector is termed H5.020hArg.
  • H ⁇ .HOCMVhArg and H5.020hArg were purified through three rounds of plaque isolation and have been found to express arginase activity in 293 cells. These vectors are useful in liver-directed gene therapy as described above with respect to the OTC-containing vectors.
  • Example 8 Recombinant Adenoviruses Containing Human Carbamyl phosphate synthetase (CPS) cDNA
  • Human CPS cDNA was obtained from human liver tissue by RT-PCR using primers generated using the published sequence [Y. Haraguchi et al, Gene. 107:335-340 (1991)].
  • the primer sequences are as follows. 5• primer: 5'-AAGTCTTATCACACAATCTCATTAA-3' [SEQ ID NO:3] 3' primer: 5'-GCCCTGTTAAAGTGTCCTGAG-3' [SEQ ID NO:4].
  • pGEM-T vector Promega, Madison, Wl
  • pGEM-CPS is then restricted with Sail and SphI, and cloned into pAd.link (a plasmid containing the human Ad5 sequences, map units 0 to 16, which is deleted of Ela and Elb as described in X. Ye et al, J. Biol. Chem.. 271:3639-3634 (1996)).
  • the resulting plasmid, pAd.CMVhCPS is linearized and co-transfected into 293 cells with Clal-digested dl327 virus DNA to generate H5.030CMVhCPS.
  • adenovirus containing human CPS under control of another promoter, e.g., a mouse albumin promoter.
  • Suitable plasmids can be readily obtained or constructed by one of skill in the art.
  • pGEM-CPS can be restricted with Sail and SphI as described above and cloned into pAd.albBcl2 backbone [kindly provided by Shujen Chen] in which Bcl2 cDNA has been removed by BamHI digestion.
  • the new plasmid, pAd.albhCPS is linearized by Nhil and cotransfected with Clal-digested dl327 virus DNA into 293 cells to generate H5.030albhCPS.
  • the human CPS cDNA generated is removed from pGEM-CPS as described above and inserted into the Nhrul site of pMT-LCR expression vector [kindly provided by R. Palmiter, University of Washington] .
  • the hCPS mini-gene which includes MT promoter, hCPS cDNA and human growth hormone (hGH) polyadenylation sequence is removed by EcoRI and inserted into pAd.link, as described above.
  • the resulting plasmid, pAd.MThCPS is linearized by Nhel and co ⁇ transfected into 293 cells with Clal-digested dl327 virus DNA to produce H5.030MThCPS.
  • the resulting hCPS vectors are assayed for activity in 293 cells as described in T. Nuzum and P.J. Snodgrass, The Urea Cycle (ed. S. Grisolia et al.), pp. 325-355, John Wiley and Sons, New York. These vectors are useful in liver-directed gene therapy as described above with respect to the OTC-containing vectors.
  • Human AL cDNA is obtained from human liver tissue by RT-PCR using primers generated using the published sequence [W. E. O'Brien et al, Proc. Natl. Acad. Sci. USA. 8.3:7211-7215 (1986)].
  • the human AL cDNA is then cloned into pGEM-T vector (Promega, Madison, Wl) essentially as described in the examples above.
  • the hAL cDNA is then inserted into pAd.CMVlink-1 [X. Ye et al, J. Biol. Chem.. 121:3639-3646 (1996)].
  • the resulting plasmid, pAd.CMVhAL is linearized with Nhel and co ⁇ transfected into 293 cells with Clai digested H5.110 or H5.020 backbone.
  • the resulting hAL vectors are assayed for activity in 293 cells as described in T. Nuzum and P.J. Snodgrass, The Urea Cycle (ed. S. Grisolia et al.), pp. 325-355, John Wiley and Sons, New York. These vectors are useful in liver-directed gene therapy as described above with respect to the OTC- containing vectors.
  • the experiment illustrates the method of this invention for using gene therapy in the treatment of an hyperammonemia in a human patient with a defect in the argininosuccinate synthetase (AS) gene of the urea cycle.
  • the recombinant virus H5.H0CMVhAS of Example 6 is administered intravenously to the adult human patient experiencing an acute episode of hyperammonemia due to the above-noted defect in a 1 ml dosage, i.e. a recombinant virus concentration of about 1 x IO 11 pfu/ml of saline solution.
  • a dosage of 1 ⁇ g/ml saline solution of anti-CD40L (Bristol-Myers Squibb Co.] is administered iv to the patient to suppress unwanted immune responses to the recombinant virus.
  • the levels of expression of the encoded urea cycle AS enzyme in serum are monitored periodically by conventional methods to determine the selection, adjustment or frequency of dosage administration and to assess the goal of reversing underlying metabolic derangements within 72 hours.
  • the H5.HOCMVhArgr and H5.020hArg vectors of example 7 may be utilized in the treatment of a human patient with a defect in the arginase (ARG) gene of the urea cycle;
  • the H5.030CMVhCPS, H5.030albhCPS, or H5.030MThCPS vectors of example 8 may be utilized in the treatment of a human patient with a defect in the carbamyl phosphate synthetase (CPS) gene of the urea cycle;
  • the H5.110hAL or H5.020hAL vector of example 9 may be utilized in the treatment of a human patient with a defect in the argininosuccinate lyase (AL) gene of the urea cycle.
  • MOLECULE TYPE other nucleic acid
  • MOLECULE TYPE other nucleic acid
  • MOLECULE TYPE other nucleic acid
  • SEQUENCE DESCRIPTION SEQ ID NO:3: AAGTCTTATC ACACAATCTC ATTAA 25 (2) INFORMATION FOR SEQ ID NOM:
  • MOLECULE TYPE other nucleic acid
  • SEQUENCE DESCRIPTION SEQ ID NO:4: GCCCTGTTAA AGTGTCCTGA G 21

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Abstract

Ce procédé permet de renforcer l'uréogenèse chez un sujet, un mammifère de préférence, qui le nécessite. Il consiste à lui administrer une quantité efficace d'un virus recombiné qui exprime in vivo au moins une enzyme propre au cycle de l'urée. Il peut aussi consister à administrer des adénovirus recombinés supplémentaires qui expriment d'autres enzymes propres au cycle de l'urée, ou un seul adénovirus recombiné qui exprime plusieurs enzymes. Ce procédé comprend aussi l'administration d'un modulateur immun avec le virus recombiné.
PCT/US1997/001564 1996-02-13 1997-02-13 Procede de traitement des troubles du foie WO1997030167A1 (fr)

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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999063067A1 (fr) * 1998-06-01 1999-12-09 Incyte Pharmaceuticals, Inc. Homologue de carbamoyl phosphate synthase
WO2000024916A1 (fr) * 1998-10-27 2000-05-04 Crucell Holland B.V. Production amelioree de vecteurs de virus associes aux adenovirus
WO2001085981A2 (fr) * 2000-05-05 2001-11-15 Research Foundation Of City University Of New York Methodes de stimulation de la regeneration et de la reparation du systeme nerveux par regulation de l'activite de l'arginase 1 et de la synthese des polyamines
US7118913B2 (en) * 2000-08-25 2006-10-10 Chung-Ang University Industry Academic Cooperation Foundation Expression vector containing urea cycle enzyme gene, transformant thereof, and use of transformant for protein over-expression
US8173706B2 (en) 2009-04-03 2012-05-08 Ocera Therapeutics, Inc. L-ornithine phenyl acetate and methods of making thereof
US8389576B2 (en) 2004-11-26 2013-03-05 Ucl Business Plc Compositions comprising ornithine and phenylacetate or phenylbutyrate for treating hepatic encephalopathy
US8946473B2 (en) 2010-10-06 2015-02-03 Ocera Therapeutics, Inc. Methods of making L-ornithine phenyl acetate
US10039735B2 (en) 2014-11-24 2018-08-07 Ucl Business Plc Treatment of diseases associated with hepatic stellate cell activation using ammonia-lowering therapies
US10835506B2 (en) 2015-08-18 2020-11-17 Ocera Therapeutics, Inc. Treatment and prevention of muscle loss using L-ornithine in combination with at least one of phenylacetate and phenylbutyrate
US11066352B2 (en) 2017-05-11 2021-07-20 Ocera Therapeutics, Inc. Processes of making L-ornithine phenylacetate
US11266620B2 (en) 2009-06-08 2022-03-08 Ucl Business Ltd Treatment of portal hypertension and restoration of liver function using L-ornithine phenylacetate

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995027512A2 (fr) * 1994-04-11 1995-10-19 Baylor College Of Medicine Compositions et procedes de therapie genique pour traiter des maladies
WO1996012406A1 (fr) * 1994-10-19 1996-05-02 Genetic Therapy, Inc. Therapie genique par administration concurrente et repetee d'adenovirus et d'agents immunodepresseurs

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995027512A2 (fr) * 1994-04-11 1995-10-19 Baylor College Of Medicine Compositions et procedes de therapie genique pour traiter des maladies
WO1996012406A1 (fr) * 1994-10-19 1996-05-02 Genetic Therapy, Inc. Therapie genique par administration concurrente et repetee d'adenovirus et d'agents immunodepresseurs

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
CAVARD C. ET AL.: "correction of mouse ornithine transcarbamylase deficiency by gene transfer into the germ line", NUCLEIC ACID RESEARCH, vol. 16, no. 5, 1988, OXFORD, GB, pages 2099 - 2110, XP000671680 *
DEMARQUOY J.: "Retroviral mediated gene therapy for the treatment of citrullinemia. Transfer and expression of argininosuccinate synthetase in human hematopoietic cells", EXPERIENTIA, vol. 49, no. 4, 1993, BASEL CH, pages 345 - 348, XP000674276 *
JONES S.N. ET AL.: "ectopic correction of ornithine transcarbamylase deficiency in Sparse Fur mice", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 265, no. 24, 25 August 1990 (1990-08-25), MD US, pages 14684 - 14690, XP000673285 *
SCHOFIELD J.P., CASKEY C.T.: "Gene therapy of autosomal recessive carbamoyl phosphate synthetase (CPSase I) deficiency", JOURNAL OF CELLULAR BIOCHEMISTRY, SUPPLEMENT, KEYSTON SYMPOSIUM ON GENE THERAPY AND MOLECULAR MEDICINE, 1995, NEW YORK, US, XP000197553 *
SMITH I. ET AL.: "methabolic correction in adult ortnithine transcarbamylase deficient (otcd)sparse fur (spf/Y) mice with E1 deleted, E2a defective recombinant adenovirus", PEDIATRIC RESEARCH, vol. 37, no. 4/2, April 1995 (1995-04-01), BALTIMORE, US, pages 155a, XP000671587 *
YANG Y. ET AL: "CELLULAR IMMUNITY TO VIRAL ANTIGENS LIMITS E1-DELETED ADENOVIRUSES FOR GENE THERAPY", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, vol. 91, no. 10, 1 May 1994 (1994-05-01), WASHINGTON US, pages 4407 - 4411, XP002009850 *

Cited By (27)

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Publication number Priority date Publication date Assignee Title
WO1999063067A1 (fr) * 1998-06-01 1999-12-09 Incyte Pharmaceuticals, Inc. Homologue de carbamoyl phosphate synthase
WO2000024916A1 (fr) * 1998-10-27 2000-05-04 Crucell Holland B.V. Production amelioree de vecteurs de virus associes aux adenovirus
WO2001085981A2 (fr) * 2000-05-05 2001-11-15 Research Foundation Of City University Of New York Methodes de stimulation de la regeneration et de la reparation du systeme nerveux par regulation de l'activite de l'arginase 1 et de la synthese des polyamines
WO2001085981A3 (fr) * 2000-05-05 2002-03-14 Res Foundation Of City Univers Methodes de stimulation de la regeneration et de la reparation du systeme nerveux par regulation de l'activite de l'arginase 1 et de la synthese des polyamines
AU2001259453B2 (en) * 2000-05-05 2006-08-31 Beth Israel Deaconess Medical Center Methods for stimulating nervous system regeneration and repair by regulating arginase 1 and polyamine synthesis
US7741310B2 (en) 2000-05-05 2010-06-22 Research Foundation Of The City University Of New York Methods for stimulating nervous system regeneration and repair by regulating arginase I and polyamine synthesis
US8673594B2 (en) 2000-05-05 2014-03-18 Research Foundation Of City University Of New York Methods for stimulating nervous system regeneration and repair by regulating arginase I and polyamine synthesis
US7118913B2 (en) * 2000-08-25 2006-10-10 Chung-Ang University Industry Academic Cooperation Foundation Expression vector containing urea cycle enzyme gene, transformant thereof, and use of transformant for protein over-expression
US9566257B2 (en) 2004-11-26 2017-02-14 Ucl Business Plc Compositions comprising ornithine and phenylacetate or phenylbutyrate for treating hepatic encephalopathy
US10610506B2 (en) 2004-11-26 2020-04-07 Ucl Business Ltd Compositions comprising ornithine and phenylacetate or phenylbutyrate for treating hepatic encephalopathy
US8389576B2 (en) 2004-11-26 2013-03-05 Ucl Business Plc Compositions comprising ornithine and phenylacetate or phenylbutyrate for treating hepatic encephalopathy
US10173964B2 (en) 2009-04-03 2019-01-08 Ocera Therapeutics, Inc. L-ornithine phenyl acetate and methods of making thereof
US8492439B2 (en) 2009-04-03 2013-07-23 Ocera Therapeutics, Inc. L-ornithine phenyl acetate and methods of making thereof
US9034925B2 (en) 2009-04-03 2015-05-19 Ocera Therapeutics, Inc. L-ornithine phenyl acetate and methods of making thereof
US11161802B2 (en) 2009-04-03 2021-11-02 Ocera Therapeutics, Inc. L-ornithine phenyl acetate and methods of making thereof
US8785498B2 (en) 2009-04-03 2014-07-22 Ocera Therapeutics, Inc. L-ornithine phenyl acetate and methods of making thereof
US9604909B2 (en) 2009-04-03 2017-03-28 Ocera Therapeutics, Inc. L-ornithine phenyl acetate and methods of making thereof
US8173706B2 (en) 2009-04-03 2012-05-08 Ocera Therapeutics, Inc. L-ornithine phenyl acetate and methods of making thereof
US10550069B2 (en) 2009-04-03 2020-02-04 Ocera Therapeutics, Inc. L-ornithine phenyl acetate and methods of making thereof
US11266620B2 (en) 2009-06-08 2022-03-08 Ucl Business Ltd Treatment of portal hypertension and restoration of liver function using L-ornithine phenylacetate
US8946473B2 (en) 2010-10-06 2015-02-03 Ocera Therapeutics, Inc. Methods of making L-ornithine phenyl acetate
US9260379B2 (en) 2010-10-06 2016-02-16 Ocera Therapeutics, Inc. Methods of making L-ornithine phenyl acetate
US10525029B2 (en) 2014-11-24 2020-01-07 Ucl Business Ltd Treatment of diseases associated with hepatic stellate cell activation using ammonia-lowering therapies
US10039735B2 (en) 2014-11-24 2018-08-07 Ucl Business Plc Treatment of diseases associated with hepatic stellate cell activation using ammonia-lowering therapies
US11040021B2 (en) 2014-11-24 2021-06-22 Ucl Business Ltd Treatment of diseases associated with hepatic stellate cell activation using ammonia-lowering therapies
US10835506B2 (en) 2015-08-18 2020-11-17 Ocera Therapeutics, Inc. Treatment and prevention of muscle loss using L-ornithine in combination with at least one of phenylacetate and phenylbutyrate
US11066352B2 (en) 2017-05-11 2021-07-20 Ocera Therapeutics, Inc. Processes of making L-ornithine phenylacetate

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