Patent Application
20200054682
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Aug. 13, 2019, is named 14595-001-999_SL.txt and is 12,905 bytes in size.
The present invention provides a composition of cells with reduced mitochondrial DNA and/or replacement of mitochondrial DNA, methods for their production, and methods for treating various diseases associated with genetic or age-related mitochondrial dysfunctions.
Mitochondria play a major and critical role in cellular homeostasis, and are involved in a diverse range of disease processes. They participate in intracellular signaling, apoptosis and perform numerous biochemical tasks, such as pyruvate oxidation, the Krebs cycle, and metabolism of amino acids, fatty acids, nucleotides and steroids. One crucial task is their role in cellular energy metabolism. This includes β-oxidation of fatty acids and production of ATP by means of the electron-transport chain and the oxidative-phosphorylation system. The mitochondrial respiratory chain consists of five multi-subunit protein complexes embedded in the inner membrane, comprising: complex I (NADH-ubiquinone oxidoreductase), complex II (succinate-ubiquinone oxidoreductase), complex III (ubiquinol-ferricytochrome c oxidoreductase), complex IV (cytochrome c oxidoreductase), and complex V (FIFO ATPase).
The mammalian mitochondrial genome is a small, circular, double-stranded molecule containing 37 genes, including 13 protein-encoding genes, 22 transfer RNA (tRNA) genes and two ribosomal RNA (rRNA) genes. Of these, 24 (22 tRNAs and two rRNAs) are needed for mitochondrial DNA translation, and 13 encode subunits of the respiratory chain complexes. In addition, nuclear DNA (nDNA) encodes most of the approximately 900 gene products in the mitochondria.
Mitochondrial disease or disorders are a clinically heterogeneous group of disorders that are characterized by dysfunctional mitochondria. Disease onset can occur at any age and can manifest with a wide range of clinical symptoms. Mitochondrial disease or disorders can involve any organ or tissue, characteristically involve multiple systems, typically affecting organs that are highly dependent on aerobic metabolism, and are often relentlessly progressive with high morbidity and mortality. Mitochondrial disease or disorders are the most common group of inherited metabolic disorders and are among the most common forms of inherited neurological disorders.
Mitochondrial disease or disorders can be caused by mutations in genes in the nuclear DNA (nDNA) and/or mitochondrial DNA (mtDNA) that encode structural mitochondrial proteins or proteins involved in mitochondrial function. While some mitochondrial disorders only affect a single organ (e.g., the eye in Leber hereditary optic neuropathy [LHON]), many involve multiple organ systems and often present with prominent neurologic and myopathic features. Even though tissues with high energy demand, such as brain, muscle, and eye, are more frequently involved, patients' phenotype can be extremely varied and heterogeneous. This variation is due in part because of several factors, such as, the dual genetic control (nDNA and mtDNA), level of heteroplasmy (percentage of mutated DNA in single cells and tissues), tissue energy demand, maternal inheritance, and mitotic segregation.
Many patients with a mitochondrial disease or disorder have a mixture of mutated and wild-type mtDNA (known as heteroplasmy); the proportion of mutated and wild-type mtDNA is a key factor that determines whether a cell expresses a biochemical defect. The majority of pathogenetic mtDNA mutations are heteroplasmic, with a mixture of mutated and wild-type mtDNA inside an individual cell. High levels of heteroplasmy refer to cells with high levels of mutant mtDNA and low levels of wild-type mtDNA, whereas low levels of heteroplasmy refer to cells with low levels of mutant mtDNA and high levels of wild-type mtDNA. Studies in single cells from patients with mitochondrial disease or disorders have shown that the level of mutated and wild-type mtDNA is very important for determining the cellular phenotype. For example, cells become respiratory deficient if they contain high levels of mutated mtDNA and low levels of wild-type mtDNA (that is, high levels of heteroplasmy). The threshold at which this deficiency occurs depends on the precise mutation and the cell type. Typically, high percentage levels of mutated mtDNA (>50%) are required to result in cellular defects, but some mtDNA mutations only generate a deficiency if present at very high levels (typically mt tRNA mutations) and others (such as single, large-scale mtDNA deletions) produce a deficiency when there is ˜60% deleted mtDNA. For example, in individuals harboring the m.8993T>G pathogenic variant, higher percentage levels of mutated mtDNA are seen in those presenting with Leigh syndrome than in those presenting with neurogenic weakness with ataxia and retinitis pigmentosa (NARP). In addition, clinical phenotypes in MELAS and MERRF correlate with heteroplasmy (see, e.g., Chinnery, P. F., et al., Brain 120 (Pt 10), 1713-1721 (1997)).
Advances in next-generation sequencing technology have revealed many mutations that cause mitochondrial disease or disorders. In addition, investigations into other organisms, such as C. elegans, have revealed some of the proteins involved in heteroplasmy. For example, a recent study using C. elegans demonstrated that mitochondrial unfolded protein response (UPRmt) functions to maintain the heteroplasmy and propagate mutated mtDNA following a disturbance of the original mtDNA (see, e.g., Lin, Y. F. et al. Nature 533, 416-419, doi:10.1038/nature17989 (2016)). However, the mechanism related to heteroplasmy maintenance and propagation in mammalian cells remains unknown.
The management and treatment of patients with mitochondrial disease or disorders remains challenging. For the vast majority of patients, the condition is relentlessly progressive leading to considerable morbidity and, in those most severely affected, death. The classical method to remove endogenous mtDNA involves long term treatment of cells with low concentrations of ethidium bromide (EtBr), a known carcinogen and teratogen, limiting its application for therapeutic purposes. In addition to the potential for unwanted side effects, the EtBr protocol can take several months, which further limits its clinical use. Moreover, mitochondrial transfer protocols generally involve a complete depletion of endogenous mtDNA, termed rho (ρ) 0 cells, before transfer of exogenous mitochondria. This complete depletion of mtDNA severely hinders the ability of a cell to ingest exogenous mitochondria.
Other mitochondrial transfer protocols have attempted to add mitochondria without depletion of endogenous mtDNA, but this approach has been found to be inefficient or harmful to a cell. For example, mitochondrial transfer using simple coincubation has been reported to be ineffective and not equally efficient among different cell types. Additional techniques to transfer have involved injection using invasive instruments, which caused harm to the recipient cell, or other invasive instruments, such as nanoblades, but all were less efficient than coincubation (Caicedo et al, Stem Cells International, (2017), vol. 2017, Article ID 7610414, 23 pages).
Accordingly, current methods of mitochondrial transfer are not only impractical for the clinical setting, but they are also inefficient, harmful to recipient cells and/or time intensive. Thus, there is a significant unmet need to develop improved methods for mitochondrial transfer that can be optionally used in the treatment of a subject having or suspected having mitochondrial disease or disorders, and diseases or disorders associated with impaired or dysfunctional mitochondria, as well as improved models for studying mitochondrial disease or disorders.
In one aspect, provided herein is 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 from a healthy donor, for a sufficient period of time to non-invasively transfer exogenous mitochondria into the recipient cell, thereby generating a mitochondria replaced cell.
In another aspect, provided herein is a method of treating a subject in need of mitochondrial replacement, 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, thereby generating a mitochondria replaced cell; and (b) administering a therapeutically effective amount of the mitochondria replaced recipient cell from step (a) to the subject in need of mitochondrial replacement.
In yet another aspect, provided herein is a method of treating a subject having or suspected of having an age-related disease, the method 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, thereby generating a mitochondria replaced cell; and (b) administering a therapeutically effective amount of the mitochondria replaced recipient cell from step (a) to the subject having or suspected of having an age-related disease.
In a further aspect, provided herein is a method of treating a subject having or suspected of having a mitochondrial disease or disorder, the method comprising: (a) generating a mitochondria replaced recipient 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, thereby generating a mitochondria replaced cell; and (b) administering a therapeutically effective amount of the mitochondria replaced recipient cell from step (a) to the subject having or suspected of having a mitochondrial disease or disorder.
In some embodiments of the methods provided herein, the exogenous mitochondria is a functional mitochondria. In certain embodiments, the exogenous mitochondria comprises wild-type mtDNA. In specific embodiments, the exogenous mitochondria is isolated mitochondria. In further embodiments, the isolated mitochondria is an intact mitochondria. In some embodiments, the exogenous mitochondria is allogeneic.
Also provided herein is 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 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.
The disclosure also provides a method of treating a subject in need of mitochondrial replacement, 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 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; and (b) administering a therapeutically effective amount of the mitochondria replaced recipient cell from step (a) to the subject in need of mitochondrial replacement.
In another aspect, provided herein is a method of treating a subject having or suspected of having an age-related disease, the method 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 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; and (b) administering a therapeutically effective amount of the mitochondria replaced recipient cell from step (a) to the subject having or suspected of having an age-related disease.
In yet another aspect, provided herein is a method of treating a subject having or suspected of having a mitochondrial disease or disorder, the method comprising: (a) generating a mitochondria replaced recipient 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 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; and (b) administering a therapeutically effective amount of the mitochondria replaced recipient cell from step (a) to the subject having or suspected of having a mitochondrial disease or disorder.
In certain embodiments of the methods provided herein, 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. In some embodiments, the small molecule is a nucleoside reverse transcriptase inhibitor (NRTI). In other embodiments, the polynucleotide is comprised of messenger ribonucleic acid (mRNA) or deoxyribonucleic acid (DNA). In further embodiments, the recipient cell transiently expresses the fusion protein. In yet further embodiments, the endonuclease is selected from the group consisting of XbaI, EcoRI, BamHI, HindIII, PstI, Cas9, zinc finger nuclease (ZFN), and transcription activator-like effector nuclease (TALEN). In some embodiments, the MTS targets a mitochondrial matrix protein. In specific embodiments, the mitochondrial matrix protein is selected from the group consisting of cytochrome c oxidase subunit IV, cytochrome c oxidase subunit VIII, and cytochrome c oxidase subunit X.
In some embodiments of the methods provided herein, the agent that reduces endogenous mtDNA copy number reduces about 5% to about 99% of the endogenous mtDNA copy number. In certain embodiments, the agent that reduces endogenous mtDNA copy number reduces about 30% to about 70% of the endogenous mtDNA copy number. In further embodiments, the agent that reduces endogenous mtDNA copy number reduces about 50% to about 95% of the endogenous mtDNA copy number. In yet further embodiments, the agent that reduces endogenous mtDNA copy number reduces about 60% to about 90% of the endogenous mtDNA copy number. In some embodiments, the agent that reduces endogenous mtDNA copy number reduces mitochondrial mass.
Also provided herein is a method of generating a mitochondria replaced cell, comprising: (a) contacting a recipient cell with an agent that reduces mitochondrial function; (b) incubating the recipient cell for a sufficient period of time for the agent to partially reduce the endogenous mitochondrial function in the recipient cell; and (c) co-incubating (1) the recipient cell from step (b) in which the endogenous mitochondrial function 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, thereby generating a mitochondria replaced cell.
The present disclosure also provides a method of generating a mitochondria replaced cell, comprising: (a) contacting a recipient cell with an agent that reduces mitochondrial function; (b) incubating the recipient cell for a sufficient period of time for the agent to partially reduce the endogenous mitochondrial function in the recipient cell; and (c) co-incubating (1) the recipient cell from step (b) in which the endogenous mitochondrial function has been partially reduced, and (2) 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.
In some embodiments of the methods provided herein, the agent that reduces mitochondrial function transiently reduces endogenous mitochondrial function. In other embodiments, the agent that reduces mitochondrial function permanently reduces endogenous mitochondrial function.
In certain embodiments of the methods provided herein, 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. In some embodiments, the neurodegenerative disease is 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. In specific embodiments, the retinal disease is selected from the group consisting of age-related macular degeneration, macular edema and glaucoma.
In some embodiments of the methods provided herein, 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. In certain embodiments of the methods provided herein, the metabolic disease is diabetes. In further embodiments, the neurodegenerative disease is Alzheimer's disease, or Parkinson's disease. In yet further embodiments, the genetic disease is selected from the group consisting of Hutchinson-Gilford Progeria Syndrome, Werner Syndrome, and Huntington's disease.
In certain embodiments of the methods provided herein, the mitochondrial disease or disorder is caused by mitochondrial DNA abnormalities, nuclear DNA abnormalities, or both. In specific embodiments, the mitochondrial disease or disorder caused by mitochondrial DNA abnormalities is 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. In yet other specific embodiments, 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 (LB SL), 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).
In some embodiments of the methods provided herein the endogenous mtDNA encodes for a dysfunctional mitochondria. In specific embodiments, the endogenous mtDNA comprises mutant mtDNA. In other embodiments, the endogenous mtDNA in the recipient cell comprises wild-type mtDNA. In yet further embodiments, the endogenous mtDNA comprises mtDNA associated with a mitochondrial disease or disorder. In some embodiments, the endogenous mtDNA is heteroplasmic. In specific embodiments, the recipient cell has endogenous mitochondria that is dysfunctional.
In certain embodiments of the methods provided herein, 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.
In some embodiments, the recipient cell is an animal cell or a plant cell. In certain embodiments, the animal cell is a mammalian cell. In specific embodiments, the recipient cell is a somatic cell. In other embodiments, the recipient cell is a bone marrow cell. In some embodiments, the bone marrow cell is a hematopoietic stem cell (HSC), or a mesenchymal stem cell (MSC). In other embodiments, the recipient cell is a cancer cell. In further embodiments, the recipient cell is a primary cell. In yet further embodiments, the recipient cell is an immune cell. In specific embodiments, the immune cells is selected from the group consisting of a T cell, a phagocyte, a microglial cell, and a macrophage. In further embodiments, the T cell is a CD4+ T cells. In other embodiments, the T cell is a CD8+ T cells. In certain embodiments, the T cell is a chimeric antigen receptor (CAR) T cell.
In another embodiment of the methods provided herein, the exogenous mitochondria and/or exogenous mtDNA is stable. In some embodiments, the exogenous mtDNA alters heteroplasmy in the recipient cell.
In some aspects of the methods provided herein, the method further comprises delivering a small molecule, a peptide, or a protein.
The disclosure also provides methods provided herein, further comprising contacting the recipient cell with a second active agent prior to co-incubating the recipient cell with exogenous mitochondria and/or exogenous mtDNA. In certain embodiments, the second active agent is 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. In some embodiments, the activator of endocytosis is a modulator of cellular metabolism. In specific embodiments, the modulator of cellular metabolism comprises nutrient starvation, a chemical inhibitor, or a small molecule. In yet further embodiments, the chemical inhibitor or the small molecule is an mTOR inhibitor. In even further embodiments, the mTOR inhibitor comprises rapamycin or a derivative thereof.
The disclosure also provides 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 from a healthy donor, for a sufficient period of time to non-invasively transfer exogenous mitochondria into the recipient cell, thereby generating a mitochondria replaced cell, wherein said mitochondria replaced cell comprises greater than 5% of exogenous mtDNA.
The disclosure further provides a composition of 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 mtDNA from healthy donor, for a sufficient period of time to non-invasively transfer exogenous mtDNA into the recipient cell, thereby generating a mitochondria replaced cell, wherein said mitochondria replaced cell comprises greater than 5% of exogenous mtDNA. In some embodiments of the compositions provided herein, the one or more mitochondria replaced cells 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.
In another aspect, provided herein is a composition for use in a method of generating one or more mitochondria replaced cells comprising an agent that reduces endogenous mtDNA copy number, and a second active agent. In some embodiments, the composition further comprising one or more recipient cells, or a combination thereof. In certain embodiments, the composition further comprising exogenous mtDNA exogenous mtDNA and/or exogenous mitochondria.
In certain embodiments of the compositions provided herein, the agent that reduces endogenous mtDNA copy number is a small molecule or a fusion protein. In some embodiments, the small molecule is a nucleoside reverse transcriptase inhibitor (NRTI). In other embodiments, the fusion protein comprises an endonuclease that cleaves mtDNA and a mitochondrial target sequence (MTS). In some embodiments, the endonuclease cleaves wild-type mtDNA. In specific embodiments, the endonuclease is selected from the group consisting of XbaI, EcoRI, BamHI, HindIII, PstI, Cas9, zinc finger nuclease (ZFN), and transcription activator-like effector nuclease (TALEN). In some embodiments, the MTS targets a mitochondrial matrix protein. In further embodiments, the mitochondrial matrix protein is cytochrome c oxidase subunit IV, cytochrome c oxidase subunit VIII, and cytochrome c oxidase subunit X. In specific embodiments, the fusion protein is transiently expressed.
In some embodiments of the compositions provided herein, the reduction of endogenous mtDNA copy number is a partial reduction. In certain embodiments, the partial reduction is a reduction of about 5% to about 99% of endogenous mtDNA. In specific embodiments, the partial reduction is a reduction of about 50% to about 95% of the endogenous mtDNA copy number. In further embodiments, the partial reduction is a reduction of about 60% to about 90% of the endogenous mtDNA copy number.
The disclosure also provides a composition comprising one or more mitochondria replaced cells obtained by the method of: (a) contacting a recipient cell with an agent that reduces mitochondrial function; (b) incubating the recipient cell for a sufficient period of time for the agent to partially reduce endogenous mitochondrial function in the recipient cell; and (c) co-incubating (1) the recipient cell from step (b) in which the endogenous mitochondrial function 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, thereby generating a mitochondria replaced cell, wherein said mitochondria replaced cell comprises greater than 5% of exogenous mtDNA.
In another aspect, provided herein is a composition of one or more mitochondria replaced cells obtained by the method of: (a) contacting a recipient cell with an agent that reduces mitochondrial function; (b) incubating the recipient cell for a sufficient period of time for the agent to partially reduce endogenous mitochondrial function in the recipient cell; and (c) co-incubating (1) the recipient cell from step (b) in which the endogenous mitochondrial function has been partially reduced, and (2) exogenous mtDNA from healthy donor, for a sufficient period of time to non-invasively transfer exogenous mtDNA into the recipient cell, thereby generating a mitochondria replaced cell, wherein said mitochondria replaced cell comprises greater than 5% of exogenous mtDNA. In some embodiments, the one or more mitochondria replaced cells 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.
The disclosure also provides a composition for use in a method of generating one or more mitochondria replaced cells comprising an agent that reduces mitochondrial function, and a second active agent. In some embodiments, the composition further comprises an exogenous mitochondria, one or more recipient cells, or a combination thereof. In yet further embodiments, the composition further comprises exogenous mtDNA.
In some embodiments of the compositions provided herein, the one or more mitochondria replaced cells comprise wild-type exogenous mtDNA.
Also provided herein are compositions further comprising a second active agent. In some embodiments, the second active agent is 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. In specific embodiments, the activator of endocytosis is an activator of a clathrin-independent endocytosis pathway. In some embodiments, the activator of endocytosis is an activator of a clathrin-independent endocytosis pathway. In further embodiments, the clathrin-independent endocytosis pathway is 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. In yet further embodiments, the clathrin-independent endocytosis pathway is macropinocytosis. In specific embodiments, the activator of endocytosis comprises nutrient stress, and/or an mTOR inhibitor. In some embodiments, the mTOR inhibitor comprises rapamycin or a derivative thereof.
In certain embodiments, the disclosure further provides a composition where the total mtDNA copy number of the one or more mitochondria replaced cells comprises greater than 5% of exogenous mtDNA. In some embodiments, the total mtDNA copy number of the one or more mitochondria replaced cells comprises greater than 30% of exogenous mtDNA. In specific embodiments, the total mtDNA copy number of the one or more mitochondria replaced cells comprises greater than 50% of exogenous mtDNA. In further embodiments, the total mtDNA copy number of the one or more mitochondria replaced cells comprises greater than 75% of exogenous mtDNA.
In some embodiments of the compositions provided herein, the exogenous mitochondria is isolated mitochondria. In specific embodiments, the isolated mitochondria is intact. In some embodiments, the exogenous mitochondria and/or exogenous mtDNA is allogeneic. In specific embodiments, the exogenous mitochondria further comprises exogenous mtDNA.
In certain embodiments of the compositions provided herein, the one or more cells are animal cells or plant cells. In some embodiments, the animal cells are mammalian cells. In specific embodiments, the cells are somatic cells. In further embodiments, the somatic cells are epithelial cells. In yet further embodiments, the epithelial cells are thymic epithelial cells (TECs). In other embodiments, the somatic cells are immune cells. In certain embodiments, the immune cells are T cells. In specific embodiments, the T cells are CD4+ T cells. In other embodiments, the T cells are CD8+ T cells. In some embodiments, the T cells are chimeric antigen receptor (CAR) T cells. In other embodiments, the immune cells are phagocytic cells. In certain embodiments, the one or more mitochondria replaced cells are bone marrow cells. In specific embodiments, the bone marrow cells are a hematopoietic stem cell (HSC), or a mesenchymal stem cell (MSC).
In some embodiments of the compositions provided herein, the one or more mitochondria replaced cells are more viable than an isogenic cell having homoplasmic endogenous mtDNA. In other embodiments, the one or more mitochondria replaced cells are 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.
In certain embodiments of the compositions provided herein, the composition further comprises a small molecule, a peptide, or a protein.
Also provided herein is a composition for use in delaying senescence and/or extending lifespan in a cell comprising: (a) a senescent or near senescent cell having endogenous mitochondria; (b) isolated exogenous mitochondria from a non-senescent cell; and (c) an agent that reduces endogenous mtDNA copy number. In some embodiments the agent is a fusion protein. In certain embodiments, the fusion protein comprises an endonuclease that cleaves mtDNA and a mitochondrial target sequence (MTS). In specific embodiments, the endonuclease cleaves wild-type mtDNA. In some embodiments, the endonuclease is selected from the group consisting of XbaI, EcoRI, BamHI, HindIII, PstI, Cas9, zinc finger nuclease (ZFN), and transcription activator-like effector nuclease (TALEN). In further embodiments, the MTS targets a mitochondrial matrix protein. In yet further embodiments, the mitochondrial matrix protein is selected from the group consisting of cytochrome c oxidase subunit IV, cytochrome c oxidase subunit VIII, and cytochrome c oxidase subunit X. In certain embodiments, the fusion protein is transiently expressed in said senescent or near senescent cell.
The disclosure further provides a composition for use in delaying senescence and/or extending lifespan in a cell comprising: (a) a senescent or near senescent cell having endogenous mitochondria; (b) isolated exogenous mitochondria from a non-senescent cell; and (c) an agent that reduces mitochondrial function. In some embodiments, the agent that reduces mitochondrial function transiently reduces endogenous mitochondrial function. In other embodiments, the agent that reduces mitochondrial function permanently reduces endogenous mitochondrial function. In some embodiments, the exogenous mitochondria from the non-senescent cell has enhanced function relative to the endogenous mitochondria.
In some embodiments, the composition for use in delaying senescence and/or extending lifespan in a cell further comprises a second active agent. In specific embodiments, the second active agent is 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. In some embodiments, the activator of endocytosis is an activator of a clathrin-independent endocytosis pathway. In specific embodiments, the clathrin-independent endocytosis pathway is 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. In further embodiments, the clathrin-independent endocytosis pathway is macropinocytosis. In some embodiments, said activator of endocytosis comprises nutrient stress, and/or an mTOR inhibitor. In certain embodiments, said mTOR inhibitor comprises rapamycin or a derivative thereof.
In another aspect, the disclosure also provides a pharmaceutical composition comprising an isolated population of mitochondria replaced cells having an exogenous mitochondria from a healthy donor, wherein the cells are obtained by any of the methods provided herein for obtaining a mitochondrial replaced cell. In yet another aspect, the disclosure provides a pharmaceutical composition comprising an isolated population of mitochondria replaced cells having an exogenous mtDNA from a healthy donor, wherein the cells are obtained by any of the methods provided herein for obtaining a mitochondrial replaced cell. In some embodiments, the pharmaceutical composition comprising an isolated population of mitochondria replaced cells having an exogenous mtDNA from a healthy donor further comprises exogenous mitochondria.
For example, in some embodiments, a pharmaceutical composition comprising an exogenous mitochondria from a healthy donor are obtained by a method of generating a mitochondrial replaced cell that includes (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 from a healthy donor, for a sufficient period of time to non-invasively transfer exogenous mitochondria into the recipient cell, thereby generating a mitochondria replaced cell. In certain embodiments, the cells are obtained by a method comprising (a) contacting a recipient cell with an agent that reduces mitochondrial function; (b) incubating the recipient cell for a sufficient period of time for the agent to partially reduce the endogenous mitochondrial function in the recipient cell; and (c) co-incubating (1) the recipient cell from step (b) in which the endogenous mitochondrial function 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, thereby generating a mitochondria replaced cell.
In other embodiments, the cells are obtained by a method 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 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. In other embodiments, the cells are obtained by a method comprising: (a) contacting a recipient cell with an agent that reduces mitochondrial function; (b) incubating the recipient cell for a sufficient period of time for the agent to partially reduce the endogenous mitochondrial function in the recipient cell; and (c) co-incubating (1) the recipient cell from step (b) in which the endogenous mitochondrial function has been partially reduced, and (2) 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.
In certain embodiments of the pharmaceutical compositions provided herein, the cells are obtained by a method further comprising further comprising contacting the recipient cell with a second active agent prior to co-incubating the recipient cell with exogenous mitochondria and/or exogenous mtDNA. In some embodiments, the second active agent is 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. In specific embodiments, the activator of endocytosis is a modulator of cellular metabolism. In other embodiments, the modulator of cellular metabolism comprises nutrient starvation, a chemical inhibitor, or a small molecule. In further embodiments, the chemical inhibitor or the small molecule is an mTOR inhibitor. In yet further embodiments, said mTOR inhibitor comprises rapamycin or a derivative thereof.
In certain embodiments of the pharmaceutical compositions provided herein, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
In some embodiments of the pharmaceutical compositions provided herein, the cells are T cells. In other embodiments, the cells are hematopoietic stem cells.
Provided herein are novel and enhanced methods of generating a mitochondria replaced cell (MirC) that do not require complete removal of endogenous mtDNA, and can optionally be performed using reagents that are compatible with clinical use. In addition, in certain embodiments provided herein are methods of treatment involving administering a therapeutically effective amount of the MirC generated using the methods provided herein.
Also provided are compositions that include one or more mitochondria replaced cells obtained by the methods provided herein. In certain embodiments, the compositions can also include a second active agent that enhances the uptake of exogenous mitochondria, exogenous mtDNA, or a combination thereof, and/or an agent that reduces endogenous mtDNA copy number or reduces endogenous mitochondrial function. In further embodiments, the compositions can also include exogenous mitochondria and/or exogenous mtDNA, one or more recipient cells, or a combination thereof. In one specific embodiment, provided herein are methods and compositions for use in the treatment of a disease or disorder associated with dysfunctional mitochondria. However, it is understood that the methods and compositions provided herein can also be used to delay senescence, extend the lifespan, or enhance the function of a cell that has functional mitochondria, and is not limited to replacement of dysfunctional mitochondria. Furthermore, the methods and compositions provided herein can also be used to replace functional mitochondria with exogenous mitochondria that is dysfunctional or exhausted, for example, to generate a disease model.
Unless particularly defined otherwise, all terms including technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclatures used in this specification and the experimental methods described below are widely known and generally used in the related art.
As used herein, the term “mitochondria replaced cell” or MirC is intended to mean a cell having the substitution of endogenous mitochondria and/or mtDNA with exogenous mitochondria and/or mtDNA. For example, an exemplary mitochondria replaced cell (MirC) involves the substitution of endogenous mtDNA that encodes dysfunctional mitochondria, such as mtDNA originating from a subject having a mitochondrial disease or disorder, with exogenous mtDNA that encodes functional mitochondria, such as mtDNA originating from a healthy subject. Exemplary MirC can also include a cell with endogenous mitochondria substituted with exogenous mitochondria. However, it is understood that the substitution of the endogenous mitochondria and/or mtDNA can also include, for example, functional endogenous mtDNA from one cell, such as from an old cell, that is substituted with functional exogenous mtDNA from a different cell, such as from a healthier cell that is from a young subject. It is further understood that healthy endogenous mitochondria and/or mtDNA can also be substituted with dysfunctional exogenous mitochondria and/or exogenous mtDNA such as, for example, to mimic a mitochondrial disease or disorder. Replacement need not result in a complete substitution of all the endogenous mitochondria in a cell, and that exemplary mitochondria and/or mtDNA replacement involves substitution of about 5% of more, about 10% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, or about 95% or more of the endogenous mitochondria and/or mtDNA.
As used herein, the term “recipient cell,” “acceptor cell,” and “host cell” are interchangeable and refer to a cell receiving the exogenous mitochondria and/or mtDNA. In some embodiments, the exogenous mitochondria and/or mtDNA is from isolated mitochondria from a donor cell. In some embodiments, the donor cells and the recipient cells may be different or identical. In some embodiments, the donor cells and the recipient cells come from different or the same species. In some embodiments, the donor cells and the recipient cells come from different or the same tissues.
As used herein, the term “healthy donor” is intended to mean a donor that does not have a mitochondrial disease or disorder, age-related disease, or otherwise dysfunctional mitochondria. In preferred embodiments, a healthy donor has a wild-type mtDNA sequence, relative to the Cambridge Reference Sequence of the mitochondrial genome.
As used herein, the terms “treat,” “treating,” and “treatment” refer to reduction in severity, progression, spread, and/or frequency of symptoms, elimination of symptoms and/or underlying cause, prevention of the occurrence of symptoms and/or their underlying cause, and improvement or remediation of damage. “Treatment” is meant to include therapeutic treatment as well as prophylactic, or suppressive measures for the condition, disease or disorder.
As used herein, the term “agent” when used in reference to depleting reducing mtDNA refers to an enzyme or compound that is capable of reducing mtDNA. Preferred agents include restriction enzymes, such as XbaI, that cleave mtDNA at one or more sites, without producing toxicity in the recipient cell. However, agents can also include an enzyme or compound that inhibit mtDNA synthesis or selectively promote degradation of the mitochondria.
As used herein, the terms “reduce,” or “decrease” generally means a decrease of at least 5%, for example a decrease by at least about 10%, or at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or any decrease between 5%-99% as compared to a reference level, as that term is defined herein. It is understood that a partial reduction or an agent that partially reduces endogenous mtDNA or decrease, as used herein, does not result in a complete depletion of all endogenous mtDNA (i.e., ρ0 cells). The term “increase” as used herein generally means an increase of at least 5%, for example an increase by at least about 10%, or at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or more than 90%.
As used herein, the term “endogenous” refers to originating or derived internally. For example, endogenous mitochondria are mitochondria that are native to a cell.
As used herein, the term “exogenous” refers to cellular material (e.g., mitochondria or mtDNA) that is non-native to the host, such as cellular material that is derived externally. “Externally” typically means from a different source. For example, mitochondrial genomes are exogenous to host cells or host mitochondria when the mitochondrial genomes originate from different cell types or different species than the host cells or host mitochondria. In addition, “exogenous” can also refer to mitochondrial genomes that are removed from mitochondria, manipulated, and returned to the same mitochondria.
As used herein, the term “sufficient period of time” refers to an amount of time that produces the desired results. It is understood that the sufficient period of time will vary according to the experimental conditions, including but not limited to, the temperature, the amount of reagent used, and the cell type. Exemplary protocols are provided throughout as guidelines for the “sufficient period of time,” and a person skilled in the art would be able to identify the period of time that is sufficient without undue experimentation.
As used herein, the term “majority” is intended to mean the greatest amount, relative to the other amounts being compared. An exemplary majority when comparing two groups, is an amount that is any integer greater than about 50% or more, about 60% or more, about 70% or more, about 80% or more, or about 90% or more, or about 95% or more, of the total population, including any integer in-between. It is understood that the majority will depend on the total population being compared, and can be amounts lower than 50% when there are three or more groups being compared.
As used herein, the term “non-invasively” when used in reference to the transfer of exogenous material is intended to mean without the use of invasive instruments (e.g., nanoblade or electroporation), physical force (e.g., centrifugation), or harmful culture conditions (e.g., thermal shock). In preferred embodiments, the non-invasive transfer procedure involves co-incubation of a recipient cell and donor mitochondria.
As used herein, the term “subject in need of mitochondrial replacement,” is intended to mean a subject that has or is predisposed to having a dysfunctional mitochondria. The subject in need of mitochondrial replacement may be asymptomatic and in need of preventative care. The subject in need of mitochondrial replacement may also be symptomatic and in need of treatment. In certain embodiments, the subject in need of mitochondrial replacement has dysfunctional mitochondria that is not the result of an age-related disease or a mitochondrial disease or disorder.
As used herein, the term “subject” is intended to mean a mammal. A subject can be a human or a non-human mammal, such as a dog, cat, bovid, equine, mouse, rat, rabbit, or transgenic species thereof. It is understood that a “subject” can also refer to a “patient,” such as a human patient.
As used herein, the term “effective amount” refers to the amount of a composition of the invention effective to modulate, treat, or ameliorate any disease or disorder associated with heteroplasmy and/or dysfunctional mitochondria. As such, an effective amount can include, for example, a therapeutically effective amount, which refers to an effective amount in a therapy, or a biologically effective amount, which refers to an effective amount for a biological effect. The terms “therapeutically effective amount” and “effective amount” can encompass an amount that improves overall therapy, reduces or avoids symptoms or causes of disease or disorder, or enhances the therapeutic efficacy of another therapeutic agent. The amount of a given composition that will correspond to such an amount will vary depending upon various factors, such as the given composition, the pharmaceutical formulation, the route of administration, the type of condition, disease or disorder, the identity of the subject or host being treated, and the like, but can nevertheless be routinely determined by one skilled in the art. As defined herein, a therapeutically effective amount of an agent may be readily determined by one of ordinary skill by routine methods known in the art.
As used herein, the term “age-related disease” refers to any number of conditions attributable to advancement in age. These conditions include, without limitation, osteoporosis, bone loss, arthritis, stiffening joints, cataracts, macular degeneration, metabolic diseases including diabetes mellitus, neurodegenerative diseases including Alzheimer's Disease and Parkinson's Disease, immunosenescence, and heart disease including atherosclerosis and dyslipidemia. The phrase “age related disease” further encompasses neurodegenerative diseases, such as Alzheimer's Disease and related disorders, ALS, Huntington's disease, Parkinson's Disease, and cancer.
As used herein, the term “autoimmune disease” is intended to mean a disease or disorder arising from immune reactions directed against an individual's own tissues, organs or a manifestation thereof or a resulting condition therefrom. An autoimmune disease can refer to a condition that results from, or is aggravated by, the production of autoantibodies that are reactive with an autoimmune antigen or epitope thereof. An autoimmune disease can be tissue- or organ-specific, or it can be a systemic autoimmune disease. Systemic autoimmune diseases include connective tissue diseases (CTD), such as systemic lupus erythematosus (lupus; SLE), mixed connective tissue disease systemic sclerosis, polymyositis (PM), dermatomyositis (DM), and Sjögren's syndrome (SS). Additional exemplary autoimmune diseases further include rheumatoid arthritis, and anti-neutrophil cytoplasmic antibody (ANCA) polyangiitis.
As used herein, the term “genetic disease” refers to a disease caused by an abnormality, such as a mutation, in the nuclear genome. Exemplary genetic diseases include, but are not limited to, Hutchinson-Gilford Progeria Syndrome, Werner Syndrome, and Huntington's disease.
As used herein, the term “cancer” includes but is not limited to, solid cancer and blood borne cancer. The terms “cancer” and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth.
As used herein, the terms “mitochondrial disease or disorder” and “mitochondrial disorder” are interchangeable and refer to a group of conditions caused by inherited or acquired damage to the mitochondria causing an energy shortage within those areas of the body. Exemplary organs effected by mitochondrial disease or disorder include those that consume large amounts of energy such as the liver, muscles, brain, eye, ear, and the heart. The result is often liver failure, muscle weakness, fatigue, and problems with the heart, eyes, and various other systems.
As used herein, the term “mitochondrial DNA abnormalities” refer to mutations in mitochondrial genes whose products localize to the mitochondrion, and not observed in the cells of healthy subjects. Exemplary diseases associated with mitochondrial DNA abnormalities include, for example, chronic progressive external ophthalmoplegia (CPEO), Pearson syndrome, Kearns-Sayre syndrome (KSS), diabetes and deafness (DAD), leber hereditary optic neuropathy (LHON), LHON-plus, neuropathy, ataxia, and retinitis pigmentosa syndrome (NARP), maternally-inherited Leigh syndrome (MILS) also known as Leigh syndrome caused by mutant mtDNA, 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.
As used herein, the term “nuclear DNA abnormalities” within the context of mitochondrial disease or disorder refer to mutations or changes in the coding sequence of nuclear genes whose products localize to the mitochondrion. Exemplary mitochondrial disease or disorders associated with nuclear mutations include 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 (caused by nuclear mutations), 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).
As used herein, the term “dysfunctional mitochondria” refer to mitochondria that are in opposition to functional mitochondria. Exemplary dysfunctional mitochondria include mitochondria that are incapable of synthesizing or synthesize insufficient amounts of ATP by oxidative phosphorylation. As used herein, the term “functional mitochondria” refers to mitochondria that consume oxygen and produce ATP.
As used herein, the term “mutation” refers to any changing of the structure of a gene, resulting in a variant (also called “mutant”) form. Mutations in a gene may be caused by the alternation of single base in DNA, or the deletion, insertion, or rearrangement of larger sections of genes or chromosomes. In some embodiments, the mutation can affect the function or the resulting protein. For example, a mutation in a single nucleotide of DNA (i.e., point mutation) in the coding region of a protein can result in a codon that encodes for a different amino acid (i.e., missense mutation). It is understood that this different amino acid can alter the structure of the protein, and that in certain circumstances, as described herein, can alter the function of the organelle, such as the mitochondrion.
As used herein, the terms “heteroplasmy” and “heteroplasmic” refer to the occurrence of more than one type of mitochondrial DNA genome in an individual or sample. Varying degrees of heteroplasmy are associated with varying degrees of the physiological conditions described herein. Heteroplasmy may be identified by means known to the art, and the severity of the physiological condition associated with specific nucleotide alleles is expected to vary with the percentage of such associated alleles within the individual.
As used herein, the term “wild-type” when used in the context of mitochondrial DNA refers to the genotype of the typical form of a species as it occurs in nature. An exemplary reference genome for the wild-type human mtDNA genome includes the Cambridge Reference Sequence (CRS).
As used herein, the term “old” or “older” is intended to mean that the source of the mtDNA is from a subject that is greater in age than the recipient cell, or from a cell in a population of cells that have doubled their population a greater number of times since their culture in vitro (i.e., population doubling level, PDL) relative to the recipient cell.
As used herein, the term “young” or “younger” is intended to mean that the source of the mtDNA is from a subject that is lower in age than the recipient cell, or from a cell in a population of cells that have doubled their population a fewer number of times since their culture in vitro (i.e., population doubling level, PDL) relative to the recipient cell.
As used herein, the term “isolated” when used in reference to mitochondria refers to mitochondria that have been physically separated or removed from the other cellular components of its natural biological environment.
As used herein, the terms “intact” and “intact mitochondria” refers to mitochondria comprising an outer and an inner membrane, an inter-membrane space, the cristae (formed by the inner membrane) and the matrix. Exemplary intact mitochondria contain mtDNA. In preferred embodiments, intact mitochondria are functional mitochondria. However, it is understood that intact dysfunctional mitochondria can also be used in the present invention.
As used herein, the term “autologous” is intended to mean biological compositions obtained from the same subject.
As used herein, the term “allogeneic” is intended to mean biological compositions obtained from the same species, but a different genotype than that of the subject receiving the biological composition.
As used herein, the term “animal cell” is intended to mean any cell from a eukaryotic organism. It is understood that an animal cell can include mammalian and non-mammalian species, such as amphibians, fish, insects (e.g., Drosophila), and worms (e.g., Caenorhabditis elegans).
As used herein, the term “fusion protein” refers to a sequence of amino acids, predominantly, but not necessarily, connected to each other by peptidic bonds, wherein a part of the sequence is derived (i.e., has sequence similarity to sequences) from one origin (native or synthetic) and another part of the sequence is derived from one or more other origin. Exemplary fusion proteins can be prepared by construction of an expression vector that codes for the whole of the fusion protein (coding for both sections, such as a mitochondrial-targeted sequence and an endonuclease) so that essentially all the bonds are peptidic bonds. It is also understood that the fusion may be made by chemical conjugation, such as by using any of the known methodologies used for conjugating peptides.
As used herein, the terms “mitochondrial-targeted sequence (MTS)” and “mitochondrial targeting sequence (MTS)” are interchangeable and refer to any amino acid sequence capable of causing the transport of an enzyme, peptide, sequence, or compound attached to it into the mitochondria. In certain embodiments, the MTS is a human MTS. In another embodiment, the MTS is from another species. Non-limiting examples of such sequences are the cytochrome c oxidase subunit X (COX10) MTS (MAASPHTLSSRLLTGCVGGSVWYLERRT, SEQ ID NO: 36), and the cytochrome c oxidase subunit VIII (COX8) MTS (MSVLTPLLLRSLTGSARRLMVPRA, SEQ ID NO: 37). Additional non-limiting examples of MTS sequences are the natural MTS of each individual mitochondrial protein that is encoded by the nuclear DNA, translated (produced) in the cytoplasm and transported into the mitochondria, as well as citrate synthase (cs), lipoamide deydrogenase (LAD), and C6ORF66 (ORF). The various MTS may be exchangeable for each mitochondrial enzyme among themselves. Each possibility represents a separate embodiment of the fusion protein for use of the present invention.
As used herein, the term “small molecule” refers to a compound that affects a biological process and has molecular weight of about 900 Daltons or lower. An exemplary small molecule had a molecular weight between about 300 and about 700 daltons.
As used herein, the terms “about” or “approximately” when used in conjunction with a number refer to any number within 1, 5, 10, 15 or 20% of the referenced number.
As used herein, the term “somatic cell” refers to any differentiated cell forming the body of an organism, apart from stem cells, progenitor cells, and germline cells (i.e., ovogonies and spermatogonies) and the cells derived therefrom (e.g., oocyte, spermatozoa). For instance, internal organs, skin, bones, blood, and connective tissue are all made up of somatic cells. Somatic cells are obtained from animals, preferably human subjects, and cultured according to standard cell culture protocols available to those of ordinary skill in the art.
As used herein, the term “endocytosis pathway” refers to the cellular process in which cells take in molecules from their surroundings. The endocytosis pathway can be “clathrin-dependent,” which requires the recruitment of clathrin to help curve the plasma membrane into the vesicle which absorbs the molecules, or “clathrin-independent,” which does not require the recruitment of clathrin. An exemplary type of clathrin-independent endocytosis includes, for example, macropinocytosis. The term “activator of endocytosis,” as used herein, refers to agents that, e.g., induce or activate the endocytosis pathway, or process, such that the endocytosis pathway is increased. An exemplary “activator of endocytosis” increases mitochondrial uptake from the extracellular environment.
As used herein, the term “macropinocytosis” refers to a clathrin-independent form of endocytosis that mediates the non-selective uptake of solute molecules, nutrients and antigens.
As used herein, the term “compound” refers to a compound capable of effecting a desired biological function. The term includes, but is not limited to, DNA, RNA, protein, polypeptides, and other compounds including growth factors, cytokines, hormones or small molecules.
As used herein, the term “peptide” the terms “peptide,” “polypeptide” and “protein” are used interchangeably and in their broadest sense to refer to constrained (that is, having some element of structure as, for example, the presence of amino acids which initiate a β turn or β pleated sheet, or for example, cyclized by the presence of disulfide bonded Cys residues) or unconstrained (e.g., linear or unstructured) amino acid sequences. The amino acids making up the polypeptide may be naturally derived, or may be synthetic. The polypeptide can be purified from a biological sample. The polypeptide, protein, or peptide also encompasses modified polypeptides, proteins, and peptides, e.g., glycopolypeptides, glycoproteins, or glycopeptides; or lipopolypeptides, lipoproteins, or lipopeptides.
As used herein, the terms “modulate,” “modulation,” “modulator,” and “modulating” are intended to mean a change in the character or composition of the basal, homeostatic state.
An exemplary modulation includes altering cellular metabolism by disrupting the homeostasis, such that cellular metabolism is significantly reduced. The term “modulator” includes inhibitors and activators. Inhibitors are agents that, e.g., inhibit expression or modification of a desired protein, pathway, or process, or bind to, partially or totally block stimulation, decrease, prevent, delay activation, inactivate, desensitize, or down regulate the activity of the described target protein, pathway, or process. In certain embodiments, inhibitors are antagonists of the target protein, pathway, or process. Activators are agents that, e.g., induce or activate the expression or modification of a described target protein, pathway, or process, or bind to, stimulate, increase, open, activate, facilitate, enhance activation of inhibitor activity, sensitize or up regulate the activity of described target protein (or encoding polynucleotide), pathway, or process. In certain embodiments, an activator is an agonist of the target protein, pathway, or process. Modulators include naturally occurring and synthetic ligands, antagonists and agonists (e.g., small chemical molecules, antibodies and the like that function as either agonists or antagonists). It is further understood that modulators can be biological (e.g., antibodies), or chemical.
As used herein, the term “prior to” is intended to mean a period of time preceding the initiation of an event, such that it is a sufficient length of time to achieve and sustain a desired result (e.g., antibiotic selection) or effect (e.g., biological effect) without the desired result or effect completely dissipating before the intended event is initiated. For instance, in an exemplary situation, it is understood that modulating cellular metabolism prior to transfer of an exogenous mitochondria and/or exogenous mtDNA would involve a sufficient period of time to, for example, exhibit a desired biological effect (e.g., increase phosphorylation of S6 kinase), without the biological effect reverting back to the homeostatic state before the transfer of exogenous mitochondria and/or exogenous mtDNA occurs.
As used herein, the term “nutrient stress” refers to nutrient deficiency or nutrient starvation conditions sufficient to produce perturbations in the cellular homeostasis, such as induction of autophagy, AMPK signaling, and/or mTOR signaling pathways. Exemplary nutrient stress conditions include serum starvation, removal of essential amino acids, and/or disruption of metabolic pathways.
The terms “nucleic acid” and “polynucleotide” are used interchangeably herein to describe a polymer of any length composed of nucleotides, e.g., deoxyribonucleotides or ribonucleotides, or compounds produced synthetically, which can hybridize with naturally occurring nucleic acids in a sequence specific manner analogous to that of two naturally occurring nucleic acids, e.g., can participate in Watson-Crick base pairing interactions. As used herein in the context of a polynucleotide sequence, the term “bases” (or “base”) is synonymous with “nucleotides” (or “nucleotide”), i.e., the monomer subunit of a polynucleotide. The abbreviation “A,” when used in reference to a nucleotide is intended to mean adenine (A). The abbreviation “G,” when used in reference to a nucleotide is intended to mean Guanine (G). The abbreviation “C,” when used in reference to a nucleotide is intended to mean Cytosine (C). The abbreviation “T,” when used in reference to a nucleotide is intended to mean Thymine (T).
The term “pharmaceutically acceptable” when used in reference to a carrier, is intended to mean that the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
The practice of the embodiments provided herein will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, and immunology, which are within the skill of those working in the art. Such techniques are explained fully in the literature. Examples of particularly suitable texts for consultation include the following: Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001); Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1999); Glover, ed., DNA Cloning, Volumes I and II (1985); Gait, ed., Oligonucleotide Synthesis (1984); Hames & Higgins, eds., Nucleic Acid Hybridization (1984); Hames & Higgins, eds., Transcription and Translation (1984); Freshney, ed., Animal Cell Culture: Immobilized Cells and Enzymes (IRL Press, 1986); Kallen et al, Plant Molecular Biology—A Laboratory Manual (Ed. by Melody S. Clark; Springer-Verlag, 1997); Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); Scopes, Protein Purification: Principles and Practice (Springer Verlag, N.Y., 2d ed. 1987); and Weir & Blackwell, eds., Handbook of Experimental Immunology, Volumes I-IV (1986).
The present invention is based, in part, on the discovery that any agent that reduces the function of endogenous mitochondria, including an agent that reduces endogenous mitochondrial DNA (mtDNA), may enhance the non-invasive transfer of exogenous mitochondria. However, the complete depletion of the endogenous mtDNA, such as with ρ(0) cells, prevents this enhancement. This is because the non-invasive transfer of exogenous mitochondria is energy dependent, and a complete depletion of the endogenous mtDNA greatly limits the energy available to facilitate the non-invasive transfer process. Similarly, the non-invasive transfer of exogenous mitochondria is also inefficient when the mitochondria function and/or mtDNA is unperturbed, for example, when mitochondria is merely co-incubated (i.e., “add-on”) or added by centrifugation.
Thus, provided herein are methods of generating a mitochondria replaced cell (MirC), that can include (a) contacting a recipient cell with an agent that reduces endogenous mtDNA copy number or an agent that reduces mitochondrial function; (b) incubating the recipient cell for a sufficient period of time for the agent to partially reduce the endogenous mtDNA copy number or partially reduce the endogenous mitochondrial function in the recipient cell, respectively; and (c) co-incubating (1) the recipient cell from step (b) in which the endogenous mtDNA, or the endogenous mitochondrial function, respectively, 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, thereby generating a mitochondria replaced cell. Also provided herein, is a method of generating a mitochondria replaced cell, that includes performing steps (a) and (b) as described above, and then (c) co-incubating (1) the recipient cell from step (b) in which the endogenous mtDNA or the endogenous mitochondrial function, respectively, has been partially reduced, and (2) 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. In certain embodiments, the exogenous mtDNA is transferred via exogenous mitochondria.
The generation of a MirC can be a useful strategy for a variety of applications. By way of example, the transfer of exogenous mitochondria, exogenous mtDNA, or a combination thereof into a recipient cell can be useful in, for example, replacing endogenous mitochondria that is dysfunctional and/or comprised of mutant mtDNA with functional mitochondria, such as mitochondria comprised of wild-type mtDNA. In certain embodiments, the methods provided herein are performed in a recipient cell that has endogenous mtDNA that encodes for dysfunctional mitochondria. In specific embodiments, the endogenous mtDNA is mutant mtDNA. In certain embodiments, the endogenous mtDNA is heteroplasmic and comprised of both wild-type mtDNA and mutant mtDNA.
As described above, in certain applications, the transfer of exogenous mitochondria, exogenous mtDNA, or a combination thereof can involve the transfer of functional mitochondria or wild-type mtDNA to replace endogenous mitochondria that is, for example, dysfunctional or comprised of mutant mtDNA. Accordingly, in certain embodiments, the exogenous mtDNA is wild-type mtDNA. In other embodiments, the endogenous mitochondria of the recipient cell has wild-type mtDNA, and dysfunctional endogenous mitochondria. For example, exemplary dysfunctional mitochondria of the recipient cell with wild-type mtDNA can include mutant nuclear DNA that encode for mitochondrial proteins, or dysfunctional mitochondria that arises due to a secondary effect, such as aging or disease.
The endogenous mitochondria that is dysfunctional, comprised of mutant mtDNA, or a combination thereof can therefore be replaced using the methods described herein. Mitochondrial dysfunction can occur as a result of many factors. Non-limiting examples include mitochondrial dysfunction due to a disease (e.g., an age-related disease, a mitochondrial disease or disorder, a neurodegenerative disease, a retinal disease, a genetic disease), diabetes, a hearing disorder, or any combination thereof. Mitochondrial dysfunction can involve the function of the endogenous mitochondria being reduced by greater than 5%, greater than 10%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, or greater than 90%. Therefore, in some embodiments, the endogenous mitochondria includes mitochondria with reduced function of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%.
The methods provided herein are applicable to both homoplasmic and heteroplasmic mtDNA. In specific embodiments, the endogenous mtDNA is a single type of mtDNA (i.e., the endogenous mtDNA is homoplastic). In other specific embodiments, the endogenous mtDNA includes more than one type of mtDNA (i.e., the endogenous mtDNA is heteroplasmic). In some embodiments, the heteroplasmic mtDNA includes both wild-type mtDNA and mutant mtDNA. Generally, the proportion of mutant mtDNA determines the severity of the phenotype and can influence the degree to which mitochondrial function is reduced. For example, in some embodiments the heteroplasmic mtDNA is 5% mutant mtDNA and 95% wild-type mtDNA, and the mitochondrial function is reduced 5%. In other embodiments, the heteroplasmic mtDNA is 55% mutant mtDNA and 45% wild-type mtDNA, and the mitochondrial function is reduced 55%. However, it is understood that the percentage of mutant mtDNA need not be proportional to the mitochondrial function.
Dysfunctional mitochondria is generally characterized by a loss of efficiency in the electron transport chain and reductions in the synthesis of high-energy molecules, such as adenosine-5′-triphosphate (ATP), the leakage of deleterious reactive oxygen species (ROS), and/or disrupted cellular respiration. A person skilled in the art would understand how to evaluate mitochondrial function. For example, cell-based assays, such as the Seahorse Bioscience XF Extracellular Flux Analyzer, can used performed for the determination of basal oxygen consumption, glycolysis rates, ATP production, and respiratory capacity in a single experiment to assess mitochondrial dysfunction. Similarly, the Oroboros 02K respirometer can also be used to establish quantitative functional mitochondrial diagnosis. It is understood that the assay examples described above are exemplary and are not inclusive of all methods to evaluate mitochondrial function.
In some embodiments, functional mitochondria have an intact outer membrane. In some embodiments, functional mitochondria are intact mitochondria. In another embodiment, functional mitochondria consume oxygen at an increasing rate over time. In another embodiment, the functionality of mitochondria is measured by oxygen consumption. In another embodiment, oxygen consumption of mitochondria may be measured by any method known in the art such as, but not limited to, the MitoXpress fluorescence probe (Luxcel). In some embodiments, functional mitochondria are mitochondria which display an increase in the rate of oxygen consumption in the presence of ADP and a substrate such as, but not limited to, glutamate, malate or succinate. Each possibility represents a separate embodiment of the present invention. In another embodiment, functional mitochondria are mitochondria that produce ATP.
While the methods provided herein can be useful in generating a MirC from a recipient cell that has dysfunctional mitochondrial, mutant mtDNA, or a combination thereof, it is also understood that the MirC generation need not be performed in a recipient cell with dysfunctional mitochondria. In some embodiments, a MirC is generated using a recipient cell with functional endogenous mitochondria, wild-type mtDNA, or a combination thereof, and the exogenous mitochondria is also functional, contains wild-type mtDNA, or a combination thereof. For example, endogenous wild-type mtDNA can be reduced using the methods provided herein and exogenous wild-type mtDNA can be transferred into the recipient cell, such as mitochondrial replacement in an “old” recipient cell (e.g., a cell from an aged subject or a cell with relatively high population doubling level (PDL)) with exogenous mtDNA from a healthy donor cell (e.g., a young cell with relatively low PDL). Thus, in certain embodiments, the exogenous mtDNA is from a donor cell that is a healthy donor cell, for example a donor cell that is younger than the recipient cell. In certain embodiments, the donor and recipient cell have a difference in PDL of about 1.5 fold, about 2 fold, about 2.5 fold, about 3 fold, about 4 fold, about 5 fold, or greater than 5 fold. In other embodiments, the donor and recipient cells are from subjects that are separated in age by about 1.5 fold, about 2 fold, about 2.5 fold, about 3 fold, about 4 fold, about 5 fold, or greater than 5 fold. However, it is understood that a difference in age between the donor cell and the recipient cell is not a requirement. In some embodiments, the donor and recipient cell are the same age, and the donor cell is a heathy cell.
In yet other embodiments, the generation of a MirC is performed in a recipient cell with functional endogenous mitochondria, such as wild-type endogenous mtDNA, and the exogenous mtDNA is mutant, encodes for dysfunctional mitochondria, the exogenous mitochondria is dysfunctional, or a combination thereof. In other embodiments, the exogenous mitochondria, exogenous mtDNA, or a combination thereof is from a donor cell that is older than the recipient cell. For example, in some embodiments, a model of a mitochondrial disease or disorder can be created by replacement of functional mitochondria in a recipient cell with exogenous mtDNA from a donor cell that is mutant and/or encodes for dysfunctional mitochondria. It is understood that the examples described herein are exemplary and are not inclusive of all combinations involving mtDNA replacement.
As provided herein, the methods of generating a MirC can be practiced using either an agent that reduces endogenous mtDNA, or an agent that reduces endogenous mitochondrial function. In certain circumstances, a combination of the two agents can be used. Agents that are capable of reducing mitochondrial function are well known in the field, and are within the skillset of a person skilled in the art. Exemplary agents include inhibitors of the mitochondrial respiratory chain that block respiration in the presence of either ADP or uncouplers, such as an inhibitor of complex III (e.g., myxothiazol), an inhibitor of complex IV (e.g., sodium azide, potassium cyanide (KCN)), or an inhibitor of complex V (e.g., oligomycin); inhibitors of phosphorylation that abolish the burst of oxygen consumption after adding ADP, but have no effect on uncoupler-stimulated respiration; uncoupling agents that abolish the obligatory linkage between the respiratory chain and the phosphorylation system which is observed with intact mitochondria (e.g., dinitrophenol, CCCP, FCCP); ATP/ADP transport inhibitor, such as an adenine nucleotide translocase inhibitor (e.g., atractyloside) that either prevent the export of ATP, or the import of raw materials across the mitochondrial inner membrane; ionophores (e.g. valinomycin, nigericin) which make the inner membrane permeable to compounds which are ordinarily unable to cross; or a Krebs cycle inhibitor (e.g. arsenite, aminooxyacetate) which block one or more of the TCA cycle enzymes, or an ancillary reaction. It is understood that the agents that are capable of reducing mitochondrial function described above are non-limiting, and that a person skilled in the art can readily identify suitable agents that are capable of reducing mitochondrial function using techniques known in the art.
In specific embodiments, the agent that reduces endogenous mitochondrial function transiently reduces the endogenous mitochondrial function. In other embodiments, the agent that reduces endogenous mitochondrial function permanently reduces the endogenous mitochondrial function. In preferred embodiments, the agent that reduces endogenous mitochondrial function partially reduces the endogenous mitochondrial function.
Various agents can be used to reduce mtDNA. In certain embodiments, the agent that reduces mtDNA is selected from a nucleic acid encoding a fusion protein comprising a mitochondrial-targeted sequence (MTS) and an endonuclease, an endonuclease, or a small molecule. In certain embodiments, the small molecule is a nucleoside reverse transcriptase inhibitor (NRTI). The nucleic acid can be a messenger ribonucleic acid (mRNA) or a deoxyribonucleic acid (DNA). In certain embodiments, the agent that reduces mtDNA is a plasmid DNA expression vector cassette encoding an endonuclease. In preferred embodiments, the agent is a plasmid DNA expression vector cassette encoding an endonuclease with a MTS. Various expression vector cassettes can be used, and a person skilled in the art would understand the necessary considerations required to enable successful expression of the endonuclease depending on the host cell. For example, a mammalian expression vector, such as a vector having a cytomegalovirus (CMV) promoter, SV40 promoter, or CAG promoter, would be suitable for expression of the endonuclease in a mammalian cell, but not a non-mammalian cell. Similarly, it is understood that viral expression vectors can also be used and a person skilled in the art would understand that such viral expression vectors may require helper plasmids (i.e., envelope and packaging plasmids) to be used in tandem with the transfer plasmid. In other embodiments, the agent is an mRNA encoding an endonuclease. In other preferred embodiments, the agent is an mRNA encoding an endonuclease with a MTS. In yet further embodiments, the agent is an endonuclease that is a recombinant protein. In other embodiments, the agent is a small molecule, such as, for example, a small molecule that disrupts synthesis of mtDNA. Techniques for generating any of the expression methods are known to those skilled in the art, and can be readily performed without undue experimentation. In preferred embodiments, the agent is suitable for clinical use.
In specific embodiments, the endonuclease can be a restriction enzyme that cleaves DNA double helices into fragments at specific sites, such as XbaI, which cleaves the following sequence of DNA:
The endonuclease can also include, for example, restriction enzymes other than XbaI, such as EcoRI, BamHI, HindIII, or PstI, which all digest mtDNA at multiple sites. Endonucleases have defined recognition sites, which allows prediction of their sensitivity on mtDNA. The defined recognition sites of restriction enzymes, such as, for example, XbaI, EcoRI and SmaI, are specific to a given nucleic acid sequence. Accordingly, in some embodiments, the reduction of endogenous mtDNA can be performed using zinc fingers and transcription activator-like effectors (TALEs) that have been combined to DNA nucleases. These two types of DNA-binding proteins can be engineered to have specificities for new DNA sequences of interest. Similarly, clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 proteins can be also introduced into cells through the addition of the corresponding encoding genes. Therefore, in some embodiments, the endonuclease can be a programmable nuclease, such as a RNA-guided DNA endonuclease (e.g., Cas9), zinc finger nuclease (ZFN), or transcription activator-like effector nuclease (TALEN). It is understood that the nucleases described above are non-limiting, and that a person skilled in the art can readily identify suitable endonucleases using techniques known in the art. For example, the Cambridge Reference Sequence or similar consensus sequence can be used to identify suitable endonucleases that recognize the mtDNA sequence by, for example, an in silico analysis. In specific embodiments, the endonuclease cleaves a wild-type sequence of mtDNA. In other embodiments, the endonuclease cleaves a mutant sequence of mtDNA. It is also understood that the agent that reduces endogenous mtDNA need not be an endonuclease and that any agent capable of reducing mtDNA can be employed, including an agent that inhibits the biosynthesis of mtDNA, such as ethidium bromide. Also contemplated herein, are agents, such as, for example, Urolithin A or the small molecule p62-mediated mitophagy inducer (PMI), that induce autophagy in order to promote the selective degradation of endogenous mitochondria (i.e., mitophagy agonist). The present invention can also be practiced using a nucleoside reverse transcriptase inhibitor (NRTI) as an agent that reduces mtDNA.
In addition, in some embodiments, the expression vector cassette can include one or more antibiotic resistance genes to enable selection of a population of cells that express the expression vector cassette. For example, in some embodiments, the expression vector can include the puromycin N-acetyl-transferase gene (pac) from Streptomyces, and cells can be selected using puromycin. In circumstances where selection is performed using antibiotics, e.g., puromycin, the selection can be brief (e.g., 24-48 hours) to limit long term exposure to the drug. However, it is understood that the example provided above is merely exemplary, and that the expression vector cassette can include other antibiotics resistance genes, such as, for example, the bsr, bls, or BSD gene for selection with Blasticidin, or the hph gene for selection with hydromycin B. It is generally understood that the concentration of antibiotic used for selection will depend on the type of antibiotic and the cell type, and would be readily obtainable to one skilled in the art without undue experimentation. It is further understood that selection can be produced by any means known in the art, and need not involve antibiotic resistance. For example, in some embodiments, selection of the cells can be performed by, for example, fluorescence-activated cell sorting (FACS) of a cell surface marker or expression of a fluorescent protein encoded by the expression. In yet further embodiments, selection can be performed according to the cell's phenotype. For example, in some embodiments, the successful deletion of mutant endogenous mtDNA in a cell with heteroplasmy can result in a phenotypic response that is selectable, such as, for example, cell survival.
Accordingly, in some embodiments, the cells are selected after introducing an expression vector cassette that contains an endonuclease that degrades mtDNA. In some embodiments, the cells are selected to obtain a homogenous population of cells that express an endonuclease that degrades mtDNA. In specific embodiments, the cells are selected after introducing an expression vector cassette that contains an endonuclease that degrades mtDNA, and a homogenous, stable cell line is generated. In other embodiments, the cells are selected to enrich for a population of cells that express an endonuclease that degrades mtDNA. As described above, this enrichment by selection can involve a brief exposure to an antibiotic. The enriched cells can stably express the endonuclease or transiently express the endonuclease depending on the extent and/or manner of the selection pressure. It is understood that an enriched population need not be homogenous, and that an enriched population of cells that express an endonuclease that degrades mtDNA contains a higher percentage of cells with the endonuclease, relative to an unselected population of cells, but may also contain some cells that do not express the endonuclease.
In other embodiments, the cells are not selected after introducing an expression vector that contains an endonuclease that degrades mtDNA. In specific embodiments, the cells are not selected after introducing an expression vector that contains an endonuclease that degrades mtDNA, and the endonuclease is transiently expressed.
Various methods for introducing the plasmid DNA expression vector cassette, mRNA, and/or recombinant protein are known in the art. In some embodiments, the plasmid DNA expression vector cassette is introduced by electroporation. In specific embodiments, the electroporation method is flow electroporation, such as MaxCyte Flow Electroporation. In other specific embodiments, the electroporation method includes the nucleofection technology, such as Lonza's Nucleofector™ technology. In other embodiments, the plasmid DNA expression vector cassette is introduced by cationic lipid transfection. In yet further embodiments, the plasmid DNA expression vector cassette is introduced by viral transduction. It is understood that the methods described above for introducing the expression vector cassette are non-limiting and merely intended to be exemplary methods, and that any method known in the art can be used for introducing the DNA expression vector cassette.
Where the agent for reducing endogenous mitochondria comprises an endonuclease, expression of the endonuclease can also involve introduction of mRNA encoding the endonuclease or introducing the endonuclease as a recombinant protein. In certain embodiments, the MaxCyte electroporator can be used for mRNA transfection, particularly in the clinical setting, which has cleared the standards of Good Manufacturing Practice and Good Clinical Practice. The transfection can be performed using the MaxCyte electroporator according to the manufacturer's protocol. It is further understood that the methods described above are merely exemplary and that any means of introducing mRNA and/or recombinant protein can be used.
The specific targeting of the endonuclease to the mitochondria can be performed by incorporating a mitochondrial targeting sequence (MTS) adjacent to the endonuclease coding sequence, which will result in a fusion protein that targets the mitochondria. Strong MTSs have been identified and shown capable of targeting proteins to specific compartments when fused on their N-termini, and are termed mitochondrial targeting sequences. MTS suitable for the methods of the present invention are well known to the person skilled in the art (see, e.g., U.S. Pat. No. 8,039,587B2, which is hereby incorporated by reference in its entirety). For example, MTS to the mitochondrial matrix can be used, such as the MTS that is a targeting peptide from the cytochrome c oxidase subunit IV (COX 4), subunit VIII (COX 8), or subunit X (COX 10). In principle, any target sequence derived from any nuclear encoded mitochondrial matrix or inner membrane enzyme or an artificial sequence that is capable of rendering the fusion protein into a mitochondrial imported protein (hydrophobic moment greater than 5.5, at least two basic residues, amphiphilic alpha-helical conformation; see, e.g., Bedwell et al., Mol Cell Biol. 9(3) (1989), 1014-1025) is useful for the purposes of the present invention.
In certain embodiments, the MTS is a human MTS. In another embodiment, the MTS is from another species. Non-limiting examples of such sequences are the cytochrome c oxidase subunit X (COX 10) MTS (MAASPHTLSSRLLTGCVGGSVWYLERRT, SEQ ID NO: 36), and the cytochrome c oxidase subunit VIII (COX 8) MTS (MSVLTPLLLRSLTGSARRLMVPRA, SEQ ID NO: 37). Additional non-limiting examples of MTS sequences are the natural MTS of each individual mitochondrial protein that is encoded by the nuclear DNA, translated (produced) in the cytoplasm and transported into the mitochondria, as well as citrate synthase (cs), lipoamide deydrogenase (LAD), and C6ORF66 (ORF). The various MTS may be exchangeable for each mitochondrial enzyme among themselves. Accordingly, in some embodiments, the MTS targets a mitochondrial matrix protein. In specific embodiments, the mitochondrial matrix protein is subunit VIII of human cytochrome C oxidase. Each possibility represents a separate embodiment of the fusion protein for use of the present invention.
Upon contacting a recipient cell with an agent that reduces endogenous mtDNA copy number or an agent that reduces endogenous mitochondrial function, the recipient cell is incubated for a sufficient period of time for the agent to partially reduce the endogenous mtDNA copy number in the recipient cell or partially reduce the endogenous mitochondrial function in the recipient cell, respectively. Identifying the “sufficient period of time” to allow the agent to reduce partially reduce the endogenous mtDNA copy number or partially reduce the endogenous mitochondrial function is within the skill of those in the art. The sufficient or proper time period will vary according to various factors, including but not limited to, the particular type of cells, the amount of starting material (e.g., the number of recipient cells and/or amount of mtDNA to be reduced), the amount and type of agent(s), the plasmid promoter regulator(s), and/or the culture conditions. In various embodiments the sufficient period of time to allow a partial reduction of the endogenous mtDNA copy number in a recipient cell is about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 1-2 weeks, about 2-3 weeks or about 3-4 weeks. In preferred embodiments, the sufficient period of time will be long enough that the resulting recipient cell has a reduction in a majority of the endogenous mtDNA copy number or a reduction in the function of a majority of the endogenous mitochondria and is also substantially free of the agent that reduces endogenous mtDNA or the agent that reduces endogenous mitochondrial function before incubating the recipient cell with an exogenous mtDNA and/or exogenous mitochondria.
An important and novel aspect of the present invention is the finding that mitochondrial transfer efficiency is severely reduced in cells with a complete depletion of endogenous mitochondria (i.e., (ρ) 0 cells), but can be greatly improved when the endogenous mtDNA copy number is reduced but not completely depleted (i.e., (ρ)− cells). Furthermore, the present invention also demonstrates that simple add-on or centrifugation protocols are inefficient without partial reduction in the endogenous mtDNA copy number. Accordingly, in preferred embodiments, the reduction of the endogenous mtDNA copy number in the recipient cell is less than a 100% depletion of the endogenous mtDNA. In some embodiments, the endogenous mtDNA copy number in the recipient cell is reduced by about 5% to about 99%. In specific embodiments, the agent that reduces endogenous mtDNA copy number reduces about 30% to about 70% of the endogenous mtDNA copy number. In other embodiments, the agent that reduces endogenous mtDNA copy number reduces about 50% or more, about 60% or more, about 70% or more, about 80% or more, or about 90% or more, or about 95% or more of the endogenous mtDNA copy number. In yet further embodiments, the agent that reduces endogenous mtDNA copy number reduces about 60% to about 90% of the endogenous mtDNA copy number. It is also understood that in some embodiments, the agent that reduces endogenous mtDNA copy number reduces mitochondrial mass.
In certain embodiments, the exogenous mtDNA is contained in isolated exogenous mitochondria from a donor cell. Mitochondrial isolation may be accomplished by any of a number of well-known techniques including but not limited to those described herein, and in the cited references. In certain embodiments, the exogenous mitochondria for use in mitochondrial transfer is isolated using a commercial kit, such as, for example, the Qproteum mitochondria isolation kit (Qiagen, USA), the MITOISO2 mitochondria isolation kit (Sigma, USA), or Mitochondria Isolation Kit for Cultured Cells (Thermo Scientific). In other embodiments, the exogenous mitochondria for use in mitochondrial transfer is isolated manually. For example, an exemplary manual isolation of mitochondria includes isolating the mitochondria from donor cells by pelleting the donor cells, washing the cell pellet of 1-2 mL derived from approximately 109 cells grown in culture, swelling the cells in a hypotonic buffer, rupturing the cells with a Dounce or Potter-Elvehjem homogenizer using a tight-fitting pestle, and isolating the mitochondria by differential centrifugation. Manual isolation can also include, for example, sucrose density gradient ultracentrifugation, or free-flow electrophoresis. Without wishing to be bound by any particular method, it is understood that the kits and manual methods described herein are exemplary, and that any mitochondrial isolation method can be used, and would be within the skill set of a person skilled in the art.
In some embodiments, the isolated donor mitochondria is substantially pure of other organelles. In other embodiments, the isolated mitochondria can contain impurities and is enriched for mitochondria. For example, in some embodiments, the isolated mitochondria are about 90% pure, about 80% pure, about 70% pure, about 60%, pure, about 50%, pure, or any integer in-between. In general, it is understood that any impurities contained with the isolated donor mitochondria will not affect the viability or function of the recipient cell upon mitochondrial transfer. In specific embodiments, the transfer of the exogenous mitochondria, exogenous mtDNA, or a combination thereof does not involve transfer of non-mitochondrial organelles.
The quantity and quality of isolated mitochondria can easily be determined by a number of well-known techniques including but not limited to those described herein, and in the cited references. For example, in some embodiments, the quantity of isolated mitochondria is determined by assessment of total protein content. Various methods are available for measurement of total protein content, such as the Biuret and Lowry procedures (see, e.g., Hartwig et al., Proteomics, 2009 June; 9(11):3209-14). In other embodiments, the quantity of isolated mitochondria is determined by mtDNA copy number.
In some embodiments, the isolated mitochondria are functional mitochondria. In further embodiments, the isolated mitochondria are dysfunctional mitochondria. In some embodiments, the mitochondrial function can be assessed in the donor cell prior to isolation. In other embodiments, the mitochondrial function can be assayed from the isolated mitochondria.
The preservation of mitochondrial membrane integrity is another important factor during mitochondria isolation. In some embodiments, the mtDNA used in the methods provided herein is from intact mitochondria. In specific embodiments, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or greater than 90% of the isolated mitochondria are intact. Mitochondrial membrane integrity can be accomplished by any of a number of well-known techniques including but not limited to those described herein, and in the cited references. For example, TMRM, Rhod123, JC-1 and DiOC6 are typical probes for measurement of mitochondrial membrane potential (see, e.g., Perry et al., Biotechniques, 2011 February; 50(2):98-115). JC-1 is a widely used dye for measurement of inner-membrane potential of isolated mitochondria, and is based on electrochemical proton gradient of mitochondrial inner membrane.
In certain embodiments of the methods provided herein, the recipient having a partial reduction of endogenous mtDNA in co-incubated with exogenous mitochondria from a healthy donor for a sufficient period of time to non-invasively transfer exogenous mitochondria into the recipient cell, thereby generating a mitochondria replaced cell. In other embodiments, the recipient having a partial reduction of endogenous mtDNA in co-incubated with exogenous mtDNA from a healthy donor for a sufficient period of time to non-invasively transfer exogenous mitochondria into the recipient cell, thereby generating a mitochondria replaced cell. Identifying the “sufficient period of time” to non-invasively transfer exogenous mitochondria and/or exogenous mtDNA into the recipient cell is within the skill of those in the art. The sufficient or proper time period will vary according to various factors, including but not limited to, the particular type of cells, the amount of starting material (e.g., the number of recipient cells and/or amount of endogenous mtDNA to be replaced), the amount of donor material (e.g., the quantity, quality, and/or purity of exogenous mtDNA) and/or the culture conditions. In various embodiments the sufficient period of time to non-invasively transfer exogenous mitochondria and/or exogenous mtDNA into a recipient cell is about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 1-2 weeks, about 2-3 weeks or about 3-4 weeks. In certain embodiments, at the end of the co-incubation period, the recipient cells will have a majority of the exogenous mtDNA and be substantially free of any exogenous mitochondria organelles.
Another feature of the current invention is the finding that the total mtDNA copy number in the MirC does not substantially increase, relative to the original recipient cell. In contrast, other less efficient methods have attempted to add on mitochondria without modulating the recipient cell before the co-incubation step, or transfer exogenous mitochondria using centrifugation without modulating the recipient cell prior to the centrifugation. Consequently, the resultant cell populations using the inefficient methods tend to have large increase in the total mtDNA copy number. Thus, in certain embodiments, 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.
The use of non-invasive transfer is another unique aspect of the present invention. Previous methods have employed invasive instruments to inject exogenous mitochondria, physically force the mitochondria into the cells by centrifugation, or similar harsh conditions that are harmful to the recipient cells. In clinical settings, particularly when the recipient cell number may be limited, such as with hematopoietic stem cells or T cells, harsh manipulation of the cells in undesirable. Therefore, the use of the non-invasive transfer is a beneficial feature of this invention, which lends itself to use in the clinical setting.
As provided herein, the exogenous mitochondria, exogenous mtDNA, or a combination thereof can be autologous or allogeneic to the recipient cell. In some embodiments, the exogenous mtDNA is allogeneic, relative to the recipient cell. For example, the exogenous mtDNA can be obtained from the same species as the recipient cell, and have a different genotype than that of the recipient cell. In other embodiments, the exogenous mitochondria, exogenous mtDNA, or a combination thereof is autologous. By way of example, an exemplary autologous exogenous mtDNA can include mtDNA from a healthy donor cell, for example a “young” donor cell such as from umbilical cord blood, and the recipient cell can be from the same subject, and be an “old” recipient cell, where the terms “young” and “old” refer to the total number of times the cells in the population have doubled or the age of the subject from which the cells are taken. Another exemplary autologous exogenous mtDNA can include, for example, donor mtDNA that has been isolated from the same subject as the recipient cell and modified prior to replacing it with the recipient cell. In certain embodiments, only the mtDNA and/or mitochondria are allogenic and the recipient cell is autogenic to the subject in need of an exogenous mtDNA and/or exogenous mitochondria.
In certain embodiments, the replacement of mtDNA in the recipient cell can be evaluated by sequencing the DNA sequences of hyper variable region (HVR) of mtDNA, for example, the HV1 and/or HV2 of the D-loop, and comparing it to the sequence of both the donor mitochondria and the recipient cells. In specific embodiments, the differences in sequences between the recipient cell and the donor mitochondria can be identified by a Single Nucleotide Polymorphism assay. For example, the amplified sequences of the mtDNA from the recipient cell and the donor mitochondria can be cloned into a plasmid for use as a standard for quantification.
In some embodiments, the cells (i.e. donor cells and recipient cells) are animal cells or plant cells. In specific embodiments, the cells are mammalian cells. In some embodiments, the cells are isolated from a mammalian subject who is selected from a group consisting of: a human, a horse, a dog, a cat, a mouse, a rat, a cow and a sheep. In some embodiments, the cells are human cells. In some embodiments, the cells are cells in culture. The cells may be obtained directly from a mammal (preferably human), or from a commercial source, or from tissue, or in the form for instance of cultured cells, prepared on site or purchased from a commercial cell source and the like. In certain embodiments, the cells are primary cells (i.e., cells obtained directly from living tissue, for example, biopsy material). The cells may come from any organ including, but not limited to, the blood or lymph system, from muscles, any organ, gland, the skin, or the brain. In certain embodiments, the cells are somatic cells. In some embodiments, the cells are selected from the group consisting of epithelial cells, neural cells, epidermal cells, keratinocytes, hematopoietic cells (e.g., bone marrow cells), melanocytes, chondrocytes, hepatocytes, B-cells, T cells, erythrocytes, macrophages, monocytes, fibroblasts, muscle cells, vascular smooth muscle cells, hepatocytes, splenocytes, and pancreaticR cells.
As provided herein, in specific embodiments, the donor cells are commercially available cells cultured under Current Good Manufacturing Practices (cGMP). For example, the donor cells can be obtained from a cell repository, such as Waisman Biomanufacturing, or similar commercial resource, such as a commercial source that generates cGMP compliant cells. In some embodiments, the donor cells are cGMP manufactured bone-marrow derived Mesenchymal Stromal Cells (BM-MSCs). In other embodiments, the cells are cGMP grade human hepatocytes. As such, it is also understood that the donor cells can be frozen cells that are thawed prior to isolating the mitochondria. However, the mitochondria need not be isolated after freezing the cells, and can be isolated from fresh cells and used immediately, or, in certain embodiments, the mitochondria can be isolated and then frozen before transferring into the recipient cell.
In some embodiments, the cells are cancer cells. Typically, the cancer cells are isolated from a cancer selected from the group consisting of breast cancer, prostate cancer, lymphoma, skin cancer, pancreatic cancer, colon cancer, melanoma, malignant melanoma, ovarian cancer, brain cancer, primary brain carcinoma, head-neck cancer, glioma, glioblastoma, liver cancer, bladder cancer, non-small cell lung cancer, head or neck carcinoma, breast carcinoma, ovarian carcinoma, lung carcinoma, small-cell lung carcinoma, Wilms' tumor, cervical carcinoma, testicular carcinoma, bladder carcinoma, pancreatic carcinoma, stomach carcinoma, colon carcinoma, prostatic carcinoma, genitourinary carcinoma, thyroid carcinoma, esophageal carcinoma, myeloma, multiple myeloma, adrenal carcinoma, renal cell carcinoma, endometrial carcinoma, adrenal cortex carcinoma, malignant pancreatic insulinoma, malignant carcinoid carcinoma, choriocarcinoma, mycosis fungoides, malignant hypercalcemia, cervical hyperplasia, leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, chronic granulocytic leukemia, acute granulocytic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, hairy cell leukemia, neuroblastoma, rhabdomyosarcoma, Kaposi's sarcoma, polycythemia vera, essential thrombocytosis, Hodgkin's disease, non-Hodgkin's lymphoma, soft-tissue sarcoma, osteogenic sarcoma, primary macroglobulinemia, and retinoblastoma.
In some embodiment, the cells are stem cells. As used herein, the term “stem cell” refers to an undifferentiated cell that can be induced to proliferate. The stem cell is capable of self-maintenance or self-renewal, meaning that with each cell division, one daughter cell will also be a stem cell. Stem cells can be obtained from embryonic, post-natal, juvenile, or adult tissue. Stem cells can be pluripotent or multipotent. The term “progenitor cell,” as used herein, refers to an undifferentiated cell derived from a stem cell, and is not itself a stem cell. Some progenitor cells can produce progeny that are capable of differentiating into more than one cell type. Stem cells include pluripotent stem cells, which can form cells of any of the body's tissue lineages: mesoderm, endoderm and ectoderm. Therefore, for example, stem cells can be selected from a human embryonic stem (ES) cell; a human inner cell mass (ICM)/epiblast cell; a human primitive ectoderm cell, a human primitive endoderm cell; a human primitive mesoderm cell; and a human primordial germ (EG) cell. Stem cells also include multipotent stem cells, which can form multiple cell lineages that constitute an entire tissue or tissues, such as but not limited to hematopoietic stem cells or neural precursor cells. Stem cells also include totipotent stem cells, which can form an entire organism. In some embodiment, the stem cell is a mesenchymal stem cell. The term “mesenchymal stem cell” or “MSC” is used interchangeably for adult cells which are not terminally differentiated, which can divide to yield cells that are either stem cells, or which, irreversibly differentiate to give rise to cells of a mesenchymal cell lineage, e.g., adipose, osseous, cartilaginous, elastic and fibrous connective tissues, myoblasts) as well as to tissues other than those originating in the embryonic mesoderm (e.g., neural cells) depending upon various influences from bioactive factors such as cytokines. In some embodiments, the stem cell is a partially differentiated or differentiating cell. In some embodiments, the stem cell is an induced pluripotent stem cell (iPSC), which has been reprogrammed or de-differentiated. In specific embodiments, the recipient cell is an iPSC. In other embodiments, the recipient cell is a hematopoietic stem cell (HSC) or a MSC. Stem cells can be obtained from embryonic, fetal or adult tissues.
In other embodiments, the cells are immune cells. In specific embodiments, the recipient cell is an immune cell. In some embodiments, the immune cell is selected from the group consisting of a T cell, a phagocyte, a microglial cell, and a macrophage. In specific embodiments, the T cell is a CD4+ T cell. In other embodiments, the T cell is a CD8+ T cell. In yet further embodiments, the T cell is a chimeric antigen receptor (CAR) T cell. In specific embodiments, the recipient cell is an exhausted or near exhausted T cell in a state or near a state of T cell dysfunction.
Also provided herein are methods for the transfer of mtDNA and/or mitochondria that involve the use of a second active agent in combination with any of the methods described in Section 5.2. The transfer of mitochondria has been reported to involve the endocytosis pathway, which is an ATP-dependent process. For example, under certain cell culture conditions, mitochondria have been observed to be engulfed via macropinocytosis (see, e.g., Kitani et al., J Cell Mol Med., 2014, 18(8):1694-1703). Accordingly, the present invention also relates to the novel findings that the use of a second active agent prior to co-incubating the recipient cell with exogenous mitochondria and/or exogenous mtDNA can promote the uptake of the exogenous mitochondria and/or exogenous mtDNA.
Various types of agents can be used to promote the uptake of the exogenous mitochondria and/or exogenous mtDNA. In some embodiments, the second active agent is 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. In specific embodiments, the activator of endocytosis is a modulator of cellular metabolism. Cellular metabolism can be modulated using various methods known to one skilled in the art. In certain embodiments, modulation of cellular metabolism comprises nutrient starvation, a chemical inhibitor, or a small molecule.
As described above, transfer of intact mitochondria has been reported to occur by an endocytosis pathway. For example, the exogenous mitochondria and/or exogenous mtDNA can be transferred by uptake of intact mitochondria via the endocytosis pathway. The endocytosis pathways can be subdivided into four categories: 1) clathrin-mediated endocytosis, 2) caveolae, 3) macropinocytosis, and 4) phagocytosis. Clathrin-mediated endocytosis is mediated by small (approx. 100 nm in diameter) vesicles that have a morphologically characteristic coat made up of a complex of proteins that are mainly associated with the cytosolic protein clathrin. Accordingly, in certain embodiments, the endocytosis pathway for mitochondrial transfer is a clathrin-dependent endocytosis pathway. In other embodiments, the endocytosis pathway for mitochondrial transfer is a clathrin-independent pathway. In specific embodiments, the endocytosis pathway is macropinocytosis.
Macropinocytosis has been suggested to be an important process in nutrient-deprived environments. As a result, it was hypothesized that a shortage of cellular nutrients or an inhibition of the pathways or target molecules that are activated with sufficient nutrition such as mTOR could be a strategy to augment the cellular engulfment of intact mitochondria into the cytosol. Specifically, as provided herein, it was discovered that a suppression of mTOR can enhance the uptake of exogenous mitochondria. mTOR is an essential sensor of amino acids, energy, oxygen, and growth factors, and a key regulator of protein, lipid, and nucleotide synthesis that is involved in uptake of extracellular nutrients. Accordingly, in some embodiments, the methods provided herein further comprise contacting a recipient cell with a small compound, a peptide, or a protein that can increase macropinocytosis. In specific embodiments, the methods provided herein further comprise modulating cellular metabolism of a recipient cell prior to transfer of the exogenous mitochondria and/or exogenous mtDNA. In certain embodiments, the modulating cellular metabolism is performed using the same small compound, a peptide, or a protein that can increase macropinocytosis.
Modulating cellular metabolism may be accomplished by any of a number of well-known techniques including but not limited to those described herein, and in the cited references. For example, in some embodiments, modulating cellular metabolism is performed by nutrient starvation or nutrient deprivation. In other embodiments, modulating cellular metabolism is performed by a chemical inhibitor or small molecule. In specific embodiments, the chemical inhibitor or small molecule is an mTOR inhibitor.
Various compounds are known to inhibit mTOR, including rapamycin, also known as sirolimus (CAS Number 53123-88-9; C51H79NO13), and rapamycin derivatives (e.g., rapamycin analogs, also known as “rapalogs”). Rapamycin derivatives include, for example, temsirolimus (CAS Number 162635-04-3; C56H87NO16), everolimus (CAS Number 159351-69-6; C53H83NO14), and ridaforolimus (CAS Number 572924-54-0; C53H84NO14P). Accordingly, in some embodiments, the methods provided herein for mitochondrial transfer further comprise modulating cellular metabolism of a recipient cell prior to transfer of the exogenous mitochondria and/or exogenous mtDNA using rapamycin or a derivative thereof. It is understood that the embodiments described above for modulating cellular metabolism are non-limiting, and modulating cellular metabolism need not involve a chemical compound or small molecule.
Accordingly, in some embodiments, rapamycin or a derivative thereof, which include clinically approved drugs, can be utilized to increase the transfer efficiency of exogenous mitochondria, either as a stand-alone method or in combination with any of the methods provided herein, such as methods involving the partial reduction in the endogenous mitochondria of the recipient cells.
One skilled in the art would understand that additional methods of delivery can also be used to introduce the exogenous mitochondria and/or exogenous mtDNA, and that macropinocytosis is an exemplary pathway. In some embodiments, the mtDNA can be delivered by clathrin-dependent endocytosis, or clathrin-independent endocytosis. In specific embodiments, the clathrin-independent pathway can be, for example, CLIC/GEEC endocytic pathway, Arf6-dependent endocytosis, flotillin-dependent endocytosis, macropinocytosis, circular doral ruffles, phagocytosis, or trans-endocytosis. It is further understood that delivery of exogenous mitochondria and/or exogenous mtDNA can be enhanced by the use of any compound that stimulates mitochondrial delivery, such as an activator of endocytosis. Non-limiting exemplary compounds suitable for activating endocytosis include, for example, phorbol-12-myristate-13-acetate (PMA) (C36H56O8), 12-O-tetradecanoylphorbol 13-acetate (TPA) (C36H56O8), tanshinone IIA sodium sulfonate (TSN-SS) (C19H17O6S.Na), and phorbol-12,13-dibutyrate, or derivatives thereof. Furthermore, it is also understood that non-endocytosis mediated transfer of mtDNA and/or mitochondria can be used, including methods that bypass endocytosis and/or cell fusion.
Provided herein are various methods for the treatment of conditions associated with mutant mtDNA and/or dysfunctional mitochondria, uses of compositions for the treatment of conditions associated with mutant mtDNA and/or dysfunctional mitochondria, and uses of compositions in the manufacture of medicaments for the treatment of conditions associated with mutant mtDNA and/or dysfunctional mitochondria. Also provided are methods of treatment involving the use of exogenous mitochondria and/or exogenous mtDNA to restore or enhance the function of endogenous mitochondria, uses of compositions to restore or enhance the function of endogenous mitochondria, and uses of compositions in the manufacture of medicaments for the treatment of a subject in need of mitochondrial replacement. In certain embodiments, the treatment involves prevention of mitochondrial dysfunction.
5.4.1 Methods of Treating an Age-Related Disease
In certain embodiments, provided herein are methods of treating a subject having or suspected of having an age-related disease involving any of methods described in Section 5.2 and/or Section 5.3. In some embodiments, provided herein are methods of treating a subject having or suspected of having an age-related disease involving generating a mitochondria replaced cell ex vivo or in vitro by contacting a recipient cell with an agent that reduces endogenous mtDNA or reduces endogenous mitochondrial function, incubating the recipient cell for a sufficient period of time for the agent to partially reduce the mtDNA copy number in the recipient cell or partially reduce the endogenous mitochondrial function, co-incubating (1) the recipient cell in which the endogenous mtDNA or endogenous mitochondrial function has been partially reduced, and (2) exogenous mitochondria and/or exogenous mtDNA from a healthy donor, for a sufficient period of time to non-invasively transfer exogenous mitochondria into the recipient cell, thereby generating a mitochondria replaced cell, and then administering a therapeutically effective amount of the mitochondria replaced recipient cell to the subject having or suspected of having an age-related.
In certain embodiments, the age-related disease includes an autoimmune disease, a metabolic disease, a genetic disease, cancer, a neurodegenerative disease, and immunosenescence. The metabolic disease can include diabetes. Non-limiting examples of neurodegenerative diseases that can be treated by the methods provided herein include Alzheimer's disease, or Parkinson's disease. In addition, the genetic disease capable of being treated include Hutchinson-Gilford Progeria Syndrome, Werner Syndrome, and Huntington's disease. Additional age-related diseases that involve dysfunctional mitochondria are also contemplated.
In certain embodiments, the methods of treating a subject having or suspected of having an age-related disease involves generating a MirC, where the recipient cell used to generate the MirC is a T cell or a hematopoietic stem cell (HSC). For example, endogenous mtDNA, endogenous mitochondria, or a combination thereof in a senescent T cell or hematopoietic stem cell (HSC) can be replaced for rejuvenation. The in vitro or ex vivo mitochondrial replacement can be a feasible option for the treatment using human T cells and/or hematopoietic stem cells with diseased patients. Thus in some embodiments, the methods provided herein can be used to delay senescence and/or extending lifespan in a cell by non-invasively transferring isolated exogenous mitochondria from a healthy, non-senescent cell into a senescent or near senescent cell to rejuvenate the recipient cell, and the resulting rejuvenated MirC can then be administered to a patient having or suspected of having an age-related disease.
As demonstrated herein, the rejuvenation of senescent T cells is one possible embodiment by which the present invention can be used to treat a subject having an age-related disease, such as cancer. By way of example, an old T cell, exhibiting a Senescence Associated Secretory Phenotype (SASP) consisting of inflammatory cytokines, growth factors, and proteases, reduced and/or slower rates of cell population doublings, shortened telomeres, increased DNA damage response (DDR), or a combination thereof can be rejuvenated by using the methods provided herein to non-invasively transfer, for example, isolated mitochondria from a young, healthy T cell that is autologous to a subject having an age-related disease, such as cancer. The T cell-derived MirC with characteristics of a young, non-senescent cell can then be administered to the subject for treatment of the age-related disease.
Thus, in specific embodiments, the methods of treating a subject having or suspected of having an age-related disease involves generation of a MirC where the recipient cell is a T cell. T cell fate is regulated by the metabolic pathway, with either glycolysis or oxidative phosphorylation (OXPHOS) being responsible for providing a majority of the energy to T cells. Glycolysis dominant T cells select to differentiate into effector T cells, whereas OXPHOS dominant T cells for memory T cells. Thus, exogenous mitochondria and/or mtDNA can be used to modulate T cell fate. For example, in the case of allergy, exogenous mitochondria and/or mtDNA could be used to calm hyper-activated T cells. In other situations, such as in cancer immunotherapy, exogenous mitochondria and/or mtDNA could empower anti-tumor T cells to allow the T cells to persist for a longer time, or facilitate T cell lytic capacity and/or reduce tumor burden. Moreover, emerging treatments using chimeric antigen receptor T cells (CAR T) utilize autologous T cells. Those CAR T cells might be in fatigue due to aging or malnutrition such as cachexia which is frequently seen in a severe pathologic stage of cancer. The mitochondrial replacement technology may energize and rejuvenate CAR T to provide more ATP leading to better outcomes.
Accordingly, in certain embodiments, the methods of treating a subject involve a recipient cell that is a T cell. The T cell can be a CD4+ T cell, a CD8+ T cell, or a CAR T cell. In specific embodiments, the mitochondrial replacement in the recipient T cell results in a T cell with a prolonged lifespan. For example, the lifespan can be increased about 1.5 fold, about 2 fold, about 3 fold, about 4 fold, about 5 fold, or greater than 5 fold. In specific embodiments, the mitochondrial replacement in the recipient T cell inhibits or delays senescence of the recipient T cells, as compared to a T cell without mitochondrial replacement. As described in Section 5.2, lifespan can be prolonged by performing mtDNA replacement using exogenous mitochondria and/or exogenous mtDNA from a donor cell that is younger than the recipient cell. In certain embodiments, the donor and recipient cell have a difference in PDL of about 1.5 fold, about 2 fold, about 2.5 fold, about 3 fold, about 4 fold, about 5 fold, or greater than 5 fold. In other embodiments, the donor and recipient cells are from subjects that are separated in age by about 5 years, about 10 years, about 15 years, about 20 years, or greater than 20 years. In other specific embodiments, the mitochondrial replacement in the recipient T cell results in T cells having increased lytic capacity, relative to T cells not having mitochondrial replacement. In yet further embodiments, the mitochondrial replacement in the T cells results in reduced tumor burden.
Although in certain embodiments plasmid-based gene transfection can be used to generate a T cell with exogenous mitochondria and/or exogenous mtDNA, in other embodiments mRNA transfection can be used. The use of mRNA transfection can decrease the chance of the RNA sequence being integrated into the host genome, and can also have minimal long-term gene expression that would cause the endogenous mtDNA reduction.
In certain embodiments, the MaxCyte electroporator can be used for mRNA transfection, particularly in the clinical setting, which has cleared the standards of Good Manufacturing Practice and Good Clinical Practice. The transfection can be performed using the MaxCyte electroporator according to the manufacturer's protocol.
The methods of treating a subject having or suspected of having an age-related disease can also involve the generation of a MirC using the methods provided herein, where the recipient cell is a hematopoietic stem cell (HSC). Hematopoietic stem cells (HSC) supply not only blood cells, but also, for example, the endothelium which fixes damaged resident cells in the remote organs via trans-differentiation. Moreover, malfunctions of HSCs have been reported to be involved in senescence throughout the whole body. Therefore, it is contemplated that HSC-derived MirC can be used as a method of treatment in any age-related disease.
In addition, allogenic HSC transplantation can result in rejection of the transplant or even graft-versus-host disease. Autologous HSC transplantation is often a safer and more practical measure for disease intervention. For example, autologous HSC transplantation typically does not require the preconditioning with immunosuppressive agents, such as radiation and chemicals. Accordingly, in vitro or ex vivo generation of a MirC using exogenous mtDNA from a healthy young mitochondria in an autologous HSC that is then returned back to the patients' bodies is envisioned using the methods provided herein.
In certain embodiments, the HSC is autologous to the subject in need of mitochondrial and/or mtDNA replacement, and the exogenous mtDNA is allogenic. As provided herein, the mtDNA replacement in an HSC can result in a differentiated cell with functional mitochondria and/or a differentiated cell with improved function. Accordingly, the methods provided herein can be used in the setting of HSC transplantation.
Aging alters the biological processes and leads to development of degenerative disorders, such as Alzheimer's disease, atherosclerosis, osteoporosis, type 2 diabetes mellitus, and tissue fibrosis which is causative for chronic kidney disease and chronic obstructive pulmonary disease. Mitochondria can play a role in senescence, via reactive oxygen species generated by mitochondria, which can impact the ageing process. Mitochondrial dysfunction in aging is in a vicious cycle related to a deregulated nutrient sensing where a shortage of nicotinamide adenine dinucleotide (NAD+), caused by downregulation of nicotinamide phosphoribosyltransferase (NAMPT) and hyperactivation of poly (ADP-ribose) polymerase 1 (PARP1), leads to an inhibition of NADtdependent deacetylase sirtuin 1 (SIRT1). It then relays to the acetylation-dependent inactivation of PGC1α consequently resulting in a depressed mitochondrial biogenesis that exaggerates the NAD+ availability. The low activity of PGC1α yields downregulated the expressions not only of mitochondrial proteins encoded in nucleus but also of the mitochondrial transcription factor TFAM neighboring the mitochondrial DNA.
In addition to the two core senescence-regulating pathways including p53 and p16/Rb, senescence-associated secretory phenotype (SASP), where an array of inflammatory cytokines, chemokines, and proteases such as IL-1, IL-6/VEGF, IL-8, and CXCL9/MMP are released, is one of the most characterized phenomena in senescence. The transcription factor GATA4 is degraded with the association of the autophagic adaptor p62 by selective autophagy under normal condition, whereas DNA damage response (DDR) kinases ATM (ataxia telangiectasia mutated) and ATR (ataxia telangiectasia and Rad3-related) received senescence signals facilitate the dissociation between GATA4 and p62 and stabilize GATA4, in turn activate NF-kB through TRAF3IP2 (tumor necrosis factor receptor-associated factor interacting protein 2) and ILIA and support SASP. SASP is completely hampered in rho0 cells (mtDNA free cells established by a forced mitophagy). Mitochondrial replacement of oocytes derived from the old in an experimental IVF surely promoted the success rate for zygote formation, development and embedding of embryo, and bearing offspring.
Impaired proteostasis (protein homeostasis) is another characteristics in aging. The integrity of proteostasis is strictly maintained by translational regulation, protein folding chaperon, ubiquitin-proteasome system (UPS), and the autophagy-lysosome system. Because chaperones depend upon ATP, decrease of bioenergy with aging jeopardize the function to correct protein folding. Both UPS and the autophagy-lysosome system, including mitophagy, decline with time. The alternations of these three systems generate aggregates which are not recycled in cytosol leading to degenerative disorders. In mitochondrial matrices, the accumulation of aberrant proteins not only actuates the system to degrade them but also offers an opportunity to recover the mitochondrial function communicating to nucleus termed as mitochondrial unfolded protein response (UPR′). All the above mentioned pathways involve mitochondria. The mitochondrial replacement in somatic cells could break the deleterious worsening cycle of aging, slow the senescent process, and even rejuvenate cells.
Thus, the methods provided herein offer clinically viable methods to treat heteroplasmy, and/or treat various diseases, such as diseases associated with senescence, by replacing endogenous dysfunctional mitochondria, such as endogenous mitochondria with mutant mtDNA, with young and/or healthy mitochondria that can have either an autologous or allogeneic origin.
In some embodiments, the methods provided herein for mitochondria replacement can be used for the treatment of mitochondrial disease or disorder, as well as senescence, cancer, and immune system deficiencies.
5.4.2 Methods of Treating a Mitochondrial Disease or Disorder
Also provided herein, are methods of treating a subject having or suspected of having mitochondrial disease or disorder according to any of methods described in Section 5.2 and/or Section 5.3. In some embodiments, the methods of treating a subject having or suspected of having a mitochondrial disease or disorder include generating a MirC according to any of the methods described in Section 5.2 and/or Section 5.3, and then administering a therapeutically effective amount of the mitochondria replaced recipient cell to the subject having or suspected of having a mitochondrial disease or disorder.
Various mitochondrial diseases or disorders are known, and all are capable of being treated using the methods provided herein. For example, the mitochondrial disease or disorder capable of being treated using the methods provided herein can be a Complex I deficiency (OMIM:252010). Complex I deficiency can be caused by a mutation in any of the subunits thereof. In another embodiment, the Complex I deficiency is caused by a mutation in a gene selected from the group consisting of NDUFV1 (OMIM:161015), NDUFV2 (OMIM:600532), NDUFS1 (OMIM:157655), NDUFS2 (OMIM:602985), NDUFS3 (OMIM:603846), NDUFS4 (OMIM:602694), NDUFS6 (OMIM:603848), NDUFS7 (OMIM:601825), NDUFS8 (OMIM:602141), and NDUFA2 (OMIM:602137).
In addition, the mitochondrial disease or disorder capable of being treated using the methods provided herein can be a Complex IV deficiency (cytochrome c oxidase; OMIM:220110). Complex IV deficiency can be caused by a mutation in any of the subunits thereof. In certain circumstances the Complex IV deficiency is caused by a mutation in a gene selected from the group consisting of MTCO1 (OMIM:516030), MTCO2 (OMIM:516040), MTCO3 (OMIM:516050), COX10 (OMIM:602125), COX6B1 (OMIM:124089), SCO1 (OMIM:603644), FASTKD2 (OMIM:612322), and SCO2 (OMIM:604272).
Mitochondrial diseases or disorders can be caused by or associated with a mutation. The mutation can be a point mutation, a missense mutation, a deletion, and an insertion. It is understood that the identification of mutations in mtDNA or nDNA is within the skill of those in the art, and exemplary methods are provided herein, such as, for example, a single nucleotide polymorphism (SNP) assay or a droplet digital PCR.
Non-limiting examples of specific types of mitochondrial diseases or disorders capable of being treated using the methods provided herein include Ornithine Transcarbamylase deficiency (hyperammonemia) (OTCD), Carnitine 0-palmitoyltransferase II deficiency (CPT2), Fumarase deficiency, Cytochrome c oxidase deficiency associated with Leigh syndrome, Maple Syrup Urine Disease (MSUD), Medium-Chain Acyl-CoA Dehydrogenase deficiency (MCAD), Acyl-CoA Dehydrogenase Very Long-Chain deficiency (LCAD), Trifunctional Protein deficiency, Progressive External Ophthalmoplegia with Mitochondrial DNA Deletions (POLG), DGUOK, TK2, Pyruvate Decarboxylase deficiency, and Leigh Syndrome (LS). In another embodiment, the mitochondrial disease or disorder is selected from the group consisting of Alpers Disease; Barth syndrome; (3-oxidation defects; carnitine-acyl-camitine deficiency; carnitine deficiency; co-enzyme Q10 deficiency; Complex II deficiency (OMIM:252011), Complex III deficiency (OMIM:124000), Complex V deficiency (OMIM:604273), LHON-Leber Hereditary Optic Neuropathy; MM-Mitochondrial Myopathy; LIMM-Lethal Infantile Mitochondrial Myopathy; MMC-Maternal Myopathy and Cardiomyopathy; NARP-Neurogenic muscle weakness, Ataxia, and Retinitis Pigmentosa; Leigh Disease; FICP-Fatal Infantile Cardiomyopathy Plus, a MELAS-associated cardiomyopathy; MELAS-Mitochondrial Encephalomyopathy with Lactic Acidosis and Strokelike episodes; LDYT-Leber's hereditary optic neuropathy and Dystonia; MERRF-Myoclonic Epilepsy and Ragged Red Muscle Fibers; MHCM-Maternally inherited Hypertrophic CardioMyopathy; CPEO-Chronic Progressive External Ophthalmoplegia; KSS-Kearns Sayre Syndrome; DM-Diabetes Mellitus; DMDF Diabetes Mellitus+Deafness; CIPO-Chronic Intestinal Pseudoobstruction with myopathy and Ophthalmoplegia; DEAF-Maternally inherited DEAFness; PEM-Progressive encephalopathy; SNHL-SensoriNeural Hearing Loss; Encephalomyopathy; Mitochondrial cytopathy; DEMCHO-Dementia and Chorea; AMDF-Ataxia, Myoclonus; ESOC Epilepsy; Optic atrophy; FBSN Familial Bilateral Striatal Necrosis; FSGS Focal Segmental Glomerulosclerosis; LIMM Lethal Infantile Mitochondrial Myopathy; MDM Myopathy and Diabetes Mellitus; MEPR Myoclonic Epilepsy and Psychomotor Regression; MERME MERRF/MELAS overlap disease; MHCM Maternally Inherited Hypertrophic CardioMyopathy; MICM Maternally Inherited Cardiomyopathy; MILS Maternally Inherited Leigh Syndrome; Mitochondrial Encephalocardiomyopathy; Multisystem Mitochondrial Disorder (myopathy, encephalopathy, blindness, hearing loss, peripheral neuropathy); NAION Nonarteritic Anterior Ischemic Optic Neuropathy; PEM Progressive Encephalopathy; PME Progressive Myoclonus Epilepsy; RTT Rett Syndrome; SIDS Sudden Infant Death Syndrome; and MIDD Maternally Inherited Diabetes and Deafness.
The methods provided herein for treating a mitochondrial disease or disorder can also include, in specific embodiments, a mitochondrial disease or disorder caused by mitochondrial DNA abnormalities, where the mitochondrial DNA abnormalities are selected from the group consisting of chronic progressive external ophthalmoplegia (CPEO), Pearson syndrome, Kearns-Sayre syndrome (KSS), diabetes and deafness (DAD), 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.
Mutations in mtDNA are thought to be associated with numerous clinical disorders. In adults, these include neurological diseases (e.g., migraine, strokes, epilepsy, dementia, myopathy, peripheral neuropathy, diplopia, ataxia, speech disturbances, and sensorineural deafness), gastrointestinal diseases (e.g., constipation, irritable bowel, and dysphagia), cardiac diseases (e.g., heart failure, heart block, and cardiomyopathy), respiratory diseases (e.g., respiratory failure, nocturnal hypoventilation, recurrent aspiration, and pneumonia), endocrine diseases (e.g., diabetes, thyroid disease, parathyroid disease, and ovarian failure), ophthalmological diseases (e.g., optic atrophy, cataract, ophthalmoplegia, and ptosis). In children, disorders thought to be associated with mtDNA mutations include neurological diseases (e.g., epilepsy, myopathy, psychomotor retardation, ataxia, spasticity, dystonia, and sensorineural deafness), gastrointestinal diseases (e.g., vomiting, failure to thrive, and dysphagia), cardiac diseases (e.g., biventricular hypertrophic cardiomyopathy and rhythm abnormalities), respiratory diseases (e.g., central hypoventilation and apnea), hematological diseases (e.g., anemia and pancytopenia), renal diseases (e.g., renal tubular defects), liver diseases (e.g., hepatic failure), endocrine diseases (e.g., diabetes and adrenal failure), and ophthalmological diseases (e.g., optic atrophy). Accordingly, the methods and compositions provided herein are contemplated for use in treating or preventing diseases and disorders associated with mutations in mtDNA.
In other specific embodiments, the methods provided herein allow for treating a mitochondrial disease or disorder where the mitochondrial disease or disorder is caused by nuclear DNA abnormalities, and the nuclear DNA abnormalities are 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).
Many individuals with a mutation of mtDNA display a cluster of clinical features that fall into a discrete clinical syndrome, such as the Kearns-Sayre syndrome (KSS), chronic progressive external ophthalmoplegia (CPEO), mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS), myoclonic epilepsy with ragged-red fibers (MERRF), neurogenic weakness with ataxia and retinitis pigmentosa (NARP), or Leigh syndrome (LS). However, considerable clinical variability exists and many individuals do not fit neatly into one particular category, which is well-illustrated by the overlapping spectrum of disease phenotypes (including mitochondrial recessive ataxia syndrome (MIRAS) resulting from mutation of the nuclear gene POLG, which has emerged as a major cause of mitochondrial disease or disorder.
Exemplary diseases where mitochondrial impairment is known to play an important role include, but are not limited to, the pathogenesis of many neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis. In addition, mitochondrial disease or disorders are subtyped into a number of syndromes according to the symptoms rather than the types of mutations. For example, mitochondrial syndromes include Mitochondrial myopathy, Encephalomyopathy, Lactic acidosis, Stroke-like symptoms (MELAS), Myoclonic Epilepsy with Ragged Red Fibers (MERRF), and Leigh syndrome.
5.4.3 Methods of Treating a Subject in Need of Mitochondrial Replacement
Also provided herein, are methods of treating a subject in need of mitochondrial replacement according to any of methods described in Section 5.2 and/or Section 5.3. In some embodiments, the methods of treating a subject in need of mitochondrial replacement include generating a MirC according to any of the methods described in Section 5.2 and/or Section 5.3, and then administering a therapeutically effective amount of the mitochondria replaced recipient cell to the subject in need of mitochondrial replacement.
A subject in need of mitochondrial replacement includes any subject that has a dysfunctional mitochondria. In certain embodiments, the subject in need of mitochondrial replacement has 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. Neurodegenerative diseases that can benefit from mitochondrial replacement include, but are not limited to, amyotrophic lateral sclerosis (ALS), Huntington's disease, Alzheimer's disease, Parkinson's disease, Friedreich's ataxia, Charcot Marie Tooth disease and leukodystrophy. The retinal disease can be wet or dry age-related macular degeneration, macular edema, or glaucoma. Other exemplary diseases, such as age-related diseases, and/or mitochondrial disease or disorder are described in more detail in Section 5.4.1 and 5.4.2.
A subject in need of mitochondrial replacement can also include a subject that is predisposed to mitochondrial dysfunction, and is asymptomatic. For example, the subject may have mutant mtDNA, but be without manifestations of, for example, a mitochondrial disease because the disease is an adult-onset disease. Therefore, the methods provided herein can be used to also prevent any of the diseases described herein by treating a subject in need of mitochondrial replacement.
The current invention also provides methods, as described in Section 5.2 and Section 5.3, for producing or enhancing the production of an induced pluripotent stem cell (iPSC) from a non-pluripotent cell. iPSCs have been demonstrated to be produced from non-pluripotent cells using exogenous expression of stemness factors, such as Oct3/4, Klf4, Sox2, and c-Myc. In addition, low amount of mitochondrial DNA (mtDNA) copies have been detected in undifferentiated ESCs, while this number increases upon differentiation together with the level of mitochondrial maturation (Facucho-Oliveira J M, et al, J Cell Sci 2007; 120(Pt 22):4025-4034). Thus, the present invention has also identified that the methods provided herein can be used to enhance the generation of iPSC by reducing endogenous mtDNA in a non-pluripotent by contacting a recipient non-pluripotent cell with an agent that reduces endogenous mtDNA, incubating the recipient non-pluripotent cell for a sufficient period of time for the agent to partially reduce the endogenous mtDNA in the non-pluripotent cell, and then introducing one or more expression cassettes for expression of Oct3/4, Klf4, Sox2, and c-Myc. In certain embodiments, exogenous mtDNA and/or exogenous mitochondria is non-invasively transferred into the recipient cells.
It is understood that the introduction of the one or more expression cassettes for expression of Oct3/4, Klf4, Sox2, and c-Myc can occur prior to, during, or after introduction of the agent that reduces endogenous mtDNA. Accordingly, in some embodiments, the methods for producing an iPSC includes introducing one or more expression cassettes for expression of Oct3/4, Klf4, Sox2, and c-Myc, contacting a recipient non-pluripotent cell with an agent that reduces endogenous mtDNA, and incubating the recipient non-pluripotent cell for a sufficient period of time for the agent to partially reduce the endogenous mtDNA in the recipient cell.
In certain embodiments, the method further comprises incubating the recipient cell with an exogenous mitochondria and/or exogenous mtDNA for a sufficient period of time to non-invasively transfer the exogenous mitochondria and/or exogenous mtDNA into a recipient cell. In specific embodiments, the method further comprises incubating the recipient cell with an exogenous mitochondria and/or exogenous mtDNA for a sufficient period of time to replace a majority of the endogenous mtDNA. The methods of producing an iPSC from a non-pluripotent cell that include transferring exogenous mitochondria and/or exogenous mtDNA and/or exogenous mitochondria can also include any of the embodiments described in Section 5.3.
Because low amounts of mitochondrial DNA (mtDNA) copies have been detected in undifferentiated embryonic stem cells (ESCs), the methods provided herein can also be used to promote pluripotency in non-pluripotent stem cells, and reduce the number of exogenous genes that are required to generate an iPSC. For example, in some embodiments, the methods provided herein can be used to generate an iPSC by reducing endogenous mtDNA in a non-pluripotent by contacting a recipient non-pluripotent cell with an agent that reduces endogenous mtDNA, incubating the recipient non-pluripotent cell for a sufficient period of time for the agent to partially reduce the endogenous mtDNA in the non-pluripotent cell, and then introducing one or more of Oct3/4, Klf4, Sox2, and c-Myc into the non-pluripotent cell, thereby generating a pluripotent stem cell. In some embodiments, the iPSC can even be generated using only small molecule agents and no exogenous factors.
In certain embodiments, the iPSC contains mutant mtDNA. For example, the mutant mtDNA can contain a point mutation, such as, for example, a point mutation in tRNA (e.g., MELAS). The mutant mtDNA can also include mtDNA with a long deletion of mtDNA. In other embodiments, the non-pluripotent cell for use in producing iPSC is heteroplasmic. The incorporation of mutant mtDNA can facilitate, for example, generation of disease models.
In some embodiments, the non-pluripotent recipient cells are somatic cells. In specific embodiments, the non-pluripotent cells are fibroblasts.
Culture conditions, identification, and establishment of iPSCs is within the skill of those in the art. For example, methods include those provided in U.S. Pat. Nos. 8,058,065, and 8,278,104, which are hereby incorporated by reference in their entireties.
As disclosed previously, mutant mtDNA and/or heteroplasmy can result in dysfunctional mitochondria. Therefore, assays useful for assessing mitochondrial function and/or mtDNA mutations in connection with the methods provided herein for mtDNA replacement include any assays known to a person skilled in the art that can be used to determine or predict the functionality of mitochondria and/or mtDNA mutations.
By way of example, assays to determine mitochondrial function include, for example, measurement of any one of the following: secretory factors associated with senescence (e.g., pro-inflammatory cytokines, proteases, and growth and angiogenesis factors, such as IL-1, IL-6/VEGF, IL-8, and CXCL9/MMP); mitochondria function by using Oroboros; Mitophagy by using Keima-Red; mitochondrial permeability; mitochondrial membrane potential; cytochrome c levels; reactive oxygen species; cell respiration; transcriptomics and proteomics for measurement of activated innate immunity, rescission of hyperactivated glycolysis, mitigation of ER stress, repression of mTOR-S6 pathway, and activation of cell cycle; mitochondria dynamics, such as fission and fusion, observed by superfine microscopy, and quantified by a specialized software; or any assay known in the art that measures mitochondrial function
Various sequencing methods can be used in combination of any of the methods provided herein to (1) detect mutant mtDNA, (2) quantify heteroplasmy, and/or (3) evaluate or confirm transfer of exogenous mitochondria and/or exogenous mtDNA. A stretch of roughly 1,100 nucleotides is gene-free that been called D-Loop, Displacement Loop, and Control Region. The D-Loop contains two regions within which mutations accumulate more frequently than anywhere else in the mitochondrial genome. The regions are called hypervariable regions HV1 and HV2, respectively. Accordingly, in some embodiments, mtDNA mutations can be identified in connection with the methods provided herein, by sequencing the hypervariable regions (HV) (i.e., HV1 and/or HV2) of the D-loop of mtDNA. mtDNA sequencing can be performed using any sequencing method known in the art. In specific embodiments, the sequencing method comprises a single nucleotide polymorphism (SNP) assay. In other embodiments, the sequencing method comprises digital PCR. In specific embodiments, the digital PCR is droplet digital PCR.
Also provided herein are compositions of cells obtained by any of the methods described in Sections 5.2-5.5. In certain embodiments, provided herein is 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 for a sufficient period of time to non-invasively transfer the exogenous mitochondria into the recipient cell, thereby generating a mitochondria replaced cell, wherein said mitochondria replaced cell comprises greater than 5% of exogenous mtDNA. In other aspects, provided herein is 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 mtDNA from 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, wherein said mitochondria replaced cell comprises greater than 5% of exogenous mtDNA.
The compositions can also be obtained by a method that involves contacting a cell with an agent that reduces mitochondrial function, and then incubating the recipient cell for a sufficient period of time for the agent to partially reduce endogenous mitochondrial function in the recipient cell. In some embodiments, the recipient cell having partially reduced endogenous mitochondrial function can then be co-incubated with either exogenous mitochondria from a healthy donor, for a sufficient period of time to non-invasively transfer exogenous mitochondria into the recipient cell, thereby generating a mitochondria replaced cell. In other embodiments, the recipient cell having partially reduced endogenous mitochondrial function can then be co-incubated with either exogenous mtDNA from a healthy donor, for a sufficient period of time to non-invasively transfer exogenous mitochondria into the recipient cell, thereby generating a mitochondria replaced cell. In some embodiments, the mitochondria replaced cell generated by the methods described above comprises greater than 5% of exogenous mtDNA.
As described above, the exogenous mitochondria can be comprised of exogenous mtDNA. Therefore, in some embodiments both exogenous mitochondria and exogenous mtDNA are transferred to the recipient cell and the MirC has both exogenous mitochondria and exogenous mtDNA. In other embodiments, the exogenous mtDNA is transferred to the recipient cell via exogenous mitochondria, and then the exogenous mtDNA is delivered to the endogenous mitochondria. Under certain circumstances the exogenous mitochondria is removed from the cell after the exogenous mtDNA is delivered to the endogenous mitochondria. Accordingly, in some embodiments, the MirC have exogenous mtDNA and does not have exogenous mitochondria.
Because the endogenous mtDNA of the recipient cell is partially degraded, the MirC that includes exogenous mitochondria, exogenous mtDNA, or a combination thereof can contain both exogenous mtDNA and endogenous mtDNA. Similarly, in scenarios where the exogenous mitochondria is transferred to the recipient cell, the MirC can contain both exogenous mitochondria and endogenous mitochondria. Thus, in specific embodiments, the compositions of one or more mitochondria replaced cells obtained by the methods provided herein have a mixture of endogenous and exogenous mitochondria. In other embodiments, the compositions of one or more mitochondria replaced cells obtained by the methods provided herein have a mixture of endogenous mtDNA and exogenous mtDNA (i.e., heteroplasmic mtDNA). In yet further embodiments, the one or more mitochondria replaced cells have a total mtDNA copy number that is 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.
The present invention also includes compositions for use in a method of generating mitochondria replaced that includes an agent that reduces endogenous mtDNA or an agent that reduces mitochondrial function, and a second active agent. In certain embodiments, the composition can further include an exogenous mitochondria, one or more recipient cells, or a combination thereof. In yet further embodiments, the composition can further include exogenous mtDNA.
As described in Section 5.3, various second active agents can be used in the methods for generating one or more mitochondria replaced cells. For example, in some embodiments, the second active agent includes large molecules, small molecules, or cell therapies, and the second active agent is optionally selected from 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.
The use of an activator of endocytosis was found to enhance the uptake of exogenous mitochondria in cells treated with the MTS-XbaIR plasmid, but had no effect on promoting uptake with “add on” or mock transfected cells, which indicated that this mechanism of transferring exogenous mitochondria was unique to the invention provided herein. Non-limiting exemplary compounds suitable for activating endocytosis include, for example, phorbol-12-myristate-13-acetate (PMA) (C36H56O8), 12-O-tetradecanoylphorbol 13-acetate (TPA) (C36H56O8), tanshinone IIA sodium sulfonate (TSN-SS) (C19H17O6S.Na), and phorbol-12,13-dibutyrate, or derivatives thereof. In some embodiments, the activator of endocytosis comprises a modulator of cellular metabolism.
Modulating cellular metabolism may be accomplished by any of a number of well-known techniques including but not limited to those described herein, and in the cited references. For example, in some embodiments, modulating cellular metabolism is performed by nutrient starvation or nutrient deprivation. In other embodiments, modulating cellular metabolism is performed by a chemical inhibitor or small molecule. In specific embodiments, the chemical inhibitor or small molecule is an mTOR inhibitor.
Various compounds are known to inhibit mTOR, including rapamycin, also known as sirolimus (CAS Number 53123-88-9; C51H79NO13), and rapamycin derivatives (e.g., rapamycin analogs, also known as “rapalogs”). Rapamycin derivatives include, for example, temsirolimus (CAS Number 162635-04-3; C56H87NO16), everolimus (CAS Number 159351-69-6; C53H83NO14), and ridaforolimus (CAS Number 572924-54-0; C53H84NO14P). Accordingly, in some embodiments, the compositions provided herein comprise rapamycin or a derivative thereof. It is understood that the embodiments described above for modulating cellular metabolism are non-limiting, and modulating cellular metabolism need not involve a chemical compound or small molecule, and can include modulation of other pathways beyond mTOR. It is also understood that the compositions can optionally comprise activators of endocytosis, and that it is not a required component. In addition, in some embodiments the invention provided herein can involve non-endocytosis mediated transfer of mtDNA and/or mitochondria, such as in non-clinical settings.
As described in Section 5.5, the present invention also provides, in certain embodiments, a composition for use in a method of producing an induced pluripotent stem cells (iPSC) from a non-pluripotent cell that includes an agent that reduces endogenous mtDNA, one or more expression cassettes for expression of Oct3/4, Klf4, Sox2, and c-Myc, and a recipient cell, wherein the recipient cell is a non-pluripotent cell, wherein the agent that reduces endogenous mtDNA is present in an amount effective to increase efficiency of producing an induced pluripotent stem cells (iPSC) from a non-pluripotent cell, as compared to a non-pluripotent cell not treated with an agent that reduces endogenous mtDNA. In some embodiments, the agent that reduces endogenous mtDNA is present in an amount effective to increase efficiency of producing an induced pluripotent stem cells (iPSC) from a non-pluripotent cell, as compared to a non-pluripotent cell not treated with an agent that reduces endogenous mtDNA. This is based in part on the observation that pluripotent cells have a reduction in mtDNA copy number. In specific embodiments, the composition for use in a method of producing an iPSC further comprises exogenous mitochondria and/or exogenous mtDNA.
The present invention also includes pharmaceutical compositions for use in the treatment of an age-related disease, a mitochondrial disease or disorder, a neurodegenerative disease, diabetes, a genetic disease, or any subject in need of mitochondrial replacement, as described in Section 5.4. In certain embodiments, provided herein are pharmaceutical compositions that include an isolated population of mitochondria replaced cells that have exogenous mitochondria from a healthy donor, and the cells are obtained by the methods described herein, such as in Sections 5.2-5.3. In other embodiments, the pharmaceutical composition includes an isolated population of mitochondria replaced cells with exogenous mitochondria and/or exogenous mtDNA from a healthy donor, and the cells are obtained by the methods described herein, such as in Sections 5.2-5.3. For example, in some embodiments, the mitochondria replaced cells that have exogenous mtDNA can optionally further include exogenous mitochondria. In other embodiments, the exogenous mtDNA is transferred into the cell via exogenous mitochondria, delivered to the endogenous mitochondria, and then the exogenous mitochondria is removed from the recipient cell.
The disclosure also provides a pharmaceutical composition comprising an isolated population of mitochondria replaced cells having an exogenous mitochondria from a healthy donor, wherein the cells are obtained by any of the methods provided herein for obtaining a mitochondrial replaced cell. In yet another aspect, the disclosure provides a pharmaceutical composition comprising an isolated population of mitochondria replaced cells having an exogenous mtDNA from a healthy donor, wherein the cells are obtained by any of the methods provided herein for obtaining a mitochondrial replaced cell. In some embodiments, the pharmaceutical composition comprising an isolated population of mitochondria replaced cells having an exogenous mtDNA from a healthy donor further comprises exogenous mitochondria.
For example, in some embodiments, a pharmaceutical composition comprising an exogenous mitochondria from a healthy donor are obtained by a method that involves contacting a cell with an agent that reduces mtDNA copy number, and then incubating the recipient cell for a sufficient period of time for the agent to partially reduce endogenous mtDNA copy number in the recipient cell. In some embodiments, the recipient cell having partially reduced endogenous mtDNA copy number can then be co-incubated with either exogenous mitochondria from a healthy donor, for a sufficient period of time to non-invasively transfer exogenous mitochondria into the recipient cell, thereby generating a mitochondria replaced cell. In other embodiments, the recipient cell having partially reduced endogenous mtDNA copy number can then be co-incubated with either exogenous mtDNA from a healthy donor, for a sufficient period of time to non-invasively transfer exogenous mitochondria into the recipient cell, thereby generating a mitochondria replaced cell. In some embodiments, the mitochondria replaced cell generated by the methods described above comprises greater than 5% of exogenous mtDNA.
In other embodiments, the cells are obtained by a method that involves contacting a cell with an agent that reduces mitochondrial function, and then incubating the recipient cell for a sufficient period of time for the agent to partially reduce endogenous mitochondrial function in the recipient cell. In some embodiments, the recipient cell having partially reduced endogenous mitochondrial function can then be co-incubated with either exogenous mitochondria from a healthy donor, for a sufficient period of time to non-invasively transfer exogenous mitochondria into the recipient cell, thereby generating a mitochondria replaced cell. In other embodiments, the recipient cell having partially reduced endogenous mitochondrial function can then be co-incubated with either exogenous mtDNA from a healthy donor, for a sufficient period of time to non-invasively transfer exogenous mitochondria into the recipient cell, thereby generating a mitochondria replaced cell. In some embodiments, the mitochondria replaced cell generated by the methods described above comprises greater than 5% of exogenous mtDNA. The agent that reduces mitochondrial function can either transiently or permanently reduce mitochondrial function. It is within the skillset of a person skilled in the art to be able to determine whether the agent would transiently (e.g., reversible inhibitor) or permanently (e.g., irreversible inhibitor) reduces mitochondrial function.
In certain embodiments of the pharmaceutical compositions provided herein, the cells are obtained by a method further comprising further comprising contacting the recipient cell with a second active agent prior to co-incubating the recipient cell with exogenous mitochondria and/or exogenous mtDNA. In some embodiments, the second active agent is 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. In specific embodiments, the activator of endocytosis is a modulator of cellular metabolism. In other embodiments, the modulator of cellular metabolism comprises nutrient starvation, a chemical inhibitor, or a small molecule. In further embodiments, the chemical inhibitor or the small molecule is an mTOR inhibitor. In yet further embodiments, said mTOR inhibitor comprises rapamycin or a derivative thereof.
As described above in Section 5.2, various types of cells can be used as recipient cells and donor cells. For example, the present disclosure describes numerous examples where the recipient cells are mammalian cells. However, it is also understood that any cell with a mitochondria can be a recipient cell. Therefore, the recipient cell can also be a plant cell.
In some embodiments, the animal cells are mammalian cells. In specific embodiments, the cells are somatic cells. In further embodiments, the somatic cells are epithelial cells. In yet further embodiments, the epithelial cells are thymic epithelial cells (TECs).
The present disclosure also provides compositions where the somatic cells are immune cells. For example, the compositions can comprise immune cells where the immune cells are T cells, such as exhausted T cells. In some embodiments, the composition includes rejuvenated T cells that contain exogenous mitochondria and/or exogenous mtDNA. For example, senescent T cells or near senescent T cells (e.g., immunosenescent) can serve as a recipient cell and a T cell-derived MirC can be generated using the methods provided herein to produce a T cell with healthy exogenous mitochondria and/or exogenous mtDNA. In specific embodiments, the T cells are CD4+ T cells. In other embodiments, the T cells are CD8+ T cells. In some embodiments, the T cells are chimeric antigen receptor (CAR) T cells. For example, in some embodiments the disclosure provides a MirC that is CAR-T cell, which is efficacious in killing a cancer cell. The MirC derived CART can have prolonged survival to enable increased immunosurveillance, and enhanced cancer cell killing. In other embodiments, the immune cells are phagocytic cells.
As described above, the compositions provided herein can also include a composition for use in delaying senescence and/or extending lifespan in a cell. The composition can include a senescent or near senescent cell having endogenous mitochondria, isolated exogenous mitochondria from a non-senescent cell, and an agent that reduces endogenous mtDNA copy number. The composition can also include a senescent or near senescent cell having endogenous mitochondria, isolated exogenous mitochondria from a non-senescent cell, and an agent that reduces mitochondrial function.
Also provided herein are compositions that include one or more mitochondria replaced cells that are derived from recipient cells that are bone marrow cells. In specific embodiments, the bone marrow cells are a hematopoietic stem cell (HSC), or a mesenchymal stem cell (MSC). For example, an HSC or MSC can be isolated from a subject having or suspected of having a mitochondrial disease, an age-related disease, or otherwise be in need of mitochondrial replacement, and have the endogenous mitochondria replaced with exogenous mitochondria. Subsequently, the HSC or MSC derived MirC can then be transplanted back into the subject in need of mitochondrial replacement. In yet further embodiments, the recipient cells are iPS cells. The compositions can be used in the clinical setting and can be efficacious in treating an age-related disease, treating a mitochondrial disease or disorder, treating a neurodegenerative disease, treating diabetes, or a genetic disease. For example, in some embodiments, the iPSC can be differentiated into a particular cell type prior to administering back into the subject, using methods known in the art.
In other embodiments, provided herein are pharmaceutical compositions that include an isolated population of pluripotent cells having a reduced amount of endogenous mtDNA, wherein the cells are obtained by any of the embodiments described in Section 5.5. In specific embodiments, the isolated population of pluripotent cells are iPS cells.
Administration of cells or compounds described herein is by any of the routes normally used for introducing pharmaceuticals. The pharmaceutical compositions of the invention may comprise a pharmaceutically acceptable carrier. In a specific embodiment, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized foreign pharmacopeia for use in animals, and more particularly in humans. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there are a wide variety of suitable formulations of pharmaceutical compositions of the present invention (see, e.g., Remington's Pharmaceutical Sciences, 17th ed. 1985).
Formulations suitable for administration include aqueous and non-aqueous solutions, isotonic sterile solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. In the practice of this invention, compositions can be administered, for example, orally, nasally, topically, intravenously, intraperitoneally, intrathecally or into the eye (e.g., by eye drop or injection). The formulations of compounds can be presented in unit dose or multi-dose sealed containers, such as ampoules and vials. Solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.
The dose administered to a patient, in the context of the present invention should be sufficient to induce a beneficial response in the subject over time, i.e., to prevent, ameliorate, or improve a condition of the subject. The optimal dose level for any patient will depend on a variety of factors including the efficacy of the specific modulator employed, the age, body weight, physical activity, and diet of the patient, and on a possible combination with other drug. The size of the dose also will be determined by the existence, nature, and extent of any adverse side-effects that accompany the administration of a particular compound or vector in a particular subject. Administration can be accomplished via single or divided doses.
The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.
All patents, applications, published applications, and other publications cited herein are incorporated herein by reference in their entirety. In the event that any description of terms set forth conflicts with any document incorporated herein by reference, the description of term set forth herein shall control.
Throughout this application various publications have been referenced. The disclosures of these publications in their entireties are hereby incorporated by reference in this application in order to more fully describe the state of the art to which this invention pertains. Although the invention has been described with reference to the examples provided above, it should be understood that various modifications can be made without departing from the spirit of the invention.
The examples in this section are offered by way of illustration, and not by way of limitation. The following examples are presented as exemplary embodiments of the invention. They should not be construed as limiting the broad scope of the invention.
A scheme of the method used to generate a mitochondria replaced cell (MirC) is provided in
XbaIR is one of the most powerful endonucleases and a standard sequence of mtDNA named under Cambridge reference sequence (CRS) in human mitochondria genome has as many as five recognition sites targeted by the particular endonuclease (
The gene transfer protocol of plasmid DNA to cells was optimized using Normal Human Dermal Fibroblast (NHDF) cells that expressed enhanced green fluorescent protein (EGFP) by using the Nucleofector electroporation-based transfection method. Following one day of 2 μg/ml puromycin exposure, an efficacy of more than 90% and viability of more than 90% was established (
To specifically evaluate the effectiveness of the MTS targeting sequence, a plasmid carrying MTS fused with EGFP was generated by subcloning the EGFP gene in place of the XbaIR gene to generate the pCAGGS-MTS-EGFP-PuroR plasmid (
Taken together, these results demonstrated that XbaI could be used to digest mitochondrial DNA, the cells could be efficiently transfected without effecting cell viability, and the expression vectors containing a MTS could effectively target mitochondria.
The efficiency and efficacy of the MTS-XbaIR expression vector relative to the conventional method that employs ethidium bromide (EtBr) was evaluated according to the scheme illustrated in
The enhanced degradation of endogenous mtDNA in the MTS-XbaIR treated group, relative to the EtBr group was further confirmed by microscopy of the DsRed labeled mitochondria (
The kinetics of the expression of XbaIR following plasmid gene transfer was examined by qPCR. On day 3, the expression reached the peak and then declined to zero on day 7 (
These results demonstrated that the reduction of mtDNA copy number in the XbaI endonuclease treated group was superior to that of the group treated with the conventional method of EtBr, and did not completely delete all of the endogenous mtDNA. Moreover, a brief selection using puromycin enabled significant enrichment of the cells expressing the MTS construct.
To evaluate whether exogenous mitochondria from a healthy donor cell could be transferred to a recipient cell with XbaI mediated depletion of mtDNA, NHDF cells were transfected with the MTS-GFP or MTS-XbaIR plasmids and selected using puromycin after 48 hours. After 6 days post-transfection, isolated mitochondria from human cell lines originated from the endothelium of the uterus (named as EPC100) that were labeled with DsRed were transferred to the donor cells. A scheme of the protocol is shown in
Following transfection with MTS-GFP or MTS-XbaI and selection with puromycin the mitochondria content was evaluated by TMRM staining. As shown in
The reduction of mitochondrial DNA was further confirmed by quantifying the number of mitochondrial DNA copies by qPCR of 12S-rRNA after adjusting with β-actin (Actb) in the nucleus (
Mitochondria were isolated from DsRed-Mt EMCs by differential centrifugation. In brief, the cells were harvested from culture dishes with homogenization buffer [HB; 20 mM HEPES-KOH (pH 7.4), 220 mM mannitol and 70 mM sucrose] containing a protease inhibitor mixture (Sigma-Aldrich, St. Louis, Mo., USA). The cell pellet was resuspended in HB and incubated on ice for 5 min. The cells were ruptured by 10 strokes of a 27-gauge needle on ice. The homogenate was centrifuged (400 g, 4° C., 5 min.) two times to remove unbroken cells. The mitochondria were harvested by centrifugation (6000 g, 4° C., 5 min.) and resuspended in HB. The amounts of isolated mitochondria were expressed as protein concentration using a Bio-Rad protein assay kit (Bio-Rad, Richmond, Calif., USA). Mitochondrial transfer was conducted by co-incubating isolated mitochondria with cells in 2 ml of standard medium at 37° C. under 5% CO2 for 24 h. Importantly, the co-incubation of isolated mitochondria with ρ(−) cells on day 12 resulted in a significant increase in mtDNA copy number, similar to the levels of control NHDF cells (
Consistent with the results shown in
Comparison between existing methods demonstrated that the endonuclease method of the present invention was more efficacious in generating a mitochondria replaced cell having exogenous mitochondria (
Previously established methods used in mitochondria biology utilized cells with a complete deletion of mitochondria ρ(0) cells (see, e.g., U.S. patent application Ser. No. 12/747,771, filed Sep. 23, 2010, and published as US 2011-0008778 A1, which is incorporated by reference herein in its entirety). However, the ρ(0) cells failed to engulf exogenous mitochondria (
In addition, the uptake of DsRed labeled exogenous mitochondria was monitored in ρ(−) cells treated with or without exogenous mitochondria, untransfected cells (add on Mt), or cells treated with the mock MTS-GFP plasmid. The fluorescent intensities of DsRed was quantified every 24 hours using NIH image software. The relative values to the initial intensities were depicted in bar graph (
These results demonstrate that the MTS-XbaI expression vector can generate ρ(−) cells that have a partial deletion of endogenous mitochondria, and the mitochondrial content can be rescued by transferring isolated exogenous mitochondria from donor cells. As described herein, the methods of the current invention provide improved efficiency of mitochondrial transfer, relative to previously described methods, such as those performed in combination with centrifugation, or simple “adding on” the mitochondria without partially reducing the endogenous mtDNA. However, mitochondrial transfer was unable to be performed in cells with a complete degradation of endogenous mitochondria (ρ(0) cells), which indicated that the uptake of exogenous mitochondria likely requires energy.
To further elucidate how mitochondrial transfer of intact mitochondria occurs, the fate of transferred mitochondria into cells was investigated separately on outer and inner membrane and nucleoid. In certain circumstances, transient intermitochondrial fusion events have been observed, where two mitochondria came into close apposition, exchanged soluble inter-membrane space and matrix proteins and re-separated, preserving the original morphology (see, e.g., Liu X et al., EMBO J. 2009; 28(20):3074-3089; Huang X et al. Proc Natl Acad Sci US A. 2013; 110(8):2846-2851). Therefore, transient intermitochondrial fusion events were analyzed under the conditions described herein.
Isolated mitochondria from EPC100 donor cells was labeled with DsRed, and recipient cells with EGFP-marked mitochondria were used. A diagram of the protocol employed is illustrated in
Mitochondrial transfer was performed according to the protocol illustrated in
These results demonstrate that donor mitochondria were transferred into the mitochondrial matrices in the recipient cells and dominate under the reduction of pre-existing mitochondria. Moreover, according to these experiments, almost all isolated mitochondria were engulfed. On the other hand, add-on type of mitochondrial transfer and mock-transfectant did not exhibit the rigorous engulfment, but aggregated major part of these exogenous mitochondria onto the cellular surface.
In summary, the results from Examples III and IV demonstrate that ρ(−) cells degraded the engulfed mitochondria (
Accordingly, it is hypothesized that the exogenous mitochondria are able to briefly interact with the endogenous mitochondria, and transport mtDNA during the brief contacts. Then, the exogenous mitochondrial membrane complexes can be degraded in the cytosol to provide building blocks for the reconstituted mitochondria. The mitochondria of the recipient cell that receive the exogenous mitochondria are able to gradually reconstitute the mitochondrial membrane complex, and demonstrate the functional recovery.
To assess the origin of mtDNA following the mtDNA replacement, the different nucleotides identified between NHDF and EPC100 by sequencing the hypervariable region 1 and 2 were used (
The heteroplasmy in the mitochondria replaced NHDF was further evaluated by the single nucleotide polymorphism assay to detect the difference between the recipient NHDF and the donor EPC100 (
These results demonstrate that the methods provided herein for replacement of mtDNA with exogenous mitochondria and/or exogenous mtDNA is completely novel, and an improvement over the existing technology. As described herein, the methods provided demonstrate that the transfer of mitochondria after MTS-XbaI mediated degradation of endogenous mitochondria can result in exogenous mtDNA being the predominant mtDNA.
Whether the replaced mitochondria work to generate energy was investigated by using Oroboros 02k according to the manufacturer's instructions. Representative oxygen consumption rate curves with native control cells, the ρ(−) cells, and the mitochondria replaced cells were generated and then the respiratory flow and control ratio was calculated (
In addition, the phenotypic recovery of the mitochondria replaced cells (MirC) was demonstrated by their proliferative capability (
These results demonstrated that this methodology offers mtDNA replacement with clinically applicable materials, and results in cells with functional mitochondria that enable phenotypic recovery of the mitochondria replaced cells (MirC).
To determine a method for increasing the ability of a cell to undergo MirC, the mechanisms that regulate the macropinocytosis of exogenous mitochondria were investigated. Since ρ(−) cells are exhausted of ATP as a result of the reduced mitochondria, it was hypothesized that the intracellular energetic state of ρ(−) cells was similar to a starved state. To this effect, two molecular pathways were investigated: mammalian target of rapamycin complex 1 (mTORC1) and AMP-activated protein kinase (AMPK). mTORC1 is an essential sensor of amino acids, energy, oxygen, and growth factors, and a key regulator of protein, lipid, and nucleotide synthesis. AMPK is a sensor of AMP levels, and the activation results in autophagy, mitochondrial biogenesis, glycolysis, and lipolysis. Both pathways are involved in uptake of extracellular nutrients.
As illustrated in
Treatment with PA or rapamycin demonstrated that although AMPK pathway was not significantly activated in ρ(−) cells (
Next, we examined the effects of rapamycin and palmitic acid upon mitochondria engulfment by treating the cells with rapamycin or palmitic acid simultaneously during the mitochondria co-cultivation. A scheme of the protocol is illustrated in
As shown in
These results indicate that activation of mTORC1 by rapamycin during mitochondrial transfer can enhance macropinocytosis of mitochondria. Further, these methods demonstrate that rapamycin, which is a clinically available drug, can be used to increase the efficiency of macropinocytosis for MirC generation.
To investigate whether mitochondrial diseased cells could be corrected by using the in-vitro mtDNA replacement technique, primary fibroblasts (7SP) derived from a patient diagnosed with Leigh syndrome having a mtDNA T10158C mutation were used as recipient cells (
The kinetics of the content of mtDNA in 7S fibroblasts was almost the same as in NHDF following the mitochondria replacement (
Importantly, the number of mtDNA copies following the mitochondria replacement recovered to the same value with that of the original 7S fibroblasts on day 12 (
Whether the mitochondria in 7S fibroblasts contained exogenous and healthy mtDNA was examined by sequencing the mitochondrial genome fragment that included the 10158 nucleotide. As shown in
To generate quantitative information, a single nucleotide polymorphism (SNP) assay was performed to estimate the heteroplasmy generated using this technology. The ND3 region of mitochondrial DNA was amplified using hmt10085-F primer (5′-CAACACCCTCCTAGCCTTACTACTAA-3′ (SEQ ID NO: 17)) and hmt10184-R primer (5′-GTCGAAGCCGCACTCGTA-3′ (SEQ ID NO: 20)), and the EPC100 specific probe (5′-ACATAGAAAAATCCACCC-3′ (SEQ ID NO: 18)) or the 7SP specific probe (5′-CTACATAGAAAAATCCAC-3′ (SEQ ID NO: 19)) (
Taken together these results demonstrate that the methods of generating a mitochondrial replaced cell described herein that use MTS-XbaI to partially reduce endogenous mitochondria can be effectively used in cells from a subject with a mitochondrial disease or disorder to improve the heteroplasmy level and reduce the amount of mutant mtDNA.
The functional activity of mitochondrial replaced 7SP fibroblasts was evaluated. As shown in
In addition, the mitochondrial replaced 7SP fibroblasts (ρ(−) Mt) cells demonstrated a dramatic extension of lifespan, up to about the 63th population doubling level (PDL) while the doubling time was over 120 hours, which is the threshold of growth arrest (
The functional effect of mitochondrial transfer in 7S fibroblasts was further evaluated by measuring the cell size (
Consistent with the functional recovery of 7SP cells after the mtDNA replacement, the notable increase in cell size observed in the early PDL in 7SP fibroblasts was inhibited following the mitochondria replacement (
In order to confirm that the observed improvement in cell function after mitochondria replacement was not due to contamination with other cell types, a short tandem repeat (STR) assay was performed that can definitely discriminate cells with different origins (
Taken together, these results demonstrated that mitochondrial transfer of exogenous isolated mitochondria having wild-type mtDNA into 7SP cells, which are derived from a patient with Leigh Syndrome, increased the lifespan of 7SP cells, and improved the cellular function. Importantly, the transfer did not transform the mitochondrial replaced 7SP cells into cancer cells.
The functional effect of mitochondrial replacement in the 7SP fibroblasts was further evaluated by analyzing the cells' respiratory function by using Oroboros 02k (
These results demonstrated that transfer of exogenous mitochondria into fibroblasts derived from a patient with a mitochondrial disease or disorder can yield functional mitochondria.
In order to characterize the properties of 7SP fibroblast-derived MirC, both a reperfusion and a starvation model under a culture condition were used for 7SP fibroblast-derived MirC, the original 7SP fibroblast, and NHDF as a control. These stress conditions induce apoptosis in cultured cells, the extent of which can be quantified by AnnexinV as an early marker and propidium iodide (PI) as a late marker. Among environmental insults, the reperfusion injuries are mainly attributed to mitochondrial dysfunction. Cells predisposed to mitochondrial dysfunction due to mtDNA mutation are more fragile to the reperfusion injuries than healthy cells.
Cells were seeded on 6 well plate at 1×105 cells per well. The next day, 600 μM H2O2 (FUJIFILM Wako Pure Chemical) was added to cells for the reperfusion model or essential amino-acid-free (“-EAA”) DMEM (FUJIFILM Wako Pure Chemical) without serum was used as the culture media for the starvation model. After 3 h H2O2 or 48 h starvation treatment, cells were washed with PBS and collected to centrifugal tube. Annexin V-FITC and PI solution were added in cells and allowed to react for 30 minutes at room temperature protecting from light. Then cells were rapidly subjected to FCM analysis using 488 and 561 nm laser lines. Fluorescence data were collected using SH800 (Sony). The flow cytometry files were analyzed by using FlowJo software (TreeStar).
The results indicated that 7SP cells, which originate from a subject with Leigh syndrome, were highly susceptible to both forms of stress (i.e., H2O2 and starvation). As shown in
The same trends as the reperfusion were recognized using the starvation model (
These results demonstrated that the transfer of exogenous mitochondria from a healthy cell into a cell with mutant mtDNA can improve the function of the recipient cell.
This example demonstrates that transfer of exogenous mitochondria into recipient cells reverted early stage senescence-associated secretory phenotype (SASP). A SASP consisting of inflammatory cytokines, growth factors, and proteases is a characteristic feature of senescent cells.
To determine whether transfer of exogenous mitochondria into senescent cells could revert the SASP, the expression levels of the representative SASP cytokines, IL-6 and IL-8, chemokine, CXCL-1, and growth factor, ICAM1 were quantitatively measured at the transcript levels for NHDF, 7SP fibroblast, and 7SP fibroblast-derived MirC cells, whose PDLs were almost the same, about 15 to 20 (
Taken together, these data demonstrate that mitochondria replacement is able to not only treat mitochondrial diseases with mutations of mtDNA, but also rejuvenate senescent cells, such as cells involved in an array of diseases, including neurodegenerative, cardiovascular, metabolic, and autoimmune diseases, and even cancers.
To determine whether inducible pluripotent stem cells (iPSCs) could be generated using cells derived from patients with a long deletion of mtDNA, we attempted to build iPSC from 7SP fibroblasts using the standard methods with Sendai virus carrying Oct3/4, Klf4, Sox2, and c-Myc (OKSM), which worked well for NHDF. A diagram of the protocol design is provided in
Alkaline phosphatase staining (AP staining), which detects iPSCs colonies at the early stage, demonstrated that the original 7SP fibroblasts-derived colonies seemed to be with crumbling appearances, whereas the mitochondria replaced 7SP fibroblasts-derived colonies were solid on day 21 (
The iPSC generated by the methods described herein were further compared to the commercially available KYOU-DXR0109B Human Induced Pluripotent Stem (IPS) Cells [201B7]. Importantly, the mitochondria replaced 7SP fibroblasts showed the same level of efficiency with the iPS generation with that of healthy fibroblasts. Additionally, in agreement with previous studies, qPCR of 12S-rRNA, normalized to nuclear (3-actin, demonstrated that the iPS cells generated by mitochondrial replaced 7SP fibroblasts exhibited half of the mtDNA contents relative to control, and the mtDNA levels were similar to that of the 201B7 iPSC standard (
Moreover, the hmt10158 heteroplasmy level was less than 10% in the generated iPSCs (
These results demonstrate that iPSCs can be generated using the mitochondrial replacement technology provided herein, and could be applicable in the clinical field because this whole procedure used only materials adaptable to clinics.
This example demonstrates that mtDNA replacement can alter the lifespan of the recipient cell. In order to validate the hypothesis that the mitochondria replacement can rejuvenate senescent cells, two models were estimated in respect of cell cycle capabilities, such as doubling time and PDL at the growth arrest.
NHDFs and TIG1 embryonic lung cells with early PDL (around 5 to 10, called “young”) and late PDL (around 40 to 45, called “old”) were utilized to design the models. One model involved young cells replaced with mitochondria derived from old cells, designated as “02Y,” and another model involved old cells replaced with mitochondria derived from young cells, designated as “Y20” (
The extent of the exchange of mtDNA was evaluated by TaqMan SNP genotyping assay, based on the difference of the single nucleotide of mtDNA at the 16145 position between NHDF and TIG1, which are A and G, respectively (
In addition, the Y20 model clearly demonstrated a regain of the lifespan in old cells to around 65 PDLs (
This example describes the generation of mitochondria replaced Cell (MirC) from human primary T cells by using mRNA transfection.
Prior to the experiments, use of human primary T cells were approved by our institutional ethical committee. Peripheral blood was drawn from a healthy volunteer and centrifuged using percoll with a specific gravity of 1.077 at 400 g for 35 minutes at 20 degree to separate lymphocytes. Isolated lymphocytes of 1×106 cells per ml were seeded onto a 96-well flat plate coated with anti-CD3 and anti-CD28 antibodies. The plate was prepared by incubating with 5 μg/ml of anti-CD3 and 1 μg/ml of anti-CD28 of overnight and pre-warmed at 37° C. 2 hours prior to the seeding. On the next day of the seeding, IL-7 and IL-15 were added to the medium at a concentration of 20 μg/ml and 10 μg/ml, respectively. Medium was changed every third day with IL-7 and IL-15 at the same concentration as the initial addition.
Transfection was performed using the MaxCyte electroporator, which meets the standard of GMP/GCP, according to the manufacturer's protocol. mRNA was created according to the manufacturer's protocol in mMESSAGE mMACHINE T7 Ultra Kit (Thermo Fisher), with slight modifications. Briefly, a DNA template for mRNA was prepared from the plasmid carrying the DNA sequence without refining the fragment following endonuclease digestion, in order to reduce the possibility of mixing RNase up as lower as possible (
The results indicated that the non-refining DNA template for mRNA creation of EGFP led to nearly a 100% gene transfer efficiency with high expression and high viability at 24 hours after the gene transfection (
In order to determine the optimal protocol with respect to the timing of isolated mitochondria co-incubation, fluorescent images of human primary T cells that received mRNA of GFP by electroporation using MaxCyte ATX were taken over an 8-day period, as indicated (
The protein expression of GFP in cells receiving the MTS-GFP mRNA was evaluated by western blotting analysis using a capillary electrophoresis. The peak expression occurred at day 4, and expression was lost by day 6, as illustrated in the western blot (
Taken together, these results demonstrate that mRNA transfection of an endonuclease, such as XbaI, fused to an MTS can efficiently degrade the host mtDNA, and can be used to generate a mitochondria replaced Cell (MirC) from human primary T cells.
After determining the optimal time point for performing mitochondrial transfer in human primary T cells from Example XV, mitochondria co-incubation was performed on day 7 to prevent digestion of exogenous mtDNA by any remaining endonuclease. A scheme of the MirC protocol for human primary T cells is shown in
In order to determine the heteroplasmy of mtDNA in the recipient human primary T cells following mitochondria replacement, the difference of mtDNAs between the donor mitochondria and the recipient cells was determined by TaqMan SNP genotyping assay. Sequencing of the D-loop of mtDNA in normal human primary T cells and EPC100 (mitochondria donor cells) showed a difference in 2 nucleotide positions (nucleotides 218 and 224 mtDNA), which were C/C and T/T for T cells and EPC100 cells, respectively (
Next, to evaluate the effect that the mitochondrial transfer had on the function of the MirC human T cells, respirometry experiments were performed using Oroboros 02k. The results demonstrated a recovery of ATP production and coupling efficiency in human T cell-derived MirC on day 7, whereas ρ(−) human T cells that were generated by XbaIR mRNA transfer with electroporation maintained the loss of ATP production throughout the experiment (
These results demonstrated that human primary T cells are capable of mitochondria replacement to generate MirC using GMP graded electroporator, such as the electroporator produced by MaxCyte Inc.
Further characterizations of T cell-derived MirC were executed for murine T cells. The isolation of murine T cells from suspension solutions obtained from the spleen was performed using the EasySep Mouse Isolation Kit (STEM CELL Technologies, Inc.), which provides highly purified T cell population by negative selection using magnets. Isolated murine T cells (1×106 cells per ml) were seeded onto 96-wells plate with Dynabeads mouse T-Activator CD3/CD28 (Invitrogen, Inc.) at a bead-to-cell ratio of 1:1 and recombinant IL-2 at 30 U/ml. The medium to cultivate murine T cells was determined with respect to cell growth and CD3 expression, and RPMI1640 was found to be superior to TexMACS (
Next, electroporation of murine T cells was performed using the Nucleofector machine and mRNA. The kinetics of GFP expression following mRNA transfer were similar to human T cells (
Transfection of the MTS-XbaI mRNA indicated that murine T cells exhibited a milder decline of XbaIR transcript expression relative to human T cells, and persisted even at a low level on day 6 (
This example demonstrates that mitochondria replacement was successful in murine T cells, and rejuvenated senescent T cells.
To evaluate whether exogenous mitochondria could be successfully transferred to generate murine T cell-derived MirC, mtDNA heteroplasmy levels were of BL6 (recipient) cells and NZB (donor) cells. Two-consecutive polymorphisms at 2766 and 2767 mtDNA for ND1 was verified (
Quantification of the mitochondria replacement (XbaIR Mt) in BL6 cells that were transfected with MTS-XbaI and co-incubated with mitochondria isolated from NZB mice, demonstrated an overwhelming domination of exogenous mtDNA, whereas mock transfectants that were coincubated with isolated mitochondria following electroporation under the absence of mRNA of the endonuclease did not engulf any exogenous mtDNA (
Because the results described herein demonstrated fibroblast-derived MirC could undergo rejuvenation in vitro (
In addition, SASP was evaluated using the same representative set of makers described previously (
Moreover, senescent T cells have been found to exhibit higher DNA damage response (DDR), compared with young T cells. Therefore, DDR was measured using the histone 2 A (H2A) phosphorylation antibody, for the MirC and the original T cells. The results indicated that the positive fraction for DDR was lower in the MirC (1.53%), compared with the original T cells (4.75%) (
These in vitro results support that the somatic mitochondria replacement was verified in the MirC mtDNA, and the replacement resulted in numerous changes that were indicative of a reversal of senescence in the MirC T cells.
To examine the functional potential of the mitochondria replacement that rejuvenated senescent cells, an adoptive cell transplantation (ACT) experiment was performed. The AE17 mesothelioma cell line derived from the peritoneal cavity of C57BL/6J mice injected with asbestos fibers was used to develop tumor formation in mice. Previous experiments using this model have shown that tumor growth is mitigated by ACT of young syngeneic T cells, but not by ACT of old syngeneic T cells (Jackaman et al. Oncolmmunology 2019; 8(4): 1-16).
To determine whether the rejuvenated old T cells, generated by transferring isolated exogenous mitochondria from a young mouse to a T cell from an old mouse, exhibited functional activity, AE17 cells were subcutaneous injected into three groups of old mice (Group 1: old mice with ACT of T cells from young mouse; Group 2: old mice; or Group 3: old mice with ACT of MirC derived from a T cell of an old mouse transferred with exogenous mitochondria from a young mouse (
The body weight in each group did not show significant differences (
To verify the presence of infused T cells in the animals, T cells derived from GFP transgenic mice were transplanted into the syngeneic C57BL/6 mice, the peripheral blood and the spleen were examined to track the donor cells (
These results clearly demonstrate that ex vivo MirC generation using mitochondria from young mice into T cells from an old mouse can effectively function in vivo, and reduce tumor burden at similar levels as T cells from young mice.
To date, gene transfer methods for hematopoietic stem cells have mainly involved the use of viral vectors, because the targets were mainly genetic disorders that require a sustainable gene expression of the deficient gene. Consequently, electroporation is not used in current protocols of gene transfer of hematopoietic stem cells because of the need to generate a permanent gene expression. In contrast, the objective of the mitochondrial replacement technology provided herein is to achieve temporal high expression of the exonuclease.
Based on the experiments for fibroblasts and T cells, the condition of Nucleofector/electroporation with mRNA was adjusted, and several conditions were examined for murine fetal liver-derived Sca-1 positive cells (
Further examinations were performed by FACS analysis for the mean fluorescent intensities (MFI) on dayl following the electroporation with mRNA of GFP (
Next, we examined whether this mitochondria replacement procedure via generation of ρ(−) cells using MTS-XbaIR mRNA transfer could be applicable to hematopoietic stem cells (
The heteroplasmy changes were evaluated using the TaqMan SNP genotyping assay, as described above. A scheme of the assay is shown in
This example demonstrates that mitochondrial DNA (mtDNA) can be assayed for the presence of a specific mtDNA sequence, such as a mutation in mtDNA, using digital PCR (dPCR). Droplet Digital PCR (ddPCR) is a method for performing digital PCR that is based on water-oil emulsion droplet technology.
Primary skin fibroblasts derived from patients with mitochondrial disease were analyzed. The patient information is provided below in Table 1.
Cells from the target population were encapsulated into droplets at a concentration of one cell per droplet with the PCR mixture including primers and probes. Cell density was optimized to generate a single cell in a single droplet, and the fibroblasts were finally diluted in 1×106 cell/mL for ddPCR. After single-cell encapsulation, cell lysis and amplification of the target sequence were performed within the droplets. The number of droplets with a fluorescent signal indicated the number of cells carrying the target or reference gene.
Briefly, a 20× primer/probe mix was prepared as described below in Table 2. The standard ddPCR master mix was a 25 μL mix that includes the aforementioned primer/probe mix, template DNA and 2×ddPCR super mix.
Samples were loaded into an 8-chamber cartridge using 20 μL of the prepared qPCR sample followed by 70 μL of droplet generation oil in the adjacent wells. A rubber gasket was stretched across the top of the chambers to ensure a vacuum seal. Each 8-chamber cartridge was loaded onto the QX100 droplet generator producing 20,000 droplets per sample. Using a 50 μL multichannel pipette, 40 μL of the generated droplets were transferred to a 96-well plate and heat sealed with pierceable foil. The plate was placed in a thermal cycler using standard 2-step qPCR thermal cycling conditions with a 50% (3° C./sec) ramp rate. Prior to running thermal cycling conditions, primer/probe sets were optimized using a temperature gradient to optimize the anneal/extend temperature.
Following thermal cycling, the plate was loaded onto the QX100 droplet reader and end-point reactions were analyzed. Poisson statistical analysis of the numbers of positive and negative droplets yields absolute quantitation of the target sequence.
Before examining diseased cells, the specificity for the probes to be designed for a mutated sequence and the sensitivity for the probes to be designed for a non-mutated sequence were evaluated by using normal human dermal fibroblasts (NHDF cells) that have a non-mutated sequence (the same as Cambridge Reference Sequence) (
ddPCR of fibroblasts obtained from BK01 indicated a few percentage of double positive population, and the majority was cells with homoplasmy of mutated mtDNA (
Taken together, these results demonstrated that homoplasmy and heteroplasmy can be accurately, and quantitatively evaluated at a single cell level. In addition, the results demonstrate that the mtDNA of a subject with mitochondrial disease can be accurately measured, which could be useful for evaluating therapeutic compositions prior to transplantation in a subject or monitoring the mtDNA content prior to and/or after therapy.
This example demonstrates that hematopoietic stem or progenitor cells (HSPCs) can be ex vivo modulated using the mtDNA replacement methods provided herein for therapy.
Modulation of HSPCs can be performed ex vivo in connection with a stem cell transplant. Briefly, peripheral blood stem cells are mobilized and a blood sample is obtained from the patient. Peripheral hematopoietic stem or progenitor cells (HSPCs), e.g., CD34+ cells are isolated and sent to a manufacturing facility. At the manufacturing facility, the mitochondria is partially depleted according to the methods provided herein.
Donor mitochondria are isolated using current Good Manufacturing Practice (cGMP) manufactured bone-marrow derived Mesenchymal Stromal Cells (BM-MSCs) obtained from a cell repository (e.g., Waisman Biomanufacturing). The initial bone marrow aspirates are collected with full informed consent and in compliance with federal regulations (e.g., 21 CFR 1271). The aspirates are processed under cGMPs and banked at an early passage for subsequent expansion.
The donor mitochondria from the BM-MSCs are transferred to cultured HSPCs, changing the heteroplasmy. The modified HSPCs are sent back to the medical center for autologous transplantation (i.e., into the same subject that the HSPCs were isolated). Prior to transplantation the patient receives minimal treatment that can include a non-myeloablative regimen, such as partial irradiation or sublethal dose of anti-cancer drugs, such as busulfan. The modified HSPCs, only containing the allogenic donor mitochondria, are transfused back into the patient.
This example demonstrates that HSPCs can be ex vivo modulated using the mtDNA replacement methods provided herein for therapy that does not involve transplantation of allogenic HSPCs.
The embodiments described above are intended to be merely exemplary, and those skilled in the art will recognize, or will be able to ascertain using no more than routine experimentation, numerous equivalents of specific compounds, materials, and procedures. All such equivalents are considered to be within the scope of the invention and are encompassed by the appended claims.
This application claims the benefit of U.S. Provisional Application No. 62/718,891, filed Aug. 14, 2018, U.S. Provisional Application No. 62/731,731 filed Sep. 14, 2018, and U.S. Provisional Application No. 62/817,987 filed Mar. 13, 2019, which are incorporated herein by reference in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| 62718891 | Aug 2018 | US | |
| 62731731 | Sep 2018 | US | |
| 62817987 | Mar 2019 | US |
