Methods and compositions for treating mitochondrial disease or disorders and heteroplasmy

Abstract

The present invention provides methods and compositions for generation of mitochondria replaced cells (MirC), and therapeutic methods for using such compositions for treating a subject having an age-related disease or syndrome, mitochondrial disease or disorder, or otherwise in need of mitochondrial replacement. Also provided are methods and compositions for producing a recipient cell having a mitochondrial disease or disorder, as well as methods and compositions for producing or enhancing production of an inducible pluripotent stem cell (iPSC). In addition, methods and compositions to enhance mitochondrial transfer are also included.

1 . A method of generating a mitochondria replaced cell, comprising:
 (a) contacting a recipient cell with an agent that reduces endogenous mtDNA copy number   (b) incubating the recipient cell for a sufficient period of time for the agent to partially reduce the endogenous mtDNA copy number in the recipient cell; and   (c) co-incubating (1) the recipient cell from step (b) in which the endogenous mtDNA has been partially reduced, and (2) exogenous mitochondria or exogenous mtDNA from a healthy donor, for a sufficient period of time to non-invasively transfer the exogenous mitochondria or the exogenous mtDNA into the recipient cell, thereby generating a mitochondria replaced cell.   
     
     
         2 . A method of treating a subject in need of mitochondrial replacement, a subject having or suspected of having an age-related disease, or a subject having a mitochondrial disease or disorder, comprising:
 (a) generating a mitochondria replaced cell ex vivo or in vitro, comprising the steps of:
 (i) contacting a recipient cell with an agent that reduces mtDNA copy number; 
 (ii) incubating the recipient cell for a sufficient period of time for the agent to partially reduce the mtDNA copy number in the recipient cell; and 
 (iii) co-incubating (1) the recipient cell from step (ii) in which the endogenous mtDNA has been partially reduced, and (2) exogenous mitochondria from a healthy donor, for a sufficient period of time to non-invasively transfer exogenous mitochondria into the recipient cell or exogenous mtDNA from a healthy donor, for a sufficient period of time to non-invasively transfer exogenous mtDNA into the recipient cell, thereby generating a mitochondria replaced cell, thereby generating a mitochondria replaced cell; 
   (b) administering a therapeutically effective amount of the mitochondria replaced recipient cell from step (a) to the subject in need of mitochondrial replacement.   
     
     
         3 - 4 . (canceled) 
     
     
         5 . The method of  claim 1  or  2 , wherein
 (a) the exogenous mitochondria comprises:
 (i) a functional mitochondria; 
 (ii) wild-type mtDNA; 
 (iii) isolated mitochondria, wherein the isolated mitochondria is optionally an intact mitochondria; and/or 
 (iv) allogeneic mitochondria; and 
 
 (b) the endogenous mtDNA:
 (i) encodes for a dysfunctional mitochondria; 
 (ii) comprises mutant mtDNA; 
 (iii) comprises mtDNA associated with a mitochondrial disease or disorder; 
 (iv) is heteroplasmic; or 
 (v) comprises wild-type mtDNA; and/or 
 
 (c) the endogenous mitochondria is dysfunctional. 
 
     
     
         6 - 13 . (canceled) 
     
     
         14 . The method of  claim 1  or  2 , wherein the agent that reduces endogenous mtDNA copy number is selected from the group consisting of a polynucleotide encoding a fusion protein comprising a mitochondrial-targeted sequence (MTS) and an endonuclease, a polynucleotide encoding an endonuclease, and a small molecule,
 wherein the small molecule is optionally a nucleoside reverse transcriptase inhibitor (NRTI); 
 wherein the polynucleotide is optionally comprised of messenger ribonucleic acid (mRNA) or deoxyribonucleic acid (DNA); 
 wherein the recipient cell optionally transiently expresses the fusion protein; 
 wherein the endonuclease is optionally selected from the group consisting of XbaI, EcoRI, BamHI, HindIII, PstI, Cas9, zinc finger nuclease (ZFN), and transcription activator-like effector nuclease (TALEN); 
 wherein the MTS optionally targets a mitochondrial matrix protein; and 
 wherein the mitochondrial matrix protein is optionally selected from the group consisting of cytochrome c oxidase subunit IV, cytochrome c oxidase subunit VIII, and cytochrome c oxidase subunit X. 
 
     
     
         15 - 20 . (canceled) 
     
     
         21 . The method of  claim 1  or  2 , wherein the agent that reduces endogenous mtDNA copy number reduces
 (a) about 5% to about 99% of the endogenous mtDNA copy number; 
 (b) about 30% to about 70% of the endogenous mtDNA copy number; 
 (c) about 50% to about 95% of the endogenous mtDNA copy number; 
 (d) about 60% to about 90% of the endogenous mtDNA copy number; or 
 (e) mitochondrial mass. 
 
     
     
         22 - 29 . (canceled) 
     
     
         30 . The method of  claim 2 , wherein
 (a) the subject in need of mitochondrial replacement has a dysfunctional mitochondria; a disease selected from the group consisting of an age-related disease, a mitochondrial disease or disorder, a neurodegenerative disease, a retinal disease, diabetes, a hearing disorder, a genetic disease; or a combination thereof,
 wherein the neurodegenerative disease is optionally selected from the group consisting of amyotrophic lateral sclerosis (ALS), Huntington's disease, Alzheimer's disease, Parkinson's disease, Friedreich's ataxia, Charcot Marie Tooth disease and leukodystrophy, and 
 wherein the retinal disease is optionally selected from the group consisting of age-related macular degeneration, macular edema and glaucoma; 
   (b) the age-related disease is selected from the group consisting of an autoimmune disease, a metabolic disease, a genetic disease, cancer, a neurodegenerative disease, and immunosenescence,
 wherein the metabolic disease is optionally diabetes, 
 wherein the neurodegenerative disease is Alzheimer's disease, or Parkinson's disease, and 
 wherein the genetic disease is optionally selected from the group consisting of Hutchinson-Gilford Progeria Syndrome, Werner Syndrome, and Huntington's disease; 
   (c) the mitochondrial disease or disorder is caused by mitochondrial DNA abnormalities, nuclear DNA abnormalities, or both,
 wherein the mitochondrial disease or disorder caused by mitochondrial DNA abnormalities is optionally selected from the group consisting of chronic progressive external ophthalmoplegia (CPEO), Pearson syndrome, Kearns-Sayre syndrome (KSS), diabetes and deafness (DAD), mitochondrial diabetes, Leber hereditary optic neuropathy (LHON), LHON-plus, neuropathy, ataxia, and retinitis pigmentosa syndrome (NARP), maternally-inherited Leigh syndrome (MILS), mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS), myoclonic epilepsy and ragged-red fiber disease (MERRF), familial bilateral striatal necrosis/striatonigral degeneration (FBSN), Luft disease, aminoglycoside-induced Deafness (AID), and multiple deletions of mitochondrial DNA syndrome, and 
 wherein the mitochondrial disease or disorder caused by nuclear DNA abnormalities is selected from the group consisting of Mitochondrial DNA depletion syndrome-4A, mitochondrial recessive ataxia syndrome (MIRAS), mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), mitochondrial DNA depletion syndrome (MTDPS), DNA polymerase gamma (POLG)-related disorders, sensory ataxia neuropathy dysarthria ophthalmoplegia (SANDO), leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation (LBSL), co-enzyme Q10 deficiency, Leigh syndrome, mitochondrial complex abnormalities, fumarase deficiency, α-ketoglutarate dehydrogenase complex (KGDHC) deficiency, succinyl-CoA ligase deficiency, pyruvate dehydrogenase complex deficiency (PDHC), pyruvate carboxylase deficiency (PCD), carnitine palmitoyltransferase I (CPT I) deficiency, carnitine palmitoyltransferase II (CPT II) deficiency, carnitine-acyl-carnitine (CACT) deficiency, autosomal dominant-/autosomal recessive-progressive external ophthalmoplegia (ad-/ar-PEO), infantile onset spinal cerebellar atrophy (IOSCA), mitochondrial myopathy (MM) spinal muscular atrophy (SMA), growth retardation, aminoaciduria, cholestasis, iron overload, early death (GRACILE), and Charcot-Marie-Tooth disease type 2A (CMT2A). 
   
     
     
         31 - 45 . (canceled) 
     
     
         46 . The method of  claim 1  or  2 , wherein the mitochondria replaced cell has a total mtDNA copy number no greater than about 1.1 fold, about 1.2 fold, about 1.3 fold, about 1.4 fold, about 1.5 fold, or more, relative to the total mtDNA copy number of the recipient cell prior to contacting with the agent that reduces endogenous mtDNA copy number. 
     
     
         47 . The method of  claim 1  or  2 , wherein the recipient cell is:
 (a) an animal cell or a plant cell,
 wherein animal cell is optionally a mammalian cell, 
 wherein the mammalian cell is optionally a somatic cell or a bone marrow cell, and 
 wherein the bone marrow cell is optionally a hematopoietic stem cell (HSC), or a mesenchymal stem cell (MSC); 
 
 (b) a cancer cell; 
 (c) a primary cell; 
 (d) an immune cell,
 wherein the immune cell is optionally selected from the group consisting of a T cell, a phagocyte, a microglial cell, and a macrophage, and the T cell is optionally a CD4+ T cell, a CD8+ T cell, or a chimeric antigen receptor (CAR) T cell; or 
 
 (e) a senescent or near senescent cell. 
 
     
     
         48 - 58 . (canceled) 
     
     
         59 . The method of  claim 1  or  2 , wherein transfer of the exogenous mitochondria and/or exogenous mtDNA is stable,
 wherein the exogenous mtDNA optionally alters heteroplasmy in the recipient cell. 
 
     
     
         60 . (canceled) 
     
     
         61 . The method of  claim 1  or  2 , further comprising:
 (a) delivering a small molecule, a peptide, or a protein; and/or 
 (b) contacting the recipient cell with a second active agent prior to co-incubating the recipient cell with exogenous mitochondria and/or exogenous mtDNA, 
 wherein the second active agent is optionally selected from the group consisting of large molecules, small molecules, or cell therapies, and the second active agent is optionally selected from the group consisting of rapamycin, NR (Nicotinamide Riboside), bezafibrate, idebenone, cysteamine bitartrate (RP103), elamipretide (MTP131), omaveloxolone (RTA408), KH176, Vatiquinone (Epi743), thioctic acid, A0001 (alpha-tocopherolquinone), mitochondrial CoQ10 (MitoQ), SkQ1 (Visomitin), resveratrol, curcumin, ketogenic treatment, hypoxia, and an activator of endocytosis 
 wherein the activator of endocytosis is optionally a modulator of cellular metabolism, 
 wherein the modulator of cellular metabolism optionally comprises nutrient starvation, a chemical inhibitor, or a small molecule, 
 wherein the chemical inhibitor or the small molecule is optionally an mTOR inhibitor, and 
 wherein said mTOR inhibitor optionally comprises rapamycin or a derivative thereof. 
 
     
     
         62 - 67 . (canceled) 
     
     
         68 . A composition comprising one or more mitochondria replaced cells obtained by the method of:
 (a) contacting a recipient cell with an agent that reduces endogenous mtDNA copy number;   (b) incubating the recipient cell for a sufficient period of time for the agent to partially reduce the endogenous mtDNA copy number in the recipient cell; and   (c) co-incubating (1) the recipient cell from step (b) in which the endogenous mtDNA has been partially reduced, and (2) exogenous mitochondria or exogenous mtDNA from a healthy donor, for a sufficient period of time to non-invasively transfer the exogenous mitochondria or the exogenous mtDNA into the recipient cell, thereby generating a mitochondria replaced cell,   wherein said mitochondria replaced cell comprises greater than 5% of exogenous mtDNA,   wherein said one or more mitochondria replaced cells optionally comprise a total mtDNA copy number no greater than about 1.1 fold, about 1.2 fold, about 1.3 fold, about 1.4 fold, about 1.5 fold, or more, relative to the total mtDNA copy number of the recipient cell prior to contacting with the agent that reduces endogenous mtDNA copy number,   wherein the one or more mitochondria replaced cells optionally comprise wild-type exogenous mtDNA,   wherein the exogenous mitochondria is optionally isolated mitochondria, and the isolated mitochondria is optionally intact, and   wherein the exogenous mitochondria optionally further comprises exogenous mtDNA.   
     
     
         69 - 70 . (canceled) 
     
     
         71 . A composition comprising an agent that reduces endogenous mtDNA copy number, and a second active agent,
 wherein the composition optionally further comprises, one or more recipient cells, exogenous mtDNA, and/or exogenous mitochondria.   
     
     
         72 - 73 . (canceled) 
     
     
         74 . The composition of  claim 68  or  71 , wherein the agent that reduces endogenous mtDNA copy number is:
 (a) a small molecule,
 wherein the small molecule is optionally a nucleoside reverse transcriptase inhibitor (NRTI); or 
 
 (b) a fusion protein,
 wherein the fusion protein optionally comprises an endonuclease that cleaves mtDNA and a mitochondrial target sequence (MTS), 
 wherein the endonuclease optionally cleaves wild-type mtDNA, and is optionally selected from the group consisting of XbaI, EcoRI, BamHI, HindIII, PstI, Cas9, zinc finger nuclease (ZFN), and transcription activator-like effector nuclease (TALEN), 
 wherein the MTS optionally targets a mitochondrial matrix protein, and the mitochondrial matrix protein is optionally selected from the group consisting of cytochrome c oxidase subunit IV, cytochrome c oxidase subunit VIII, and cytochrome c oxidase subunit X, and/or 
 wherein the fusion protein is optionally transiently expressed. 
 
 
     
     
         75 - 81 . (canceled) 
     
     
         82 . The composition of  claim 68  or  71 , wherein said reduction of endogenous mtDNA copy number is a partial reduction,
 wherein the partial reduction is optionally a reduction of:
 (a) about 5% to about 99% of endogenous mtDNA; 
 (b) about 50% to about 95% of the endogenous mtDNA copy number; or 
 (c) about 60% to about 90% of the endogenous mtDNA copy number. 
 
 
     
     
         83 - 92 . (canceled) 
     
     
         93 . The composition of  claim 68  or  71 , further comprising a second active agent,
 wherein the second active agent is optionally selected from the group consisting of large molecules, small molecules, or cell therapies, and the second active agent is optionally selected from the group consisting of rapamycin, NR (Nicotinamide Riboside), bezafibrate, idebenone, cysteamine bitartrate (RP103), elamipretide (MTP131), omaveloxolone (RTA408), KH176, Vatiquinone (Epi743), thioctic acid, A0001 (alpha-tocopherolquinone), mitochondrial CoQ10 (MitoQ), SkQ1 (Visomitin), resveratrol, curcumin, ketogenic treatment, hypoxia, and an activator of endocytosis, 
 wherein the activator of endocytosis is optionally an activator of a clathrin-independent endocytosis pathway, 
 wherein the activator of endocytosis is optionally an activator of a clathrin-independent endocytosis pathway, 
 wherein the clathrin-independent endocytosis pathway is optionally selected from the group consisting of a CLIC/GEEC endocytic pathway, Arf6-dependent endocytosis, flotillin-dependent endocytosis, macropinocytosis, circular doral ruffles, phagocytosis, and trans-endocytosis, 
 wherein the clathrin-independent endocytosis pathway is optionally macropinocytosis, 
 wherein said activator of endocytosis optionally comprises nutrient stress, and/or an mTOR inhibitor, and 
 wherein said mTOR inhibitor optionally comprises rapamycin or a derivative thereof. 
 
     
     
         94 - 100 . (canceled) 
     
     
         101 . The composition of  claim 68  or  71 , wherein the total mtDNA copy number of the one or more mitochondria replaced cells comprises:
 (a) greater than 5% of exogenous mtDNA; 
 (b) greater than 30% of exogenous mtDNA; 
 (c) greater than 50% of exogenous mtDNA, or 
 (d) greater than 75% of exogenous mtDNA. 
 
     
     
         102 - 106 . (canceled) 
     
     
         107 . The composition of  claim 68  or  71 , wherein the exogenous mitochondria and/or exogenous mtDNA is optionally allogeneic. 
     
     
         108 . (canceled) 
     
     
         109 . The composition of  claim 68  or  71 , wherein the one or more cells are animal cells or plant cells,
 wherein the animal cells are optionally mammalian cells, and the mammalian cells are optionally somatic cells, and 
 wherein the somatic cells are optionally:
 (a) epithelial cells,
 wherein the epithelial cells are thymic epithelial cells (TECs), or 
 
 (b) immune cells
 wherein the immune cells are optionally phagocytic cells or T cells, and the T cells are optionally CD4+ T cells, CD8+ T cells, or chimeric antigen receptor (CAR) T cells. 
 
 
 
     
     
         110 - 119 . (canceled) 
     
     
         120 . The composition of  claim 68  or  71 , wherein the one or more mitochondria replaced cells are:
 (a) bone marrow cells,
 wherein the bone marrow cells are optionally a hematopoietic stem cell (HSC), or a mesenchymal stem cell (MSC); 
 (b) more viable than an isogenic cell having homoplasmic endogenous mtDNA; and/or 
 (c) efficacious in killing a cancer cell, treating an age-related disease, treating a mitochondrial disease or disorder, treating a neurodegenerative disease, treating diabetes, or a genetic disease. 
 
 
     
     
         121 - 123 . (canceled) 
     
     
         124 . The composition of  claim 68  or  71 , further comprising a small molecule, a peptide, or a protein. 
     
     
         125 . A composition comprising:
 (a) a senescent or near senescent cell having endogenous mitochondria;   (b) isolated exogenous mitochondria from a non-senescent cell,
 wherein the exogenous mitochondria from the non-senescent cell optionally has enhanced function relative to the endogenous mitochondria; and 
   (c) an agent that reduces endogenous mtDNA copy number,
 wherein the agent is optionally a fusion protein, 
 wherein the fusion protein optionally comprises an endonuclease that cleaves mtDNA and a mitochondrial target sequence (MTS), 
 wherein the endonuclease optionally cleaves wild-type mtDNA, and is optionally selected from the group consisting of XbaI, EcoRI, BamHI, HindIII, PstI, Cas9, zinc finger nuclease (ZFN), and transcription activator-like effector nuclease (TALEN), 
 wherein the MTS optionally targets a mitochondrial matrix protein, and the mitochondrial matrix protein is optionally selected from the group consisting of cytochrome c oxidase subunit IV, cytochrome c oxidase subunit VIII, and cytochrome c oxidase subunit X, and/or 
 wherein the fusion protein is optionally transiently expressed in said senescent or near senescent cell. 
   
     
     
         126 - 136 . (canceled) 
     
     
         137 . The composition of  claim 125 , further comprising a second active agent,
 wherein the second active agent is optionally selected from the group consisting of large molecules, small molecules, or cell therapies, and the second active agent is optionally selected from the group consisting of rapamycin, NR (Nicotinamide Riboside), bezafibrate, idebenone, cysteamine bitartrate (RP103), elamipretide (MTP131), omaveloxolone (RTA408), KH176, Vatiquinone (Epi743), thioctic acid, A0001 (alpha-tocopherolquinone), mitochondrial CoQ10 (MitoQ), SkQ1 (Visomitin), resveratrol, curcumin, ketogenic treatment, hypoxia, and an activator of endocytosis,   wherein the activator of endocytosis is optionally an activator of a clathrin-independent endocytosis pathway,   wherein the activator of endocytosis is optionally an activator of a clathrin-independent endocytosis pathway,   wherein the clathrin-independent endocytosis pathway is optionally selected from the group consisting of a CLIC/GEEC endocytic pathway, Arf6-dependent endocytosis, flotillin-dependent endocytosis, macropinocytosis, circular doral ruffles, phagocytosis, and trans-endocytosis,   wherein the clathrin-independent endocytosis pathway is optionally macropinocytosis,   wherein said activator of endocytosis optionally comprises nutrient stress, and/or an mTOR inhibitor, and   wherein said mTOR inhibitor optionally comprises rapamycin or a derivative thereof.   
     
     
         138 - 143 . (canceled) 
     
     
         144 . A pharmaceutical composition comprising an isolated population of mitochondria replaced cells having an exogenous mitochondria or an exogenous mtDNA from a healthy donor, wherein the cells are obtained by the method of  claim 1  that optionally further comprises contacting the recipient cell with a second active agent prior to co-incubating the recipient cell with exogenous mitochondria and/or exogenous mtDNA,
 wherein the cells are optionally T cells or hematopoietic stem cells. 
 
     
     
         145 . (canceled) 
     
     
         146 . The pharmaceutical composition of  claim 144 , further comprising
 (a) exogenous mitochondria, and/or   (b) a pharmaceutically acceptable carrier.   
     
     
         147 - 149 . (canceled)