EP4121120A2 - Méthodes de traitement de troubles mitochondriaux - Google Patents

Méthodes de traitement de troubles mitochondriaux

Info

Publication number
EP4121120A2
EP4121120A2 EP21771515.0A EP21771515A EP4121120A2 EP 4121120 A2 EP4121120 A2 EP 4121120A2 EP 21771515 A EP21771515 A EP 21771515A EP 4121120 A2 EP4121120 A2 EP 4121120A2
Authority
EP
European Patent Office
Prior art keywords
cells
seq
yg8r
frda
mice
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP21771515.0A
Other languages
German (de)
English (en)
Other versions
EP4121120A4 (fr
Inventor
Stephanie CHERQUI
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of California
University of California Berkeley
University of California San Diego UCSD
Original Assignee
University of California
University of California Berkeley
University of California San Diego UCSD
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US16/820,368 external-priority patent/US12011488B2/en
Application filed by University of California, University of California Berkeley, University of California San Diego UCSD filed Critical University of California
Publication of EP4121120A2 publication Critical patent/EP4121120A2/fr
Publication of EP4121120A4 publication Critical patent/EP4121120A4/fr
Pending legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/17—Monocytes; Macrophages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/20—Cellular immunotherapy characterised by the effect or the function of the cells
    • A61K40/24—Antigen-presenting cells [APC]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/414—Nervous system antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the dose, timing or administration schedule
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the invention relates generally to mitochondrial disease and more specifically to methods of treating mitochondrial diseases with hematopoietic stem and progenitor cell (HSPC) gene therapy.
  • HSPC hematopoietic stem and progenitor cell
  • Mitochondrial disease is a group of disorders caused by dysfunctional mitochondria, the organelles that are the powerhouse of the cell. Mitochondria are found in every cell of the human body except red blood cells, and convert the energy of food molecules into the ATP that powers most cell functions.
  • Mitochondrial diseases are sometimes caused by mutations in the mitochondrial DNA that affect mitochondrial function. Other causes of mitochondrial disease are mutations in genes of the nuclear DNA, whose gene products are imported into the mitochondria (mitochondrial proteins) as well as acquired mitochondrial conditions. Mitochondrial diseases take on unique characteristics both because of the way the diseases are often inherited and because mitochondria are so critical to cell function. The subclass of these diseases that have neuromuscular disease symptoms are often called mitochondrial myopathies. Symptoms associated with mitochondrial disease typically include poor growth, loss of muscle coordination, muscle weakness, visual problems, hearing problems, learning disabilities, heart disease, liver disease, kidney disease, gastrointestinal disorders, respiratory disorders, neurological problems, autonomic dysfunction and dementia.
  • Mitochondrial diseases/disorders may be caused by mutations, acquired or inherited, in mitochondrial DNA (mtDNA) or in nuclear genes that code for mitochondrial components. They may also be the result of acquired mitochondrial dysfunction due to adverse effects of drugs, infections, or other environmental causes.
  • mtDNA mitochondrial DNA
  • mtDNA mitochondrial DNA
  • nuclear genes that code for mitochondrial components They may also be the result of acquired mitochondrial dysfunction due to adverse effects of drugs, infections, or other environmental causes.
  • mtDNA mitochondrial DNA
  • mtDNA mitochondrial DNA
  • nuclear genes that code for mitochondrial components may also be the result of acquired mitochondrial dysfunction due to adverse effects of drugs, infections, or other environmental causes.
  • mtDNA mitochondrial DNA
  • nuclear genes that code for mitochondrial components may also be the result of acquired mitochondrial dysfunction due to adverse effects of drugs, infections, or other environmental causes.
  • FRDA Friedreich’s ataxia
  • FRDA is caused, in 98% of all cases, by a genetic mutation resulting in expansion of GAA repeats in the first intron of the frataxin gene (FXN).
  • FXN frataxin gene
  • the alleles may contain up to about 40 GAA repeats, whereas expanded alleles in FRDA patients can consist of 90 to 1700 repeats (SEQ ID NO: 12) (see Figure 1B).
  • the GAA repeat expansion leads to reduced expression of frataxin, a highly conserved mitochondrial protein mainly expressed in mitochondria-rich tissues including the nervous system, muscle, and heart.
  • carriers (heterozygous for the expanded allele) show ⁇ 50% reduction of frataxin mRNA and protein levels compared to normal expression, although they do not show any symptoms. While its function is not fully elucidated, frataxin is an iron binding protein participating in Fe-S cluster assembly and in its absence, iron accumulates within mitochondria leading to defective iron-mediated biosynthetic processes and increased oxidative stress.
  • Expanded GAA repeats form an intramolecular triple-helix (triplex), so-called H- DNA, in supercoiled plasmids isolated from E. coli.
  • GAA repeats are associated with a pattern of DNA methylation and histone acetylation in the adjacent regions and the formation of silenced chromatin.
  • H-DNA and higher order structures within the GAA repeats is believed to recruit chromatin-remodeling protein complexes that maintain a close chromatin structure leading to down-regulation of frataxin gene transcription.
  • Numerous data have demonstrated that analysis of GAA repeats constitute an essential part in the diagnosis of FRDA along with clinical diagnosis. Molecular genetic tests are also performed to identify carriers and in prenatal testing. Current FA diagnostic methods involve polymerase chain reaction (PCR) analysis and Southern blotting technique. The PCR test is performed by amplification of the GAA repeat-containing DNA region in the frataxin gene.
  • PCR reactions that have been employed to map GAA repeat expansions are classical PCR, long-range PCR or triplet-primed PCR (TP-PCR). In all cases, the size of the PCR fragment is analyzed using agarose-gel electrophoresis and DNA sequencing. In most cases, both PCR and Southern blot are combined to complement the results. Problems encountered during amplification of medium- and long-sized GAA repeats (i.e., number of repeats >200) using PCR have been reported. The repetitive nature of the expanded sequence and its ability to adopt H-DNA and higher order DNA structures are the two main factors causing polymerase pausing leading to false results.
  • the invention provides a method of treating a mitochondrial disease or disorder in a subject.
  • the method includes introducing ex vivo a functional human frataxin (hFXN) into hematopoietic stem and progenitor cells (HSPCs) of the subject, and transplanting the HSPCs into the subject, thereby treating the mitochondrial disease or disorder.
  • hFXN hematopoietic stem and progenitor cells
  • the step of introducing may include contacting a vector comprising a polynucleotide encoding hFXN and a FXN promoter (or other regulatory sequence that is operable with the polynucleotide and in the cell) with the HSPCs and allowing expression of hFXN.
  • the mitochondrial disease or disorder is selected from the group consisting of Friedreich’s ataxia (FRDA), diabetes, Leigh syndrome, Leber’s hereditary optic neuropathy, myoneurogenic gastrointestinal encephalopathy, and cancer.
  • the subject may be a mammal, such as a human.
  • the vector is a self-inactivating (SIN)-lentivirus vector, such as pCCL-FRDAp-FXN.
  • SI self-inactivating
  • expression of hFXN corrects neurologic, cardiac and muscular complications within about 6-12 months post-transplantation.
  • the hFXN polynucleotide is introduced into HSPCs in vivo in a subject.
  • the present invention provides a method of treating a mitochondrial disease or disorder in a subject comprising contacting cells expressing hFXN from the subject with a vector encoding a gene editing system that when transfected into the cells removes a trinucleotide extension mutation of endogenous hFXN, thereby treating the mitochondrial disease or disorder.
  • the gene editing system is selected from the group consisting of CRISPR/Cas9, zinc finger nucleases, and transcription activator-life effector nucleases.
  • the CRISPR/Cas9 system comprises one or more crRNA sequences selected from the group consisting of UP3 (SEQ ID NO: 17), UP4 (SEQ ID NO: 18), UP5 (SEQ ID NO: 19), DN3 (SEQ ID NO: 20), DN4 (SEQ ID NO: 21), and DN5 (SEQ ID NO: 22).
  • the CRISPR/Cas9 system comprises one or more guide target sequences selected from the group consisting of SEQ ID NOs: 91-95 and 96.
  • the step of contacting may include obtaining a sample of cells from the subject, transfecting or transducing the gene editing system into the sample of cells to create gene-corrected cells, and thereafter, transplanting the gene-corrected cells into the subject.
  • the sample of cells may be any cells expressing hFXN, such as blood cells and HSPCs from the subject.
  • the present invention provides an expression cassette comprising a promoter or regulatory sequence functionally linked to a polynucleotide encoding hFXN.
  • FIGS. 1A-1E are graphical and pictorial diagrams showing that systemic transplantation of WT HSPCs prevents sensory neuron degeneration and neurobehavioral deficits in YG8R mice.
  • Locomotor activity was tested using an open field, coordination using a rotarod, gait using an automated gait analysis system and muscle strength using forelimb grip strength. Data are expressed as means ⁇ sem; *P ⁇ 0.05, **P ⁇ 0.005, ***P ⁇ 0.0005; NS statistically non-significant.
  • ANOVA analysis of variance
  • Figure 1B is a representation showing intron 1 of an unaffected (top) frataxin gene (FXN) and intron 1 of FRDA (bottom) FXN, and discloses SEQ ID NOs: 15-16, respectively, in order of appearance.
  • FIG. 1D shows representative confocal images from a WT GFP + HSPC-transplanted YG8R mouse 7 months post-transplantation stained with anti-GFP and anti-NeuN.
  • Magnified image (below) demonstrates frequent close association of HSPC-derived cells with DRG neurons. Scale bar, 20 ⁇ m.
  • FIG. 1E shows confocal images of DRG and spinal cord sections of a GFP + HSPC-treated YG8R mouse. Engrafted cells (GFP) are closely associated with neurons (NeuN), and co-localization with Iba1 marker; Scale bars: 30 ⁇ m.
  • Figures 2A-2E are graphical and pictorial diagrams showing that transplanted HSPCs engraft throughout the brain and prevent frataxin-deficiency toxicity.
  • Figure 2A shows representative transverse sections of the brain of a WT GFP + HSPC-transplanted YG8R mouse 7 months post-transplantation labeled with anti-GFP and anti-NeuN. Scale bar, 1mm.
  • Magnified picture #1 of the brain shows that GFP + HSPC-derived cells are observed in periventricular regions including the corpus callosum (cc), lateral septal nuclei (LS), caudate putamen (CP), anterior cingulate area (ACA), and the somatosensory cortex (M1, S2).
  • VL lateral ventricle. Scale bar, 150 ⁇ m.
  • Magnified picture #2 of ventral striatum of the brain shows that the engrafted GFP + HSPCs are present in regions of the ventral striatum including the anterior commissure (aco), nucleus accumbens (ACB), and lateral septal nuclei (LS). CP, caudate putamen. Scale bar, 150 ⁇ m.
  • Magnified picture #3 shows that GFP + HSPC-derived cells are observed in the ventral pallidum (PAL) and the ventral striatum, including the islands of Calleja (isl) and the olfactory tubercle (OT). Scale bar, 150 ⁇ m. GFP + HSPCs were also detected through gray and white matter of the brainstem and cerebellum.
  • FIG. 1 shows a diagram of a dentate nucleus (DN) of the cerebellum and the spinal trigeminal nucleus (Sp) of the brainstem. Scale bar, 50 ⁇ m.
  • Figure 2B shows confocal image of brain labeled with anti-GFP, anti- Iba1 and anti-NeuN. Most of the bone marrow-derived GFP + cells co-localize with the microglial marker Iba1. Scale bar, 30 ⁇ m.
  • FIG. 2D shows the results of a representative Western blot showing the level of oxidation in cerebrum of one WT, one YG8R, one YG8R/YG8R HSPCs and one YG8R/WT HSPCs mouse with (+) or without (-) derivatization reagent.
  • FIGS 3A-3H are pictorial and graphical diagrams showing transplanted HSPCs engraft abundantly in heart and muscle.
  • Figure 3A shows the results of a representative Western blot showing level of oxidation in skeletal muscle of one WT, one YG8R and one YG8R/HSPCs mouse with (+) or without (-) derivatization reagent.
  • the lactate/pyruvate ratio is significantly increased in the YG8R mice compared to WT while comparable in YG8R/WT HSPCs animals.
  • Error bars indicate sem; *P ⁇ 0.05, ***P ⁇ 0.0005, NS statistically non-significant.
  • Figure 3C shows representative Perl’s staining of heart sections from 18 month old WT, YG8R control and YG8R/WT HSPCs. Characteristic staining indicates iron deposition. Scale bars, 50 ⁇ m and 15 ⁇ m (zoom).
  • FIG. 3F shows an image of a heart section from WT HSPCs transplanted YG8R mouse 7 months post-transplantation stained with anti-GFP, the cardiomyocyte marker anti- ⁇ -actinin and DAPI. GFP + cells are found in all the cardiac tissue with a highest expression in the valve suggesting that HSPCs derived cells are entering the heart by the blood flow. Scale bar, 150 ⁇ m. Magnified pictures of the heart show high level of engraftment in the left ventricle (bottom) and in the base of the aorta (top).
  • FIG. 3G shows skeletal muscle section from WT HSPCs transplanted YG8R mouse 7 months post-transplantation stained with anti-GFP, filamentous actin dye Phalloidin and DAPI. GFP + cells are engrafted homogenously in the tissue. Scale bar, 150 ⁇ m. Magnified picture of the skeletal muscle (on the left) shows that GFP + cells are localized interstitially between muscle fibers. Scale bar, 50 ⁇ m.
  • Figures 4A-4F are pictorial and graphical diagrams showing that HSPC-derived cells deliver frataxin-bearing mitochondria to the diseased cells in vitro and in vivo.
  • Figures 4A and 4B show representative frames from confocal imaging movies of YG8R-derived fibroblasts (F) co-cultured with primary macrophages (M) isolated from a DsRed Cox8- GFP transgenic mouse ( Figure 4A) or with IC21 macrophages transduced with a LV-hFXN- GFP and stained with a red MitoTracker ( Figure 4B).
  • Scale bar 10 ⁇ m.
  • Figure 4C shows a representative confocal image of brain sections from an YG8R mouse transplanted with DsRed + HSPCs (control) and brain and spinal cord sections from an YG8R mouse transplanted with DsRed + /Cox8-GFP + HSPCs at 7 months post-transplantation labelled with an anti-NeuN antibody.
  • DsRed + HSPCs control
  • DsRed + /Cox8-GFP + HSPCs at 7 months post-transplantation labelled with an anti-NeuN antibody.
  • cox8-GFP are observed in host neurons in brain and spinal cord (arrows).
  • Figure 4D shows representative confocal images of spinal cord section from an YG8R mouse transplanted with DsRed + /Cox8-GFP + HSPCs at 7 months post-transplantation labelled with an anti-NeuN antibody showing cox8-GFP within the branch extension of the DsRed + microglial cell (arrows). Scale bar, 5 ⁇ m.
  • Figure 4E shows quantification of neurons containing cox8-GFP in the cervical spinal cord gray matter of YG8R mice transplanted with DsRed + /Cox8-GFP + HSPCs at 7 months post- transplantation (for description of the automatic unbiased quantification method see Figure 8).
  • Figure 4F shows representative confocal images of brain and spinal cord sections from an YG8R mouse transplanted with DsRed + HSPCs transduced with LV-hFXN-GFP at 7 months post-transplantation and stained with anti-mcherry and anti-NeuN antibodies. In addition to the DsRed-derived bone marrow cells, frataxin-GFP are observed in host neurons. Scale bar, 10 ⁇ m.
  • Figure 5 is a pictorial diagram showing that HSPCs engraft in the peripheral nerve in YG8R mice.
  • FIG. 6A-6F are pictorial diagrams showing that HSPCs differentiate into macrophages in DRG and microglia in the spinal cord and brain.
  • Figures 6A and 6B show confocal images of DRG, spinal cord and brain sections from WT GFP + HSPC-transplanted YG8R mice labeled with anti-GFP, anti-CD68 ( Figure 6A), anti-MHCII (Figure 6B), anti- NeuN ( Figure 6A), and DAPI. Scale bars, 30 ⁇ m.
  • Figures 6C and 6D show transverse spinal cord ( Figure 6C) and brain (Figure 6D) section from WT GFP + HSPC-transplanted YG8R mouse labeled with anti-MHCII. Scale bars, 100 ⁇ m ( Figure 6C) and 300 ⁇ m ( Figure 6D).
  • Figure 6E shows a confocal image of brain section from WT GFP + HSPC- transplanted YG8R mouse labeled with anti-vwf. Scale bar, 50 ⁇ m.
  • Figure 6F shows a confocal image of choroid plexus from WT DsRed + HSPC-transplanted YG8R mouse labeled with anti-RFP and anti-Iba1. Scale bar, 100 ⁇ m.
  • Figures 7A and 7B are pictorial diagrams showing that HSPCs differentiate into macrophages in heart and muscle.
  • FIG. 8 is a pictorial diagram showing that HSPC-derived macrophages deliver mitochondria to neurons in DRG and to myocytes in heart and skeletal muscle.
  • FIG. 9A-9D are pictorial and graphical diagrams showing quantification of Cox8-GFP transfer from HSPC-derived microglia to neurons.
  • Figure 9A shows a representative transverse image of cervical spinal cord gray matter from a YG8R mouse at 7 months following transplantation with Cox8-GFP DsRed HSPCs, stained with anti-NeuN. Scale bar, 500 ⁇ m.
  • Figure 9B shows automatic outline and quantification of neurons by ImagePro software.
  • Figure 9C shows that GFP signal is only counted within the delineated neurons (arrow) and not outside (star).
  • Figure 9D shows the percentage of neurons within the gray matter of the spinal cord that contain GFP for three different animals (transplanted) and for one control. The entire gray matter from three experimental animals and one control (three sections per animal) were quantified.
  • Figures 10A-10C are pictorial and graphical diagrams showing validation of CRISPR/Cas9-mediated gene editing at the FXN intron 1 locus in human FRDA fibroblasts.
  • Figure 10A shows a list of the best six crRNAs designed following the Rule Set 2 surrounding the FXN intron 1 GAA expansion: UP3 (SEQ ID NO: 17), UP4 (SEQ ID NO: 18), UP5 (SEQ ID NO: 19), DN3 (SEQ ID NO: 20), DN4 (SEQ ID NO: 21), and DN5 (SEQ ID NO: 22).
  • Figure 10B shows the position of the crRNAs and regulatory elements surrounding the FXN intron 1 GAA expansion.
  • FIG. 11A-11H are pictorial and graphical diagrams showing GAA gene editing optimization in human FRDA lymphoblasts using the UP4/DN4 crRNA pair.
  • Figure 11A shows a schematic representing the ddPCR strategy to determine GAA gene editing efficiency from genomic DNA. Red primers can only amplify the intronic region if GAA gene editing occurs.
  • Figure 11B shows a GAA gene editing percentage measured by ddPCR in 3 different FRDA lymphoblastic cell lines 3 weeks post-electroporation with 4RNP or 4RNPenh. Data are means ⁇ SEM. *P ⁇ 0.05, **P ⁇ 0.005 and ***P ⁇ 0.0005 (student’s t-test).
  • Figure 11C shows a GAA gene editing percentage measured by ddPCR in 2 different healthy lymphoblastic cell lines 3 weeks post-electroporation with 4RNPenh. Data are means ⁇ SEM.
  • Figure 11G shows a representative Western blot showing human frataxin protein expression in healthy1, carrier1, FRDA1, FRDA1/4RNP and FRDA1/4RNPenh lymphoblasts 3 weeks post-electroporation.
  • Figure 11H shows mitochondrial activity measured in healthy, FRDA and FRDA/4RNPenh lymphoblasts in presence of succinate. Data are means ⁇ SEM. *P ⁇ 0.05, **P ⁇ 0.005 and ***P ⁇ 0.0005 (one-way Anova).
  • Figures 12A-12D are pictorial and graphical diagrams showing the impact of GAA gene editing on FRDA lymphoblasts viability and proliferative capacity.
  • Figure 12C shows a western blot representing the time course expression of p53 after 4RNPenh electroporation in FRDA lymphoblasts.
  • Figures 13A-13G are graphical diagrams showing GAA gene editing in healthy CD34 + and consequence on hematopoiesis reconstitution capacity.
  • Data are means ⁇ SEM. *P ⁇ 0.05, **P ⁇ 0.005 and ***P ⁇ 0.0005 (one-way Anova).
  • CFU-GEMM CFU-granulocyte/erythroid/macrophage/megakaryocyte
  • CFU-GM CFU- granulocyte/macrophage
  • BFU-E burst-forming unit-erythroid
  • CFU-E CFU- erythroid
  • Data are means ⁇ SEM. Non-significant (one-way Anova).
  • Figure 13D shows a GAA gene editing percentage measured by ddPCR in each colony type for each healthy donors.
  • Figure 13F shows a lineage distribution of human cells engrafted in NSG mice in the bone marrow 3 months post-transplantation determined by flow cytometry using fluorescent-labeled antibodies to human T cells (CD3), human B cells (CD19) and human myeloid cells (CD33 + ). Data are means ⁇ SEM. Non-significant (student’s t-test).
  • Figure 13G shows a GAA gene editing percentage measured by ddPCR in bone marrow (BM), spleen and thymus of transplanted NSG mice 3 months post-transplantation. Data are means ⁇ SEM.
  • Figures 14A-14G are graphical diagrams showing GAA gene editing in FRDA patient CD34 + and impact on FXN expression and hematopoiesis reconstitution.
  • Data are means ⁇ SEM. *P ⁇ 0.05, **P ⁇ 0.005 (one-way Anova).
  • Figure 14D shows a correlation curve showing the significant relation between the percentage of GAA gene editing and the increased expression of human frataxin mRNA in gene modified CD34 + from FRDA patient donors.
  • Figure 14F shows a GAA gene editing percentage measured by ddPCR in each colony type for each FRDA donors.
  • Figure 14G shows a quantification of the human mitochondrial complex subunit mRNA mtDN6 (complex I), mtCO2 (complex II) and mtATP6 (complex V) by RT-qPCR in healthy, FRDA and FRDA/4RNPenh CD34 + cells one-week post-electroporation. Data are represented as fold change relative to healthy and normalized to human tubulin. Data are means ⁇ SEM. *P ⁇ 0.05, **P ⁇ 0.005 (student’s t-test).
  • Figures 15A-15C are a series of graphical diagrams showing mitochondrial activity in lymphoblasts in presence of succinate and complex I ( Figure 15A), II ( Figure 15B) and III ( Figure 15C) inhibitors. Mitochondrial activity measured in healthy, FRDA and FRDA/4RNPenh lymphoblasts in presence of succinate that feeds complex II. Validation of the assay should result in decreased mitochondrial activity in presence of complex II (malonate and carboxin) or complex III (antimycin A and myxothiazol) inhibitors but not complex I (roterone and pyridaben) inhibitors. Data are means ⁇ SEM. *P ⁇ 0.05, **P ⁇ 0.005 and ***P ⁇ 0.0005 (one-way Anova).
  • Figure 16 is a pictorial diagram showing detection of indels within 6 potential off-target regions.
  • the 6 potential off-target regions were determined using COSMID and amplified by PCR from gDNA of unmodified (-) or gene-corrected (+) patient CD34 + .
  • the presence of indels was assessed using the T7E1 and resulting PCR fragments are represented on the agarose gel.
  • Figure 17 is a graphical diagram showing primers used to detect the presence of potential indels within edited gDNA.
  • Sequences are as follows: Z-UP (SEQ ID NO: 23), Z-DN (SEQ ID NO: 24), FXN Intron 1 Forward (SEQ ID NO: 25), FXN Intron 1 Reverse (SEQ ID NO: 26), FXN Intron 1 Probe (SEQ ID NO: 27), Gain of signal Forward (SEQ ID NO: 28), Gain of signal Reverse (SEQ ID NO: 29), Gain of signal Probe (SEQ ID NO: 30), mt-ND6 Forward (SEQ ID NO: 31), mt-ND6 Reverse (SEQ ID NO: 32), mt-CO2 (SEQ ID NO: 33), mt-CO2 (SEQ ID NO: 34), mt-ATP6 (SEQ ID NO: 35), mt-ATP6 (SEQ ID NO: 36), Tubulin Forward (SEQ ID NO: 37), Tubulin Reverse (SEQ ID NO: 38), AGAP1 Forward (SEQ ID NO: 39), AGAP1 Reverse (SEQ ID NO: 40), DGKG Forward (
  • Figure 18 shows a table of potential off-target regions using COSMID. Sequences shown are as follows: Line 1 hit (SEQ ID NO: 51), Line 1 query (SEQ ID NO: 52), Line 2 hit (SEQ ID NO: 53), Line 2 query (SEQ ID NO: 54), Line 3 hit (SEQ ID NO: 55), Line 3 query (SEQ ID NO: 56), Line 4 hit (SEQ ID NO: 57), Line 4 query (SEQ ID NO: 58), Line 5 hit (SEQ ID NO: 59), Line 5 query (SEQ ID NO: 60), Line 6 hit (SEQ ID NO: 61), Line 6 query (SEQ ID NO: 62), Line 7 hit (SEQ ID NO: 63), Line 7 query (SEQ ID NO: 64), Line 8 hit (SEQ ID NO: 65), Line 8 query (SEQ ID NO: 66), Line 9 hit (SEQ ID NO: 67), Line 9 query (SEQ ID NO: 68), Line 10 hit (SEQ ID NO: 69), Line 10 query (SEQ ID NO: 51
  • the present invention is based on the finding of complete phenotypic correction of mitochondrial disorders occurs after a single transplantation of wildtype hematopoietic stem and progenitor cells, which differentiated into phagocytic cells in the nervous system, muscle and heart leading to the neuronal and myocyte cross-correction.
  • a pressing need to identify effective therapies for mitochondrial disorders such as FRDA for which there remains no treatment.
  • preclinical studies using stem cells or gene therapy have had limited success, or have been restricted to assessment of specific tissues.
  • a self-inactivating (SIN)-lentivirus vector containing the human frataxin (hFXN) cDNA as well as the optimal promoter can be used to ex vivo gene-corrected patients’ autologous hematopoietic stem and progenitor cells (HSPCs), which can then be re-transplant in the patients to repopulate their bone marrow, which will be a reservoir of “healthy” cells for the rest of the life of the patients. These cells mobilize and integrate into the diseased tissues (brain, muscle, heart), and will lead to their rescue.
  • HSPCs autologous hematopoietic stem and progenitor cells
  • references to “the method” includes one or more methods, and/or steps of the type described herein which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.
  • the term “comprising,” which is used interchangeably with “including,” “containing,” or “characterized by,” is inclusive or open-ended language and does not exclude additional, unrecited elements or method steps.
  • the phrase “consisting essentially of” limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristics of the claimed invention.
  • the present disclosure contemplates embodiments of the invention compositions and methods corresponding to the scope of each of these phrases.
  • composition or method comprising recited elements or steps contemplates particular embodiments in which the composition or method consists essentially of or consists of those elements or steps.
  • all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, the preferred methods and materials are now described.
  • terapéuticaally effective amount means the amount of a compound or pharmaceutical composition that will elicit the biological or medical response of a tissue, system, animal or human that is being sought by the researcher, veterinarian, medical doctor or other clinician.
  • the term “therapeutically effective amount” is used herein to denote any amount of a formulation that causes a substantial improvement in a disease condition when applied to the affected areas repeatedly over a period of time. The amount will vary with the condition being treated, the stage of advancement of the condition, and the type and concentration of formulation applied. Appropriate amounts in any given instance will be readily apparent to those skilled in the art or capable of determination by routine experimentation. [0042] A “therapeutic effect,” as used herein, encompasses a therapeutic benefit and/or a prophylactic benefit as described herein. [0043] The terms “administration” or “administering” are defined to include an act of providing a compound or pharmaceutical composition of the invention to a subject in need of treatment.
  • parenteral administration and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually orally or by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular, subarachnoid, intraspinal and infrasternal injection and infusion.
  • systemic administration means the administration of a compound, drug or other material other than directly into the central nervous system, such that it enters the subject's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration.
  • a viral vector specific for the cell type is not available, the vector can be modified to express a receptor (or ligand) specific for a ligand (or receptor) expressed on the target cell, or can be encapsulated within a liposome, which also can be modified to include such a ligand (or receptor).
  • a peptide agent can be introduced into a cell by various methods, including, for example, by engineering the peptide to contain a protein transduction domain such as the human immunodeficiency virus TAT protein transduction domain, which can facilitate translocation of the peptide into the cell.
  • a protein transduction domain such as the human immunodeficiency virus TAT protein transduction domain
  • biomaterial-based technologies such as nano-cages and pharmacological delivery wafers (such as used in brain cancer chemotherapeutics) which may also be modified to accommodate this technology.
  • the viral vectors most commonly assessed for gene transfer are based on DNA- based adenoviruses (Ads) and adeno-associated viruses (AAVs) and RNA-based retroviruses and lentiviruses.
  • Lentivirus vectors have been most commonly used to achieve chromosomal integration.
  • the terms “reduce” and “inhibit” are used together because it is recognized that, in some cases, a decrease can be reduced below the level of detection of a particular assay. As such, it may not always be clear whether the expression level or activity is “reduced” below a level of detection of an assay, or is completely “inhibited.” Nevertheless, it will be clearly determinable, following a treatment according to the present methods.
  • “treatment” or “treating” means to administer a composition to a subject or a system with an undesired condition. The condition can include a disease or disorder.
  • Prevention means to administer a composition to a subject or a system at risk for the condition.
  • the condition can include a predisposition to a disease or disorder.
  • the effect of the administration of the composition to the subject can be, but is not limited to, the cessation of one or more symptoms of the condition, a reduction or prevention of one or more symptoms of the condition, a reduction in the severity of the condition, the complete ablation of the condition, a stabilization or delay of the development or progression of a particular event or characteristic, or minimization of the chances that a particular event or characteristic will occur.
  • the term “genetic modification” is used to refer to any manipulation of an organism’s genetic material in a way that does not occur under natural conditions. Methods of performing such manipulations are known to those of ordinary skill in the art and include, but are not limited to, techniques that make use of vectors for transforming cells with a nucleic acid sequence of interest. Included in the definition are various forms of gene editing in which DNA is inserted, deleted or replaced in the genome of a living organism using engineered nucleases, or “molecular scissors.” These nucleases create site-specific double-strand breaks (DSBs) at desired locations in the genome.
  • DSBs site-specific double-strand breaks
  • NHEJ nonhomologous end-joining
  • HR homologous recombination
  • TALEN transcription activator-like effector-based nucleases
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • the prokaryotic CRISPR/Cas system has been adapted for use as gene editing (silencing, enhancing or changing specific genes) for use in eukaryotes (see, for example, Cong, Science, 15:339(6121):819-823 (2013) and Jinek, et al., Science, 337(6096):816-21 (2012)).
  • gene editing stress, et al., Science, 337(6096):816-21 (2012).
  • CRISPR system refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated (“Cas”) genes, including sequences encoding a Cas gene, a tracr (trans-activating CRISPR) sequence (e.g., tracrRNA or an active partial tracrRNA), a tracr-mate sequence (encompassing a “direct repeat” and a tracrRNA-processed partial direct repeat in the context of an endogenous CRISPR system), a guide sequence (also referred to as a “spacer”, “guide RNA” or “gRNA” in the context of an endogenous CRISPR system), or other sequences and transcripts from a CRISPR locus.
  • tracr trans-activating CRISPR
  • tracrRNA or an active partial tracrRNA e.g., tracrRNA or an active partial tracrRNA
  • a tracr-mate sequence encompassing a “direct repeat” and a tracrRNA-processed
  • tracr mate sequences operably linked to a guide sequence can also be referred to as “pre-crRNA” (pre-CRISPR RNA) before processing or crRNA after processing by a nuclease.
  • pre-crRNA pre-CRISPR RNA
  • a tracrRNA and crRNA are linked and form a chimeric crRNA-tracrRNA hybrid where a mature crRNA is fused to a partial tracrRNA via a synthetic stem loop to mimic the natural crRNA:tracrRNA duplex as described in Cong, Science, 15:339(6121):819-823 (2013) and Jinek, et al., Science, 337(6096):816-21 (2012)).
  • a single fused crRNA-tracrRNA construct can also be referred to as a guide RNA or gRNA (or single-guide RNA (sgRNA)).
  • gRNA guide RNA
  • sgRNA single-guide RNA
  • the crRNA portion can be identified as the ‘target sequence’ and the tracrRNA is often referred to as the ‘scaffold’.
  • one or more vectors driving expression of one or more elements of a CRISPR system are introduced into a target cell such that expression of the elements of the CRISPR system direct formation of a CRISPR complex at one or more target sites. While the specifics can be varied in different engineered CRISPR systems, the overall methodology is similar. A practitioner interested in using CRISPR technology to target a DNA sequence can insert a short DNA fragment containing the target sequence into a guide RNA expression plasmid.
  • the sgRNA expression plasmid contains the target sequence (about 20 nucleotides), a form of the tracrRNA sequence (the scaffold) as well as a suitable promoter and necessary elements for proper processing in eukaryotic cells.
  • Such vectors are commercially available (see, for example, Addgene). Many of the systems rely on custom, complementary oligos that are annealed to form a double stranded DNA and then cloned into the sgRNA expression plasmid. Co-expression of the sgRNA and the appropriate Cas enzyme from the same or separate plasmids in transfected cells results in a single or double strand break (depending of the activity of the Cas enzyme) at the desired target site.
  • Zinc-finger nucleases are artificial restriction enzymes generated by fusing a zinc finger DNA-binding domain to a DNA-cleavage domain.
  • Zinc finger domains can be engineered to target specific desired DNA sequences and this enables zinc-finger nucleases to target unique sequences within complex genomes. By taking advantage of endogenous DNA repair machinery, these reagents can be used to precisely alter the genomes of higher organisms.
  • the most common cleavage domain is the Type IIS enzyme Fok1. Fok1 catalyzes double-stranded cleavage of DNA, at 9 nucleotides from its recognition site on one strand and 13 nucleotides from its recognition site on the other. See, for example, U.S.
  • Transcription activator-like effector nucleases have an overall architecture similar to that of ZFNs, with the main difference being that the DNA-binding domain comes from TAL effector proteins, transcription factors from plant pathogenic bacteria.
  • the DNA-binding domain of a TALEN is a tandem array of amino acid repeats, each about 34 residues long. The repeats are very similar to each other; typically they differ principally at two positions (amino acids 12 and 13, called the repeat variable diresidue, or RVD).
  • RVD repeat variable diresidue
  • Each RVD specifies preferential binding to one of the four possible nucleotides, meaning that each TALEN repeat binds to a single base pair, though the NN RVD is known to bind adenines in addition to guanine.
  • TAL effector DNA binding is mechanistically less well understood than that of zinc-finger proteins, but their seemingly simpler code could prove very beneficial for engineered-nuclease design.
  • TALENs also cleave as dimers, have relatively long target sequences (the shortest reported so far binds 13 nucleotides per monomer) and appear to have less stringent requirements than ZFNs for the length of the spacer between binding sites.
  • Monomeric and dimeric TALENs can include more than 10, more than 14, more than 20, or more than 24 repeats.
  • the nuclease activity of the genome editing systems described herein cleave target DNA to produce single or double strand breaks in the target DNA.
  • Double strand breaks can be repaired by the cell in one of two ways: non-homologous end joining, and homology-directed repair.
  • non-homologous end joining NHEJ
  • the double-strand breaks are repaired by direct ligation of the break ends to one another. As such, no new nucleic acid material is inserted into the site, although some nucleic acid material may be lost, resulting in a deletion.
  • a donor polynucleotide with homology to the cleaved target DNA sequence is used as a template for repair of the cleaved target DNA sequence, resulting in the transfer of genetic information from a donor polynucleotide to the target DNA.
  • new nucleic acid material can be inserted/copied into the site. Therefore, in some embodiments, the genome editing vector or composition optionally includes a donor polynucleotide.
  • the modifications of the target DNA due to NHEJ and/or homology-directed repair can be used to induce gene correction, gene replacement, gene tagging, transgene insertion, nucleotide deletion, gene disruption, gene mutation, etc.
  • cleavage of DNA by the genome editing vector or composition can be used to delete nucleic acid material from a target DNA sequence by cleaving the target DNA sequence and allowing the cell to repair the sequence in the absence of an exogenously provided donor polynucleotide.
  • the methods can be used to add, i.e., insert or replace, nucleic acid material to a target DNA sequence (e.g., to “knock in” a nucleic acid that encodes for a protein, an siRNA, an miRNA, etc.), to add a tag (e.g., 6xHis (SEQ ID NO: 13), a fluorescent protein (e.g., a green fluorescent protein; a yellow fluorescent protein, etc.), hemagglutinin (HA), FLAG, etc.), to add a regulatory sequence to a gene (e.g., promoter, polyadenylation signal, internal ribosome entry sequence (IRES), 2A peptide, start codon, stop codon, splice signal, localization signal, etc.), to modify a nucleic acid sequence (e.g., introduce a mutation), and the like.
  • a tag e.g., 6xHis (SEQ ID NO: 13
  • a fluorescent protein e.g., a
  • compositions can be used to modify DNA in a site-specific, i.e., “targeted” way, for example gene knock-out, gene knock-in, gene editing, gene tagging, etc., as used in, for example, gene therapy.
  • targeted for example gene knock-out, gene knock-in, gene editing, gene tagging, etc.
  • the terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer.
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, ⁇ - carboxyglutamate, and O-phosphoserine.
  • Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an ⁇ carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
  • Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
  • a “regulatory gene” or “regulatory sequence” is a nucleic acid sequence that encodes products (e.g., transcription factors) that control the expression of other genes.
  • a “protein coding sequence” or a sequence that encodes a particular protein or polypeptide is a nucleic acid sequence that is transcribed into mRNA (in the case of DNA) and is translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory sequences.
  • the boundaries of the coding sequence are determined by a start codon at the 5' terminus (N-terminus) and a translation stop nonsense codon at the 3' terminus (C-terminus).
  • a coding sequence can include, but is not limited to, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic DNA, and synthetic nucleic acids.
  • a transcription termination sequence will usually be located 3' to the coding sequence.
  • a “promoter” is defined as a regulatory DNA sequence generally located upstream of a gene that mediates the initiation of transcription by directing RNA polymerase to bind to DNA and initiating RNA synthesis.
  • a promoter can be a constitutively active promoter (i.e., a promoter that is constitutively in an active/"ON” state), it may be an inducible promoter (i.e., a promoter whose state, active/"ON” or inactive/"OFF", is controlled by an external stimulus, e.g., the presence of a particular compound or protein), it may be a spatially restricted promoter (i.e., transcriptional control element, enhancer, etc.)(e.g., tissue specific promoter, cell type specific promoter, etc.), and it may be a temporally restricted promoter (i.e., the promoter is in the "ON" state or "OFF” state during specific stages of embryonic development or during specific stages of a biological process.
  • a constitutively active promoter i.e., a promoter that is constitutively in an active/"ON” state
  • it may be an inducible promoter (i.e., a promoter whose state, active/"ON
  • the term “gene” means the deoxyribonucleotide sequences comprising the coding region of a structural gene.
  • a “gene” may also include non- translated sequences located adjacent to the coding region on both the 5' and 3' ends such that the gene corresponds to the length of the full-length mRNA.
  • the sequences which are located 5' of the coding region and which are present on the mRNA are referred to as 5' non- translated sequences.
  • the sequences which are located 3' or downstream of the coding region and which are present on the mRNA are referred to as 3' non-translated sequences.
  • the term “gene” encompasses both cDNA and genomic forms of a gene.
  • a genomic form or clone of a gene contains the coding region interrupted with non-coding sequences termed “introns” or “intervening regions” or “intervening sequences.”
  • Introns are segments of a gene which are transcribed into heterogenous nuclear RNA (hnRNA); introns may contain regulatory elements such as enhancers. Introns are removed or “spliced out” from the nuclear or primary transcript; introns therefore are absent in the messenger RNA (mRNA) transcript.
  • mRNA messenger RNA
  • the terms “functionally linked” and “operably linked” are used interchangeably and refer to a functional relationship between two or more DNA segments, in particular gene sequences to be expressed and those sequences controlling their expression.
  • a promoter/enhancer sequence including any combination of cis- acting transcriptional control elements is operably linked to a coding sequence if it stimulates or modulates the transcription of the coding sequence in an appropriate host cell or other expression system.
  • Promoter regulatory sequences that are operably linked to the transcribed gene sequence are physically contiguous to the transcribed sequence.
  • “Conservatively modified variants” applies to both amino acid and nucleic acid sequences.
  • conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent variations,” which are one species of conservatively modified variations.
  • Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid.
  • each codon in a nucleic acid except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan
  • TGG which is ordinarily the only codon for tryptophan
  • amino acid sequences one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.
  • antibody refers to polyclonal and monoclonal antibodies and fragments thereof, and immunologic binding equivalents thereof.
  • antibody refers to a homogeneous molecular entity, or a mixture such as a polyclonal serum product made up of a plurality of different molecular entities, and broadly encompasses naturally-occurring forms of antibodies (for example, IgG, IgA, IgM, IgE) and recombinant antibodies such as single-chain antibodies, chimeric and humanized antibodies and multi-specific antibodies.
  • antibody also refers to fragments and derivatives of all of the foregoing, and may further comprise any modified or derivatised variants thereof that retains the ability to specifically bind an epitope.
  • Antibody derivatives may comprise a protein or chemical moiety conjugated to an antibody.
  • a monoclonal antibody is capable of selectively binding to a target antigen or epitope.
  • Antibodies may include, but are not limited to polyclonal antibodies, monoclonal antibodies (mAbs), humanized or chimeric antibodies, camelized antibodies, single chain antibodies (scFvs), Fab fragments, F(ab') 2 fragments, disulfide-linked Fvs (sdFv) fragments, for example, as produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, intrabodies, nanobodies, synthetic antibodies, and epitope-binding fragments of any of the above.
  • mAbs monoclonal antibodies
  • sdFv single chain antibodies
  • HSCs possess the ability of multipotency (i.e., one HSC can differentiate into all functional blood cells) and self-renewal (i.e., HSCs can divide and give rise to an identical daughter cell, without differentiation). Through a series of lineage commitment steps, HSCs give rise to progeny that progressively lose self-renewal potential and successively become more and more restricted in their differentiation capacity, generating multi-potential and lineage-committed progenitor cells, and ultimately mature functional circulating blood cells.
  • hematopoietic stem and progenitor cells hematopoietic stem and progenitor cells
  • HSPCs hematopoietic stem and progenitor cells
  • a “pluripotent cell” refers to a cell derived from an embryo produced by activation of a cell containing DNA of all female or male origin that can be maintained in vitro for prolonged, theoretically indefinite period of time in an undifferentiated state that can give rise to different differentiated tissue types, i.e., ectoderm, mesoderm, and endoderm.
  • Embryonic stem cells ES cells are pluripotent stem cells derived from the inner cell mass of a blastocyst, an early-stage preimplantation embryo.
  • pharmaceutically acceptable carrier encompasses any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water and emulsions such as an oil/water or water/oil emulsion, and various types of wetting agents.
  • HSPC hematopoietic stem and progenitor cell
  • mitochondrial diseases/disorders may be caused by mutations, acquired or inherited, in mitochondrial DNA (mtDNA) or in nuclear genes that code for mitochondrial components. They may also be the result of acquired mitochondrial dysfunction due to adverse effects of drugs, infections, or other environmental causes.
  • mitochondrial diseases include, but are not limited to, mitochondrial myopathy, diabetes mellitus and deafness (DAD), Leber's hereditary optic neuropathy (LHON), Leigh syndrome, subacute sclerosing encephalopathy, Neuropathy, ataxia, retinitis pigmentosa, and ptosis (NARP), myoneurogenic gastrointestinal encephalopathy (MNGIE), Myoclonic Epilepsy with Ragged Red Fibers (MERRF), Mitochondrial myopathy, encephalomyopathy, lactic acidosis, stroke-like symptoms (MELAS), mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) and diseases due to mitochondrial complex deficiency, such as Friedreich’s ataxia (FRDA).
  • DAD diabetes mellitus and deafness
  • LHON Leber's hereditary optic neuropathy
  • Ligh syndrome Leigh syndrome
  • subacute sclerosing encephalopathy Neuropathy, ataxia, retinitis
  • FRDA is a progressively lethal multi-systemic disease. Although the exact function of FXN is still under debate, it is predicted to assist in the biogenesis of mitochrondrial iron-sulfur clusters. Thus, frataxin deficiency results in altered cellular iron metabolism, increased mitochondrial iron load, decreased mitochondrial energy production and biogenesis as well as increased oxidative stress. Clinical features include gait and limb ataxia, muscle weakness, dysarthria and also vision and hearing anomalies, diabetes and cardiomyopathy. Frataxin deficiency impacts neuronal functions particularly and this affects mainly the peripheral and central nervous systems (CNS), leading to the progressive destruction of the Dorsal Root Ganglia (DRG).
  • CNS peripheral and central nervous systems
  • DRG Dorsal Root Ganglia
  • HSPCs Hematopoietic stem and progenitor cells
  • HSPC hematopoietic stem and progenitor cell
  • HSPC transplantation using a self- inactivating (SIN)-lentivirus vector containing human CTNS cDNA under the control of the strong ubiquitous short intron-less human Elongation Factor 1 alpha (EFS) promoter in lethally irradiated Ctns -/- mice led to the abundant engraftment of HSPC-derived cells in all organs, which correlated with the dramatic reduction in tissue cystine levels (up to 94% decrease).
  • ETS Elongation Factor 1 alpha
  • cystinosin is a ubiquitous, lysosomal transmembrane protein.
  • transplanted HSPCs led to the transfer of cystinosin-bearing lysosomes via tunneling nanotubes (TNTs) after differentiating into macrophages.
  • TNTs tunneling nanotubes
  • macrophage-derived tubular extensions penetrated the dense tubular basement membrane and delivered cystinosin-containing lysosomes into the epithelia in Ctns -/- mice, so as to prevent proximal tubule degeneration.
  • the same mechanism has been demonstrated in the eye and thyroid of HSPC-transplanted Ctns -/- mice.
  • the invention provides a method of treating a mitochondrial disease or disorder in a subject.
  • the method includes introducing ex vivo a functional human frataxin (hFXN) into hematopoietic stem and progenitor cells (HSPCs) of the subject, and thereafter transplanting the HSPCs into the subject, thereby treating the mitochondrial disease or disorder.
  • the step of introducing may include contacting a vector comprising a polynucleotide encoding hFXN and an ubiquitous or endogenous FXN promoter with the HSPCs and allowing expression of hFXN.
  • the vector is a self-inactivating (SIN)-lentivirus vector, such as pCCL-EFS-FXN or pCCL- FRDAp-FXN.
  • nucleic acid sequences for human and mouse frataxin (FRDA) are known in the art. See, for example, GenBank Accession No.: U43747.1, human frataxin mRNA, complete cds, which provides the nucleic acid sequence (SEQ ID NO: 1): TTTACAGGGCATAACTCATTTTATCCTTACCACAATCCTATGAAGTAGGAACTTTT ATAAAACGCATTTTATATNCAAGGGCACAGAGAGGNTAATTAACTTGCCCTCTGGT CACACAGCTAGGAAGTGGGCAGAGTACAGATTTACACTAGGCATCCGTCTCCTGNC CCCACATANCCAGCTGCTGTAAACCCATACCGGCGGCCAAGCAGCCTCAATTTGTG CATGCACCCACTTCCCAGCAAGACAGCAGCTCCCAAGTTCCTCCTGTTTAGAATTT TAGAAGCGGCGGGCCACCAGGCTGCA
  • the method of treating a mitochondrial disease or disorder in a subject includes contacting cells expressing hFXN from the subject with a vector encoding a gene editing system that when transfected into the cells removes a trinucleotide extension mutation of endogenous hFXN, thereby treating the mitochondrial disease or disorder.
  • the gene editing system is selected from the group consisting of CRISPR/Cas, zinc finger nucleases, and transcription activator-life effector nucleases.
  • the step of contacting may be performed ex vivo by first obtaining a sample of cells from the subject, transfecting the gene editing system into the sample of cells, and thereafter transplanting the transfected cells into the subject, thereby treating the mitochondrial disease or disorder.
  • the sample of cells may be any cells expressing hFXN, such as, for example, blood cells or HSPCs of the subject.
  • hFXN hFXN
  • HSPCs of the subject.
  • mitochondria can readily be transferred via tunneling nanotubes (TNTs).
  • TNTs tunneling nanotubes
  • YG8R mice are currently considered the best animal model of FRDA as they express only the human mutated frataxin containing 280 GAA repeats (SEQ ID NO: 14), without endogenous murine frataxin, fxn -/- FXN + .
  • This mouse model exhibits a decrease of 57% frataxin expression resulting in a mild progressive phenotype including ataxia, and coordination and locomotor anomalies similar to the clinical manifestations in FRDA patients.
  • the mice display a degeneration of the large sensory neurons of DRG, and decrease in aconitase activity and increase of oxidized proteins in the brain, heart and skeletal muscle.
  • the advantages of this mouse model are that the genetic defect is similar to that of humans and that the impact of stem cell therapy is tested in the CNS, heart and skeletal muscle in the same animal model.
  • the impact of HSPC transplantation in YG8R mice has been impressive as the neurological complications and muscle weakness were fully rescued in the treated mice, with functional, histological and biochemical properties comparable to wild-type (WT) mice.
  • WT wild-type mice.
  • the present disclosure also demonstrates that HSPCs differentiated into phagocytic cells in the brain, spinal cord, DRG, muscle and heart and transferred frataxin to the adjacent disease cells.
  • the present disclosure provides a method for autologous transplantation of ex vivo gene-modified HSPCs to introduce a functional frataxin.
  • the method involves use of a pCCL SIN-LV vector or gene editing to remove a trinucleotide extension mutation of endogenous hFXN in the HSPCs.
  • this approach has proven effective in the YG8R mouse model. This represents a unique treatment approach for FRDA that should lead to a clinical trial for this disease after completing the pharmacology/toxicology studies.
  • Gene therapy approaches for FRDA have already been tested in vitro and in vivo with successful outcomes.
  • Vectors derived from lentiviruses have supplanted ⁇ -retroviral vector for gene therapy due to their superior gene transfer efficiency and better biosafety profile. Indeed, all cases of leukemogenic complications observed to date in clinical trials or animal models involved the use of retroviral vectors with LTR containing strong enhancer/promoters that can trigger distant enhancer activation. In contrast, the third- generation of lentivirus vectors, SIN-LV, with the deletions in their LTR, contains only one internal enhancer/promoter, which reduces the incidence of interactions with nearby cellular genes, and thus, decreases the risk of oncogenic integration.
  • SIN-LV are also designed to prevent the possibility of developing replication competent lentivirus (RCL) during production of viral supernatants with three packaging plasmids necessary for production.
  • Lentivirus vectors efficiently transduce HSPCs and do not alter their repopulation properties, which make this type of vector an attractive vehicle for stem cell gene therapy.
  • Clinical trials using SIN-LV to gene-correct human HSPCs are being undertaken in the U.S. and Europe for several conditions including HIV-1, ⁇ -thalassemia, immune deficiencies, metabolic diseases and cancers. For immune deficiency disorders, 35 patients have been transplanted with SIN-LV-modified HSPCs so far.
  • a clinical trial in patients with Adrenoleukodystrophy (ALD) has achieved stable gene correction in ⁇ 20% of hematopoietic cells in two patients. Cerebral demyelination was arrested without further progression over three years of follow-up, which represents a clinical outcome comparable to that observed after allogeneic transplantation; there was no evidence of clonal dominance.
  • a clinical trial for Wilskott-Aldrich syndrome was reported in three patients 32 months post-transplantation. Stable and long-term engraftment of the gene- modified HSPCs (25-50%) resulted in improved platelet counts, protection from bleeding and infections, and resolution of eczema.
  • HSPC gene therapy approach has the key advantages: i) it treats all the complications by a single infusion of stem cells, ii) gene-correction will occur ex vivo in a controlled environment allowing cell characterization prior to transplantation, iii) gene-corrected HSPCs will reside in the bone marrow niche after transplantation where they will self-renew and become a reservoir of healthy cells for the lifespan of the patients, iv) it avoids immune reaction as compared to allogeneic transplantation.
  • autologous HSPC gene therapy could provide a cure for the lethal disease FRDA for which no treatment currently exists.
  • Another innovative aspect provided herein is the use of HSPCs as delivery vehicles for functional mitochondrial genes.
  • the present disclosure demonstrates that one single systemic transplantation of WT HSPCs in young adult YG8R mice fully prevents the development of FRDA pathology including neurobehavioral deficits, muscle weakness and degeneration of DRG sensory neurons.
  • One advantage of exogenous HSPC transplantation is the capacity of these cells to permanently replace/repopulate the marrow and migrate from their niche to differentiate into phagocytic cell types within multiple diseased tissues.
  • HSPCs can even transmigrate across the blood brain barrier and engraft within the CNS as differentiated microglia. This phenomena is enhanced by tissue injury and even by the use of busulfan-mediated myeloablation, as opposed to total body irradiation, which enhances the clinical relevance of this work for the treatment of FRDA. Consistently, it has been shown that transplanted HSPCs differentiate into microglial cells within the CNS of the YG8R mice but also macrophages in DRG, peripheral nerves, skeletal muscle and heart, the primary sites of FRDA pathological complications.
  • the findings provided herein show largely upregulated genes >2 fold change in YG8R mice compared to WT (13 genes out of 84 total) while very few changes were identified between WT and YG8R/WT HSPCs mice (4 genes) and for none the difference was significant.
  • the significantly upregulated genes in YG8R vs WT include three solute mitochondrial carrier family 25 genes, Mipep, an important component of the human mitochondrial import machinery implicated in developmental delay and the fatty acid transporter Cpt1b, which is upregulated in stress and Post-Traumatic Stress Disorder.
  • cellular iron metabolism dysregulation is evidenced in FRDA by the presence of iron deposits in cardiomyocystes of patients (Lamarche, et al.
  • HSPC-derived macrophages engrafted in kidney could deliver cystinosin-containing lysosomes to proximal tubular cells via TNTs in the mouse model of cystinosis.
  • TNTs crossing the basement membrane was the only route possible across the continuous, thick, dense tubular basement membrane to access the tubular cells.
  • Transfer of mitochondria via TNTs has previously been shown in vitro in response to cellular stress, and this prompted the testing of HSPC transplantation in FRDA.
  • frataxin-bearing mitochondria could be transferred via TNT intercellular connections from macrophages to frataxin-deficient cells.
  • RNAs were shown to be transferred from graft-derived microglia to neurons via extracellular vesicles/exosome shedding; ii) Release of mitochondria-containing vesicles, this was previously shown from mesenchymal stem cells to pulmonary alveoli in acute lung injury model, or more recently from astrocytes to neurons in a cerebral ischemia model; iii) Microglia-to-neuron transfer of mitochondria via the microglial branch extensions directly in contact with neurons. While this route has not yet been considered, the data presented herein suggest that this is a possible mode of transfer.
  • the YG8R mouse replicates human FRDA neurological symptoms such as coordination deficits from three months of age with a progressive decrease in locomotor activity.
  • HSPC transplantation on performance of motor- and sensory-dependent functional tasks and on muscle strength at both 5 and 9 months of age was assessed (3 and 7 months post-transplantation, respectively).
  • No difference was observed in performance in any of the behavioral tests at either time point between untreated YG8R mice and those transplanted with mfxn -/- hFXN + HSPCs, indicating that neither irradiation nor transplantation with mfxn -/- hFXN + HSPCs ameliorate the disease phenotype.
  • YG8R mice Compared to WT mice, YG8R mice (controls) and YG8R mice transplanted with mfxn -/- hFXN + HSPCs displayed significantly reduced open field locomotor activity, impaired coordination on rotarod, and alterations in gait as well as significantly decreased forelimb grip strength at both time points (Figure 1A). In contrast, YG8R mice transplanted with WT HSPCs exhibited normal locomotor activity and muscle strength at both 3 and 7 months post-transplantation (Figure 1B). Interestingly, and in contrast to previous findings in the cystinosis model, the YG8R mouse exhibiting the lowest level of donor-derived blood cell engraftment still exhibited physiological rescue of the neurobehavioral deficits.
  • Neurodegeneration in FRDA involves primarily the sensory components of the central nervous system (CNS) and peripheral nervous system (PNS), beginning with loss of large sensory neurons in the dorsal root ganglia (DRG). Loss of sensory neurons in DRGs also occurs in YG8R mice and is characterized by the presence of large vacuoles.
  • CNS central nervous system
  • PNS peripheral nervous system
  • HSPC-derived cells were abundant in the ascending sensory axon tracts, within the dorsal and ventral roots, motor pools and dorsal spinal cord gray matter ( Figures 1C and 1D). These cells were >99% Iba1 + and CD68 + , while fewer cells expressed MHCII ( ⁇ 30 %; Figures 6A-6C) indicating their microglial identity. 3D-visualization of engrafted spinal cord subjected to tissue clearing showed that a high concentration of engrafted HSPC-derived cells was found in close proximity to perivascular regions, suggesting that these cells infiltrate the CNS via the vasculature.
  • WT HSPC transplantation restores frataxin expression and mitochondrial function in the brain of YG8R mice.
  • Murine frataxin (mFxn) expression analysis in the brain confirmed that tissue engraftment of the HSPC-derived cells correlated with partial restoration of mfxn expression in treated mice as compared to YG8R controls, although not up to WT expression levels; a residual expression was also detected in YG8R mice likely due to cross-reactivity with human FXN (Figure 2C).
  • Mitochondrial dysfunction in FRDA is associated with the presence of increased levels of oxidized proteins within tissues.
  • lactate and pyruvate levels were measured by mass spectrometry analysis of skeletal muscle biopsies, a common assay for measuring impairment in oxidative metabolism, which was shown to be elevated in some mitochondrial diseases.
  • a significant increase of lactate and lactate-to-pyruvate ratio in skeletal muscle of YG8R mice was demonstrated compared to WT mice, which was corrected in the transplanted WT HSPC-transplanted YG8R mice ( Figure 3B). These data represent further evidence of mitochondrial dysfunction in the YG8R mice, which is normalized in the treated mice.
  • FRDA patients In addition to neurological deficits, FRDA patients also develop a progressive hypertrophic cardiomyopathy.
  • Muscle strength was also observed to be significantly impaired in YG8R mice and normal in the WT HSPC-transplanted YG8R mice.
  • the expression levels were measured of two muscle-specific E3 ibiquitin lagases, Muscle RING finger 1 (MuRF-1) and F-box (MAFbx)/atrogin-1, and a member of the transforming growth factor- ⁇ superfamily, myostatin, which are increased in each type of skeletal muscle atrophy.
  • Macrophages deliver frataxin-bearing mitochondria to diseased cells via tunneling nanotubes in vitro. It has been previously reported in the context of the lysosomal storage disorder cystinosis, that HSPC-derived macrophages promote functional rescue of diseased cells through a lysosomal cross-corrective mechanism via TNTs.
  • phagocytic cells could also mediate the transfer of frataxin-bearing mitochondria into mfxn -/- hFXN + cells via similar route.
  • Fibroblasts harvested from YG8R neonate skin were co-cultured with macrophages isolated from the bone marrow of Cox8- GFP DsRed mice, ubiquitously expressing the mitochondrial Cox8 protein fused to GFP alongside the cytosolic DsRed reporter gene.
  • GFP + mitochondria were transferred from the DsRed-expressing macrophages to the mfxn -/- hFXN + fibroblasts via long tubular protusions (Figure 4A).
  • HSPC-derived microglial cells/macrophages enable neuronal and muscular cross-correction in vivo.
  • YG8R mice were transplanted with HPSCs isolated from DsRed Cox8-GFP mice.
  • Cox8-GFP punctae were detected within the DsRed-expressing microglial cells but also within neurons in brain, spinal cord and DRGs ( Figures 4C and 8). It was observed that neurons containing Cox8-GFP were in contact with one or more DsRed + microglial branch extensions (Figure 4C) and GFP + punctae were also observed within these microglial processes ( Figure 4D).
  • WPRE Woodchuck hepatitis virus Posttranslational Regulatory Element
  • the human FXN cDNA (633bp), corresponding to the canonical frataxin (isoform I, FXN I) found in mitochondria, was amplified by PCR and inserted into pCCL generating pCCL-EFS-hFXN ( Figure 5A), and upstream eGFP generating pCCL-EFS- hFXNeGFP. Additionally, a lentviral construct that carries Cas9 enzyme and guide RNA was generated to remove the expansion of GAA repeats in the first intron of frataxin gene. The integrity of the constructs was verified by sequencing and restriction enzyme digestion. LV virus particles were produced and titered as previously described.
  • YG8R fibroblasts were transduced with pCCL-EFShFXNeGFP, resulting in ⁇ 100% GFP + cells, which were tested for their functional rescue. It was reported that frataxin deficiency results in increased cell susceptibility to H 2 O 2 toxicity. Compared to WT fibroblasts, significant reduction in cell survival after exposure to H 2 O 2 was observed in YG8R fibroblasts. Improved survival was demonstrated in the FXN-GFP-transduced fibroblasts compared to YG8R controls but did not reach the WT level (Figure 5B).
  • YG8R mice with a deletion of murine Fxn gene (mFxn) and expressing mutant human FXN gene (hFXN) containing 190+90 GAA repeat expansion were generated in a C57BL/6J background as previously described (Al-Mahdawi, et al., GAA repeat instability in Friedreich ataxia YAC transgenic mice. Genomics 84, 301-310 (2004); Al- Mahdawi, et al., GAA repeat expansion mutation mouse models of Friedreich ataxia exhibit oxidative stress leading to progressive neuronal and cardiac pathology. Genomics 88, 580- 590 (2006), both of which are incorporated herein by reference).
  • Breeding pairs consisted of females heterozygous for Fxn and males heterozygous for Fxn and hemizygous for FXN (B6.Cg-Fxntm1Mkn Tg(FXN)YG8Pook/J), and were purchased from Jackson Laboratory (Bar Harbor, ME). YG8R mice and wild-type (WT) mice used as controls for these studies were obtained from these breeders.
  • Genotyping was performed using the following primers: mfxn-F: 5’-CTTCCCTCTACCCTGCCTTC-3’ (SEQ ID NO: 5) mfxn-R: 5’-GGAGAACAGTGGACACAGTAACA-3’ (SEQ ID NO: 6) PGK-NEO: 5’-CATCGCCTTCTATCGCCTTCT-3’ (SEQ ID NO: 7) FXN-F: 5’-GGGCAGATAAAGGAAGGAGATAC-3’ (SEQ ID NO: 8) FXN-R: 5’-ACGATAGGGCAACACCAATAA-3’ (SEQ ID NO: 9).
  • Transgenic mice constitutively expressing GFP C57BL/6-Tg(ACTB- EGFP)1Osb/J) or DsRed (B6.Cg-Tg(CAG-DsRed*MST)1Nagy/J) were also purchased from Jackson Laboratory.
  • the mtGFP-Tg transgenic mice (C57BL/6J-Tg(CAG- Cox8/EGFP)49Rin) expressing the Cox8-GFP mitochondrial fusion protein were purchased from the RIKEN BioResource Center through the National Bio-Resource Project of the MEXT (Wako, Saitama, Japan).
  • mtGFP-Tg mice were backcrossed with Dsred-Tg mice to produce DsRed-mtGFP-tg mice.
  • Genotyping for mt-GFP was done by PCR as previously described (Shitara, et al., Non-invasive visualization of sperm mitochondria behavior in transgenic mice with introduced green fluorescent protein (GFP). FEBS Lett 500, 7-11 (2001)). Mice were maintained in a temperature- and humidity-controlled animal facility, with a 12-h light-dark cycle and free access to water and food. Both male and female mice were used in all experiments. [0116] Frataxin-GFP lentivirus construction, production and titer.
  • the Self Inactivated (SIN)-lentivirus vector (LV), pCCL-EFS-X-WPRE-GFP (pCCL-GFP) was used for stable gene transfer in HSPCs and macrophages.
  • the vector backbone contains the intron-less human elongation factor 1a promoter to drive transgene expression.
  • the human FXN cDNA (Clone ID 5300379, GE Healthcare; 633bp) corresponding to the canonical frataxin (isoform I, FXN I) found in mitochondria (Perez-Luz, et al., Delivery of the 135 kb human frataxin genomic DNA locus gives rise to different frataxin isoforms.
  • Genomics 106, 76-82 (2015), incorporated herein by reference was amplified by PCR using the following primers: F: 5’-TTAGGATCCATGTGGACTCTCG-3’ (SEQ ID NO: 10) and R: 5’- AGAGGATCCAGCATCTTTTCCG-3’ (SEQ ID NO: 11); and inserted into pCCL at the BamH1 restriction site in phase with the GFP cDNA.
  • LV were produced and titered as previously described (Harrison, et al., Hematopoietic stem cell gene therapy for the multisystemic lysosomal storage disorder cystinosis. Mol Ther 21, 433-444 (2013), incorporated herein by reference).
  • Bone marrow cell isolation, transduction transplantation and engraftment determination Bone marrow cells were flushed from the femurs of 6-8 week old YG8R mice, GFP transgenic mice, DsRed transgenic mice or DsRed mt-GFP transgenic mice.
  • Hematopoietic stem and progenitor cells (HSPCs) were isolated by immunomagnetic separation using anti-Sca1 antibody conjugated to magnetic beads (Miltenyl Biotec, Auburn, CA).
  • Sca1 + cells were directly transplanted by tail vein injection of 1x10 6 cells re- suspended in 100 ⁇ l of PBS into lethally irradiated (7Gy; X-Rad 320, PXi) YG8R mice.
  • Sca1 + cells from the DsRed transgenic mice were first transduced with LV-hFXN-GFP using a multiplicity of infection (MOI) of 10 in presence of polybrene (4mg/mL) in retronectin-coated (20g/mL) 24-well plates at a density of 2x10 6 cells per well for 16 hours in StemSpan medium (StemCell Technologies) supplemented with SCF, TPO, FLT3 ligand (100ng/mL each), and IL6 (20ng/mL) cytokines (PeproTech).
  • MOI multiplicity of infection
  • Bone marrow cell engraftment of the transplanted cells was measured in peripheral blood 2 months post- transplantation; blood samples freshly harvested from the tails were treated with red blood cell lysis buffer (eBioscience, San Diego, CA) and subsequently analyzed by flow cytometry (BD Accuri C6, BD Biosciences) to determine the proportion of GFP- or DsRed- expressing cells. [0118] Behavioral tests.
  • WT mice, YG8R mice, YG8R mice transplanted with mfxn -/- hFXN + HSPCs, and YG8R mice transplanted with either WT GFP or DsRed/mt-GFP HSPCs were tested at both 5 and 9 months of age before being sacrificed for tissue analysis.
  • Rotarod analysis was performed using a Roto-rod Series 8 apparatus (Ugo Basille, Comerio, Italy). The rod was a knurled plastic dowel (6.0 cm diameter) set at a height of 30 cm. During training the mice were placed on the stationary rotarod for 30 sec before the trial was initiated.
  • each mouse was given 4 trials per day, with a 60 sec inter-trial interval on the accelerating rotarod (4-40 rpm over 5 min). The latency to fall was recorded for each trial.
  • Locomotor activity was measured using an automated monitoring system (Kinder Associates, San Diego, CA). Polycarbonate cages (42 x 22 x 20 cm) containing a thin layer of bedding material were placed into frames (25.5 x 47 cm) mounted with photocell beams. Each mouse was placed into the open field and all movements were recorded over a 60- second testing period. Grip strength was measured using a device consisting of a 10 cm long T-shaped bar connected to a digital dynamometer (Ugo Basile, Comerio, Italy).
  • Gait measure (stride length) was collected using an automated gait analysis system (CatWalk (Noldus Instruments)). Animals were placed at one end of the walkway and allowed to run down the length of the walkway, as two light sources illuminated the surface contact of paws with the glass floor, producing an image of a paw print. During locomotion, the glass walkway was filmed from below by a video camera.
  • the CatWalk software program was used to analyze recorded footage, define individual paw prints (e.g., left forepaw, right hindpaw), and give readouts of multiple parameters of gait. Testing was administered daily for 5 days. Only unbroken bouts of locomotion, during which animals ran down the walkway at a consistent speed, were used for analysis. [0119] Primary fibroblast and macrophage isolation, and transduction. Fibroblasts were generated from skin biopsies of neonate of YG8R mice.
  • IC-21 macrophage cell line was used (American Type Culture Collection, catalog #TIB-186) and cultured in RPMI 1640 medium (Gibco). Six-well plates were coated with retronectin (20 ⁇ l/ml; Takara Bio) following the manufacturer’s instructions. IC-21 macrophages were plated at 250,000 cells in 2 ml per well and transduced with pCCLFXN-eGFP using a MOI of 15. Media was changed 24 hours after transduction. [0120] Live imaging.
  • YG8R fibroblasts were co-cultured with DsRed Cox8-GFP or macrophages stably transduced with a lentivirus expressing hFXN-GFP as previously described (Naphade, et al., Brief reports: lysosomal cross-correction by hematopoietic stem cell-derived macrophages via tunneling nanotubes. Stem Cells 33, 301-309 (2015), incorporated herein by reference). Briefly, 75,000 fibroblasts were co-cultured with equal number of macrophages in glass-bottomed culture dishes (MatTek Corp, Ashland, MA).
  • cDNA was then prepared using iScript cDNA Synthesis kit (Bio-Rad). Commercial TaqMan probes specific to mouse frataxin were employed to quantitate expression (Applied Biosystems).
  • Oxidative stress detection Protein lysates from tissues directly snap-frozen in liquid nitrogen after dissection were prepared using RIPA buffer (Sigma) containing proteases inhibitors (Roche) as previously described (Campuzano, et al., Frataxin is reduced in Friedreich ataxia patients and is associated with mitochondrial membranes. Human molecular genetics 6, 1771-1780 (1997), incorporated herein by reference). For each assay, 20 ⁇ g of protein was used after total protein concentration was determined using the BCA assay.
  • Proteins were then derivatized by adding 1X 2,4-Dinitrophenylhydrazine (DNPH) solution contained in the OxyBlot Protein Oxidation Detection kit (Chemicon International) according to manufacturer’s instructions. Samples were applied to electrophoresis and transferred to a PVDF membrane. After blocking with 1% BSA/PBS-T, membrane was incubated with Rabbit anti-Dinitrophenyl (DNP) antibody followed by a Goat anti-rabbit HRP conjugate, and visualized using ECL kit (Pierce). Protein levels were normalized using an anti-Tubulin (ab6161, Abcam) antibody and band intensity was quantified using ImagePro software (Media Cybernetics).
  • DNPH 1,4-Dinitrophenylhydrazine
  • Lactate/Pyruvate analysis Muscle biopsies (10 mg) were homogenized in ice in 1 ml of ice cold 40% acetonitrile (containing 0.1 %formic acid)/40% methanol/20% H 2 O) using a tissue grinder (dounce), followed by centrifugation for 10 minutes at 13,000 x g.
  • the extraction solution contained stable isotope of lactate ( 13 C 3 sodium-lactate, Cambridge Isotope Laboratories, Inc.). Supernatants were removed, dried in a speed vac/lyophilizer system, and re-suspended in 150 ⁇ l 0.1% formic acid.
  • Pellets were re-dissolved in 0.1N NAOH and protein content measured using a bicinchoninic acid (BCA assay).5 ⁇ l of each resuspended supernatant was injected on a C18-pfp HPLC column (Mac-Mode Analytical, Chadds Ford, PA), as previously described (Gertsman, et al., Validation of a dual LC- HRMS platform for clinical metabolic diagnosis in serum, bridging quantitative analysis and untargeted metabolomics. Metabolomics 10, 312-323 (2014), incorporated herein by reference), and coupled to an API-4000 triple quadrupole mass spectrometer (AB Sciex).
  • BCA assay bicinchoninic acid
  • DRG Dorsal root ganglia
  • L5 lumbar level 5
  • DRG sections were stained with thionin (Nissl stain) for visualization of neuronal cell bodies.
  • Three DRGs per subject were acquired at 60x magnification using a BZ-X700 fluorescent microscope (Keyence). The presence of vacuoles in each DRG was traced and measured by a blinded experimenter in duplicate using ImageJ; vacuoles were defined as extremely circular white (Nissl negative) areas with smooth edges within DRG neurons. Number of vacuoles and area of vacuolar space relative to entire area of each DRG section was compared across genotypes.
  • tissue was cut into 20 ⁇ m sections and directly mounted to gelatin-coated slides.
  • tissue was sectioned to 30 ⁇ m and collected as free-floating sections.
  • immunofluorescence tissues were incubated with the following antibodies: rat anti-CD68 (1:100; BioLegend 137001), Biotin rat anti-MHCII (1:100; BD Pharmigen 553622), rabbit anti-GFP (1:500; Abcam ab290), chicken anti-GFP (1:1500, Abcam ab13970), rabbit anti- Iba1 (1:1500; Wako #019-19741), goat anti-mCherry (1:1000, Sicgen AB0040), mouse anti-NeuN (1:500; Millipore MAB377), rabbit anti-MBP (1:200, Millipore AB980), mouse anti-NF200 (1:500, Millipore MAB5262), mouse anti- ⁇ -Actinin (1:400; Sigma), Rabbit anti-von Willibrand factor (1:300
  • AlexaFluor-conjugated secondary antibodies were used for visualization of antigens. Images were acquired using the LSM 880 with Airyscan confocal microscope (Zeiss), a Keyence BZ-X710 digital microscope system for high resolution stitching images of tissue sections, or an Olympus FV1000 confocal microscope for live imaging. Confocal image stacks were analyzed with IMARIS Software (Bitplane, Oxford Instruments). [0128] Quantification of neuronal cross-correction.
  • a 6-mm segment of cervical spinal cord from a mouse at 3 months post-transplantation with DsRed + HSPCs was processed for optical clearing as previously described (Chung, et al., Structural and molecular interrogation of intact biological systems. Nature 497, 332-337 (2013), incorporated herein by reference). Briefly, PFA-fixed tissue was infused with hydrogel monomer solution (4% PFA, 4% acrylamide, 0.05% bis-acrylamide) and thermally polymerized. Lipids were then passively extracted in SDS-containing borate buffer at 37°C for 4 weeks, until tissue was cleared. Clarified tissue was incubated in Rapidclear CS for 1 day and mounted using a Wilco dish.
  • Oxidative stress measurements employed one-tailed t-tests with the assumption that YG8R oxidation levels would be higher. For vacuole measurements, the Mann-Whitney nonparametric test corrected for multiple testing by the Bonferroni correction was used. In vitro experiments were performed in biological triplicates. Error bars denote s.e.m. The level of significance is indicated as follows: *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.005. EXAMPLE 3 Materials and Methods [0131] Human blood cells. CD34 + HSPCs from healthy donors were obtained from 2 different sources: leukopheresis bags of G-SCF mobilized cells kindly provided by Dr.
  • HiFi Cas9 Nuclease V3 protein (IDT, 62 ⁇ M) was diluted in electroporation buffer (Buffer R, Invitrogen) or Opti-MEM to 36 ⁇ M. Equivolume of crRNA-tracrRNA and diluted HiFi Cas9 Nuclease were mixed together and incubated at room temperature for 10-20 min. [0134] Transfection. Human FRDA fibroblasts (GM03816) from Coriell Institute were cultured in EMEM, 15% FBS at 37°C, 5% CO 2 . Transfection was carried out using the lipofectamine CRISPRMAX Cas9 transfection reagent kit (Invitrogen) following manufacturer’s instructions.
  • RNP complex and CRISPRMAX reagent were each diluted in 50 ⁇ l Opti-MEM medium and mixed.
  • the RNP complex solution is then immediately added to the CRISPRMAX reagent solution and mixed. After a 5-10 min incubation, the solution is added to the cells.
  • PCR program was set up as follows: (94°C for 20sec, 65.6°C for 2min 30sec) x 20, (94°C for 20sec, 65.6°C for 2min 30sec + 15sec/cycle) x 17. PCR products were then run on a 0.7% agarose gel. [0136] Lymphoblast Electroporation.
  • ddPCR Digital droplet PCR
  • 100ng of gDNA, HindIII (NEB) and 1X ddPCR Supermixes for Probes (No dUTP) (Biorad) were used in combination with two sets of primers/probe (Figure 17) to generate the droplets using the QX200 droplet generator (Biorad).
  • the droplets were transferred to a 96 well plate and the following PCR was carried out: 95°C for 10min ramp at 2°C/sec, (94°C for 30sec ramp at 2°C/sec, 60°C for 1min ramp at 2°C/sec) x 39, 98°C for 10min ramp at 2°C/sec.
  • mice anti-frataxin antibody (Abcam, ab110328), mouse anti-p53 antibody (SCBT, sc-126), rabbit anti-PAN actin antibody (Cell Signaling, #4968S) and mouse anti-GAPDH antibody (SCBT, sc-365062) followed by goat anti-mouse or -rabbit horseradish peroxidase conjugated secondary antibodies.
  • MitoPlate I-1 Mitochondrial activity within lymphoblasts were measured using the MitoPlate I-1 (Biolog, #14104) following manufacturer’s instructions. Briefly, wells containing the different mitochondrial inhibitors were rehydrated with a solution containing Redox Dye mix, saponin and succinate for 1h at 37°C.
  • CD34 + HSPC isolation and in vitro differentiation CD34 + HSPCs from leukopheresis bags or peripheral blood were isolated using the Miltenyi Biotech MACS human CD34 Microbead kit following manufacturer’s instructions.
  • Cells were cultured in complete medium consisting of IMDM medium supplemented with fetal bovine serum, BSA, Glutamine, Penicillin/Streptomycin, hIL-3, hIl-6 and h-SCF (Peprotech) at 37°C. Cell proliferation and viability were determined using an automated cell counter (Biorad). CFU assays were performed using Methocult H4434 enriched methylcellulose (StemCell Technologies). Two days post-electroporation, 3000 cells from each condition were mixed with 3ml of Methocult, and plated in triplicate into 35mm gridded cell culture dishes. After 12-14 days of culture at 37°C, 5% CO2, the different types of hematopoietic colonies were identified and counted.
  • NSG mouse transplantation The non-obese diabetic (NOD) severe combined immunodeficiency (SCID) Il2rg-/- (NSG) mice (Jackson Laboratory) were housed in a pathogen free colony in a biocontainment vivarium and handled in laminar flow hoods.
  • NOD non-obese diabetic
  • SCID severe combined immunodeficiency
  • Il2rg-/- mice Jackson Laboratory mice
  • Newborn pups at 3-7 days of life of both genders were injected with 1x10 6 cells/pup via intrahepatic injection of unmodified or gene-edited human CD34 + cells one-day after conditioning with 1.25Gy of sub-lethal body irradiation from a x-ray energy irradiator, and allowed to engraft over 12-16 weeks (Huey et al., Production of Humanized Mice through Stem Cell Transfer. Curr Protoc Mouse Biol.2018;8(1):17-27). [0142] Flow cytometry analysis of hematopoiesis.
  • Off-target assessment Potential off-target regions were predicted using the COSMID software. Alt-R Genome Editing Detection Kit (IDT) and primers listed in Figure 17 were used to detect the presence of potential indels within edited gDNA.
  • IDT Alt-R Genome Editing Detection Kit
  • primers listed in Figure 17 were used to detect the presence of potential indels within edited gDNA.
  • crRNAs Six guide Crispr-RNAs (crRNAs) were designed following Rule Set 2 (RS2) (Doench et al., Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat Biotechnol.2016;34(2):184-191) to remove the GAA expansion within the first intron of the frataxin gene (Figure 10A), and tested in FRDA fibroblasts.
  • RS2 Rule Set 2
  • RNP ribonucleoprotein complex
  • the UP4/DN4 guide pair (4RNP) displayed the greatest gene editing efficiency excising a ⁇ 2kb DNA fragment containing the expansion ( Figures 10B and 10C). Sequencing of the ⁇ 2kb resected fragment confirmed directed deletion of the repeats. [0146]
  • the intronic repeat excision protocol was then optimized using 4RNP and electroporation in lymphoblast cell lines from healthy donors, FRDA patients, and related carriers (Table 1), and in presence or absence of electroporation enhancer (single-stranded DNA oligonucleotide designed in silico to possess no homology with human, mouse, or rat genomes) to increase RNP uptake.
  • HL-60 a lymphoblastic cell line characterized by a p53 deficiency (Wolf, et al., Major deletions in the gene encoding the p53 tumor antigen cause lack of p53 expression in HL-60 cells. Proc Natl Acad Sci U S A.1985;82(3):790-794), was similarly electroporated with 4RNPenh (63.7% ⁇ 3.1% GAA gene editing, data not shown). No delay in proliferation was observed in gene modified cells compared to non-electroporated or Cas9-only control cells ( Figure 12D). These data confirmed that the delay in cell growth observed in the repaired cells was due to p53 expression induced by DSB.
  • CD34 + /4RNPenh Cell viability of CD34 + /4RNPenh was significantly lower at 48 and 96h post-electroporation compared to CD34 + cells (CD34 + ) and Cas9-electroporated cells (CD34 + /Cas9), but still above 74% ( Figure 13B).
  • Figure 13B Cell viability of CD34 + /4RNPenh was significantly lower at 48 and 96h post-electroporation compared to CD34 + cells (CD34 + ) and Cas9-electroporated cells (CD34 + /Cas9), but still above 74% (Figure 13B).
  • Table 2 Table 2 [0152] The differentiation ability of individual CD34 + cells was verified using Colony- Forming Unit (CFU) assays showing that hematopoietic lineage colony distribution in 4RNPenh-edited CD34 + cells was similar to controls ( Figure 13C).
  • CFU Colony- Forming Unit
  • the input cells had a gene editing rate ranging from 24.3 to 49.8%, and at 3 months post-transplant, gene editing ranged from 0.15 to 18% with a mean of 5.49% in bone marrow, 0.1 to 3.90% with a mean of 1.16% in spleen, and 0.2 to 15% with a mean of 4.65 in thymus (Figure 13G).
  • Figure 13G The input cells had a gene editing rate ranging from 24.3 to 49.8%, and at 3 months post-transplant, gene editing ranged from 0.15 to 18% with a mean of 5.49% in bone marrow, 0.1 to 3.90% with a mean of 1.16% in spleen, and 0.2 to 15% with a mean of 4.65 in thymus (Figure 13G).
  • Figure 13G The input cells had a gene editing rate ranging from 24.3 to 49.8%, and at 3 months post-transplant, gene editing ranged from 0.15 to 18% with a mean of 5.49% in bone marrow, 0.1 to
  • Results showed six potential off-target sites within the following genes’ introns: AGAP1, UNC5D, LRP1B, RARB, EPHX2 and DGKG ( Figure 18). Indel formation was tested via T7 endonuclease 1 (T7E1) mismatch detection assay using gDNA isolated from the edited cells. No detectable off-target activity was found in these six regions for all the patients ( Figure 16). To confirm this result, PCR products were sequenced and then compared to PCR amplicons from corresponding non-edited FRDA CD34 + cells. Deconvolution analysis using the ICE Synthego software (ice.synthego.com) exhibited no indel formation in any of the edited gDNA compared to the non-edited (Table 5).
  • the ribonuclease complex 4RNPenh generate 4 DSBs to remove the GAA expansion in FXN, and showed was overexpression of p53 at 24-hour post-electroporation in healthy and FRDA lymphoblasts.
  • cell proliferation was not affected in p53-knockout HL60 lymphoblasts.
  • human CD34 + cells were are transplanted in NSG mice only 24 hours after electroporation, p53-mediated proliferation delay observed after 4RNPenh-mediated gene editing could explain the drop of efficiency rate between the input cells and the in vivo bone marrow cells.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Epidemiology (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Wood Science & Technology (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Medicinal Chemistry (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Cell Biology (AREA)
  • Mycology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)

Abstract

L'invention concerne des méthodes pour le traitement d'une maladie ou d'un trouble associé(e) à un dysfonctionnement mitochondrial par l'introduction ex vivo d'une molécule d'acide nucléique dans des cellules souches et progénitrices hématopoïétiques (CSPH) suivie par la transplantation des CSPH chez un sujet qui a besoin d'un tel traitement. La molécule d'acide nucléique peut comprendre une frataxine humaine fonctionnelle (hFXN) ou peut comprendre un système d'édition de gènes qui, lorsqu'il est transfecté dans les cellules, élimine une mutation par expansion des trinucléotides de la hFXN endogène.
EP21771515.0A 2020-03-16 2021-03-11 Méthodes de traitement de troubles mitochondriaux Pending EP4121120A4 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US16/820,368 US12011488B2 (en) 2016-03-23 2020-03-16 Methods of treating mitochondrial disorders
PCT/US2021/021850 WO2021188349A2 (fr) 2020-03-16 2021-03-11 Méthodes de traitement de troubles mitochondriaux

Publications (2)

Publication Number Publication Date
EP4121120A2 true EP4121120A2 (fr) 2023-01-25
EP4121120A4 EP4121120A4 (fr) 2024-07-31

Family

ID=77771484

Family Applications (1)

Application Number Title Priority Date Filing Date
EP21771515.0A Pending EP4121120A4 (fr) 2020-03-16 2021-03-11 Méthodes de traitement de troubles mitochondriaux

Country Status (4)

Country Link
EP (1) EP4121120A4 (fr)
AU (1) AU2021239908A1 (fr)
CA (1) CA3171432A1 (fr)
WO (1) WO2021188349A2 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN119770654A (zh) * 2025-01-08 2025-04-08 南京大学 Parp1抑制剂在制备治疗和/或缓解弗里德赖希氏共济失调的药物中的应用

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080050393A1 (en) * 1998-12-03 2008-02-28 Tang Y Tom Novel nucleic acids and polypeptides
EP1613774A2 (fr) * 2003-03-10 2006-01-11 Applera Corporation Polymorphismes genetiques associes a la stenose, procedes de detection et utilisations associees
CA2817256A1 (fr) * 2010-11-12 2012-05-18 The General Hospital Corporation Arn non codants associes a polycomb
CA2921457A1 (fr) * 2013-08-16 2015-02-19 Rana Therapeutics, Inc. Arn non codant formant de l'heterochromatine
AU2015234204A1 (en) * 2014-03-20 2016-10-06 Universite Laval CRISPR-based methods and products for increasing frataxin levels and uses thereof
ES2905181T3 (es) * 2015-05-01 2022-04-07 Prec Biosciences Inc Deleción precisa de secuencias cromosómicas in vivo
CN109476716A (zh) * 2016-03-23 2019-03-15 加利福尼亚大学董事会 治疗线粒体障碍的方法
CA3045335A1 (fr) * 2016-12-01 2018-06-07 Universite Laval Traitement base sur crispr de l'ataxie de friedreich

Also Published As

Publication number Publication date
AU2021239908A1 (en) 2022-10-06
WO2021188349A2 (fr) 2021-09-23
EP4121120A4 (fr) 2024-07-31
WO2021188349A3 (fr) 2021-10-21
CA3171432A1 (fr) 2021-09-23

Similar Documents

Publication Publication Date Title
US12012437B2 (en) Methods of treating mitochondrial disorders
Ferrari et al. Gene therapy using haematopoietic stem and progenitor cells
JP7365374B2 (ja) ヌクレアーゼ介在性遺伝子発現調節
US20240293578A1 (en) Methods of treating mitochondrial disorders
US20230330195A1 (en) Methods and compositions for the treatment of neurologic disease
US9757420B2 (en) Gene editing for HIV gene therapy
JP6722176B2 (ja) 造血幹細胞におけるヌクレアーゼ媒介性ゲノム工学および補正のための方法および組成物
KR102377189B1 (ko) C9orf72 유전자좌에서 파괴를 갖는 비-인간 동물
JP2022512674A (ja) 人工トランス活性化因子による選択
EP4121120A2 (fr) Méthodes de traitement de troubles mitochondriaux
US20240424139A1 (en) Magnetic-Assisted Nanoparticle Delivery and Gene Editing Systems and Methods of Use
CN107429261A (zh) 在细胞中迅速产生单一或多重报告基因的方法和组合物
US20260027157A1 (en) Methods for treating alzheimer's disease
JP2026505771A (ja) グリア細胞の競合的置換
Yokoo et al. Stem Cell Therapy Against Oxidative Stress and Hypoxia

Legal Events

Date Code Title Description
STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE INTERNATIONAL PUBLICATION HAS BEEN MADE

PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: REQUEST FOR EXAMINATION WAS MADE

17P Request for examination filed

Effective date: 20221005

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

DAV Request for validation of the european patent (deleted)
DAX Request for extension of the european patent (deleted)
REG Reference to a national code

Ref country code: HK

Ref legal event code: DE

Ref document number: 40087105

Country of ref document: HK

A4 Supplementary search report drawn up and despatched

Effective date: 20240703

RIC1 Information provided on ipc code assigned before grant

Ipc: A61K 35/28 20150101ALI20240627BHEP

Ipc: C12N 15/11 20060101ALI20240627BHEP

Ipc: C12N 15/63 20060101ALI20240627BHEP

Ipc: C12N 15/86 20060101ALI20240627BHEP

Ipc: A61K 48/00 20060101AFI20240627BHEP