Alternative oxidase and uses thereof

Abstract

The invention relates to a method for combating disorders affecting the mitochondrial oxidative phosphorylation system by allotopic expression of the cyanide-insensitive alternative oxidase (AOX) in human cells. The successful expression of AOX in human cells and in Drosophila has been shown to confer spectacular cyanide-resistance to mitochondrial substrate oxidation, alleviate oxidative stress, apoptosis susceptibility and metabolic acidosis. AOX is well tolerated when expressed ubiquitously in the whole organism. AOX expression is a valuable tool to limit the deleterious consequences of respiratory chain deficiency.

1 - 2 . (canceled) 
     
     
         3 . The polynucleotide of  claim 11 , wherein the polypeptide is derived from an organism from a phylum selected from the group consisting of Mollusca, Nematoda and Chordata. 
     
     
         4 . The polynucleotide of  claim 11 , wherein the polypeptide is derived from an organism from subphylum Urochordata. 
     
     
         5 . The polynucleotide of  claim 11 , wherein the polypeptide is derived from an organism from class Ascidiacea. 
     
     
         6 . The polynucleotide of claim  1 , wherein the polypeptide is derived from an organism from order Eiiterogona. 
     
     
         7 . The polynucleotide of  claim 11 , wherein the polypeptide is derived from an organism from family Cionidae. 
     
     
         8 . The polynucleotide of  claim 11 , wherein the polypeptide is derived from  Ciona intestinalis.    
     
     
         9 - 10 . (canceled) 
     
     
         11 . An isolated or purified polynucleotide comprising a nucleotide sequence that encodes a_polypeptide that comprises an amino acid sequence that is at least 75% identical to an amino acid sequence selected from the group consisting of:
 (a) SEQ ID NO: 2; and   (b) fragments of (a) that have alternative oxidase activity;   wherein the polypeptide has alternative oxidase activity.   
     
     
         12 . The polynucleotide of  11 , wherein the polynucleotide comprises a nucleotide sequence at least 85% identical to the nucleotide sequence set forth in SEQ ID NO: 1. 
     
     
         13 . A polynucleotide that comprises a nucleotide sequence that encodes a chimeric polypeptide that comprises an amino acid sequence of a mitochondrial transit peptide of animal origin fused to an amino acid sequence of a polypeptide with alternative oxidase activity of plant, fungal, or protist origin. 
     
     
         14 - 18 . (canceled) 
     
     
         19 . A polynucleotide that comprises a nucleotide sequence that encodes a chimeric polypeptide that comprises an amino acid sequence of a mitochondrial transit peptide of vertebrate origin and an amino acid of a polypeptide with alternative oxidase activity of invertebrate origin. 
     
     
         20 - 21 . (canceled) 
     
     
         22 . A polynucleotide according to  claim 11 , further comprising a promoter sequence that promotes expression of the polynucleotide in a mammalian cell. 
     
     
         23 . A polynucleotide according to  claim 22 , wherein the promoter is of mammalian origin. 
     
     
         24 . A polynucleotide according to  claim 23 , wherein the promoter is a promoter of a nuclear gene that encodes a mitochondrial protein. 
     
     
         25 . A vector comprising the polynucleotide of  claim 11 . 
     
     
         26 . A vector comprising the polynucleotide of  claim 11  operably linked to an expression control sequence. 
     
     
         27 . A vector of  claim 26  selected from the group consisting of replication deficient adenoviral vectors, adeno-associated viral vectors, and lentivirus vectors. 
     
     
         28 . A composition comprising the polynucleotide of  claim 11  and a pharmaceutically acceptable carrier, diluent or excipient. 
     
     
         29 . A composition comprising the vector of  claim 27  and a pharmaceutically acceptable carrier, diluent or excipient. 
     
     
         30 . A cell transformed or transfected with the polynucleotide of any one of  claims 11 ,  13 ,  18  and  19 , or with the vector of  claim 26 , wherein the cell expresses the polypeptide and exhibits one or more of the following properties compared to a wild type cell:
 (a) increased resistance to antimycin A;   (b) increased resistance to cyanide (CN − ); and   (c) increased resistance to oligomycin.   
     
     
         31 - 32 . (canceled) 
     
     
         33 . A vertebrate cell transformed or transfected with a polynucleotide that comprises a nucleotide sequence that encodes a polypeptide with alternative oxidase activity, wherein the cell expresses the polypeptide and exhibits one or more of the following properties compared to a wild type cell:
 (a) increased resistance to antimycin A;   (b) increased resistance to cyanide (CN − ); and   (c) increased resistance to oligomycin.   
     
     
         34 . A vertebrate cell transformed or transfected with a polynucleotide that comprises a nucleotide sequence that encodes a polypeptide with alternative oxidase activity,
 wherein the cell expresses the polypeptide;   wherein the cell exhibits oxidative phosphorylation to produce ATP through the cytochrome metabolic pathway; and   wherein, in the presence of an inhibitor of said oxidative phosphorylation, the cell oxidizes ubiquinol through the alternative oxidase pathway.   
     
     
         35 . A vertebrate cell transformed or transfected with a polynucleotide that comprises a nucleotide sequence that encodes a polypeptide with alternative oxidase activity,
 wherein the cell expresses the polypeptide;   wherein the cell exhibits oxidative phosphorylation to produce ATP through the cytochrome metabolic pathway; and   wherein the alternative oxidase activity is allosterically regulated by pyruvate to inhibit metabolic acidosis under conditions favoring metabolic acidosis.   
     
     
         36 . An isolated or purified cell according to any one of  claims 33 - 35 . 
     
     
         37 . An isolated cell according to  claim 36  that is a stem cell. 
     
     
         38 . An isolated cell according to  claim 37 , wherein the stem cell is selected from the group consisting of an embryonic stem cell, an adult stem cell and a neural stem cell. 
     
     
         39 . (canceled) 
     
     
         40 . A method for decreasing metabolic acidosis in a mammalian cell, wherein the method comprises transforming or transfecting the cell with an agent selected from the group consisting of:
 (a) a polynucleotide that comprises a nucleotide sequence that encodes a polypeptide with alternative oxidase activity; and   (b) a vector comprising said polynucleotide.   
     
     
         41 . (canceled) 
     
     
         42 . A method for palliating oxidative stress in a mammalian cell, wherein the method comprises transforming or transfecting the cell with an agent selected from the group consisting of:
 (a) a polynucleotide that comprises a nucleotide sequence that encodes a polypeptide with alternative oxidase activity; and   (b) a vector comprising said polynucleotide.   
     
     
         43 . (canceled) 
     
     
         44 . A method for decreasing metabolic acidosis in a mammalian subject, wherein the method comprises administering to a mammalian subject in need of treatment for metabolic acidosis a composition comprising an agent selected from the group consisting of:
 (a) a polynucleotide that comprises a nucleotide sequence that encodes a polypeptide with alternative oxidase activity;   (b) a vector comprising said polynucleotide; and   (c) a cell according to any one of  claims 33 - 35 ;   wherein the composition is administered in an amount effective to reduce metabolic acidosis in cells of the mammalian subject.   
     
     
         45 . (canceled) 
     
     
         46 . A method for decreasing oxidative stress in a mammalian subject, wherein the method comprises administering to a mammalian subject in need of treatment for oxidative stress a composition comprising an agent selected from the group consisting of:
 (a) a polynucleotide that comprises a nucleotide sequence that encodes a polypeptide with alternative oxidase activity;   (b) a vector comprising said polynucleotide; and   (c) a cell according to any one of  claims 33 - 35 ;   wherein the composition is administered in an amount effective to reduce oxidative stress in cells of the mammalian subject.   
     
     
         47 . The method of  claim 46 , wherein the mammalian subject is a human. 
     
     
         48 . The method of  claim 47 , wherein the human subject has a disease or condition of impaired mitochondrial respiratory function selected from the group consisting of Leigh syndrome; MERRF syndrome; Parkinson's Disease and related conditions; Mitochondrial encephalomyopathies, including progressive external opthalmoplegia, Kearns-Sayre syndrome and MELAS syndrome; diverse, multisystem pediatric disorders affecting organs such as liver, kidney, the CNS, heart, skeletal muscle, and the endocrine and sensorineural systems; diseases whose pathogenesis is known or believed to involve excessive production of reactive oxygen species in mitochondria, including amyotrophic lateral sclerosis, Alzheimer's disease, Friedreich ataxia and forms of cardiovascular disease attributable to defects in antioxidant defenses; other ataxias and neurological conditions resulting from genetics defects in POLG, c10orf2 (Twinkle) or other components of the system of mitochondrial DNA maintenance; mitochondrial hearing impairment, both syndromic and nonsyndromic; forms of diabetes mellitus attributable to defects of the mitochondrial OXPHOS system; side-effects of antiretroviral therapies that impact the mitochondrial OXPHOS system; intractable obesity and other metabolic disorders resulting from disturbances in the mobilization of food resources; NARP syndrome; Alpers-Huttenlocher disease; sensorineural deafness; benign infantile myopathy; fatal infantile myopathy; pediatric myopathy; adult myopathy; Rhabdmyolysis; Leber Hereditary Optic Neuropathy; cardiomyopathy; Barth syndrome; Fanconi syndrome; mtDNA depletion syndrome; Pearson syndrome; Diabetes mellitus and Lactic acidemia. 
     
     
         49 . The method of  claim 44 , wherein the composition is administered locally to a tissue or an organ comprising cells affected by metabolic acidosis or oxidative stress. 
     
     
         50 . The method of  claim 44 , wherein the composition is administered systemically. 
     
     
         51 . A method for reducing mitochondrial sensitivity to cyanide, wherein the method comprises introducing a polynucleotide that comprises a nucleotide sequence that encodes a polypeptide with alternative oxidase and a vector comprising said polynucleotide and allowing allotopic expression of AOX in a human or other metazoan cell. 
     
     
         52 . A method for treating pathological manifestations arising from a defect in the mitochondrial oxidative phosphorylation system, wherein the method comprises introducing a polynucleotide that comprises a nucleotide sequence that encodes a polypeptide with alternative oxidase and a vector comprising said polynucleotide and allowing allotopic expression of AOX encoded by the polynucleotide in a human or other metazoan cell.