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WO2017081033A1 - Différentiation ou reprogrammation cellulaire à l'aide de fezf2 et de lmo4 - Google Patents

Différentiation ou reprogrammation cellulaire à l'aide de fezf2 et de lmo4 Download PDF

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WO2017081033A1
WO2017081033A1 PCT/EP2016/077029 EP2016077029W WO2017081033A1 WO 2017081033 A1 WO2017081033 A1 WO 2017081033A1 EP 2016077029 W EP2016077029 W EP 2016077029W WO 2017081033 A1 WO2017081033 A1 WO 2017081033A1
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cell
nucleic acid
reprogrammed
lmo4
cells
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Michèle STUDER-MENEGHELLO
Kawssar HARB
Christian ALFANO
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Centre National de la Recherche Scientifique CNRS
Institut National de la Sante et de la Recherche Medicale INSERM
Universite de Nice Sophia Antipolis UNSA
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Centre National de la Recherche Scientifique CNRS
Institut National de la Sante et de la Recherche Medicale INSERM
Universite de Nice Sophia Antipolis UNSA
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    • 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
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4705—Regulators; Modulating activity stimulating, promoting or activating activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/30—Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C12N5/0619—Neurons
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/998—Proteins not provided for elsewhere
    • 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
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/08—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from cells of the nervous system
    • 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
    • C12N2510/00—Genetically modified cells

Definitions

  • the present invention relates to the field of medicine and more particularly to gene and cellular therapy.
  • Inventors herein identify for the first time active molecules, Fezf2 and Lmo4, whose combined expression orients the differentiation of a cell and allows the reprogramming of a cell exhibiting a particular phenotype into a reprogrammed cell exhibiting a different phenotype.
  • the present invention in particular relates to products, uses and methods for obtaining a subcortical projection neuron whose axon projects to the striatum, to the thalamus, to the brainstem and/or to the spinal cord once implanted in vivo, from a mature or immature cell.
  • the invention also relates to the thereby obtained differentiated or reprogrammed cells, the population of cells comprising said differentiated and/or reprogrammed cells, as well as the compositions and kits comprising said cells or population of cells, and to uses thereof.
  • neurodegenerative disease covers a range of conditions that primarily affect the neurons in the human nervous system. Neurons are the building blocks of the nervous system, which includes the brain and spinal cord. Neurons normally don't reproduce or replace themselves, so when they become damaged or die, the body cannot replace them. Examples of neurodegenerative diseases include motor neuron diseases (MND) such as Amyotrophic lateral sclerosis (ALS), Parkinson's, Alzheimer's, Huntington's disease, spinocerebellar ataxia (SCA) and spinal muscular atrophy (SMA). Neurodegenerative diseases are incurable and debilitating conditions that result in progressive degeneration and/or death of nerve cells. This causes problems with movement (called ataxias), or mental functioning (called dementias).
  • MND motor neuron diseases
  • ALS Amyotrophic lateral sclerosis
  • Parkinson's Alzheimer's
  • Huntington's disease spinocerebellar ataxia
  • SMA spinal muscular atrophy
  • Neurodegenerative diseases are incurable and debilitating conditions that result in progressive degeneration
  • Amyotrophic lateral sclerosis is a particular devastating neurodegenerative disorder, affecting the motor neurons of the central nervous system (cortex, brainstem) and the peripheral motor neurons (spinal cord).
  • the disease destroys the nerve cells that control voluntary movement and is characterized by progressive muscle weakening, paralysis and death, usually within 2 to 5 years after the appearance of the first clinical sign.
  • the disease affects limbs, tongue, pharynx and larynx muscles. Onset usually occurs after age 45, and the rate and pattern of disease progression vary widely.
  • Parkinson's disease also known as idiopathic or primary parkinsonism, hypokinetic rigid syndrome (HRS), or paralysis agitans
  • the motor symptoms of Parkinson's disease result from the death of dopamine-generating cells in the substantia nigra, a region of the midbrain. The cause of this cell death is poorly understood.
  • the most obvious symptoms are movement- related; these include shaking, rigidity, slowness of movement and difficulty with walking and gait. Later, thinking and behavioral problems may arise, with dementia commonly occurring in the advanced stages of the disease, whereas depression is the most common psychiatric symptom.
  • Other symptoms include sensory, sleep and emotional problems.
  • Parkinson's disease is more common in older people, with most cases occurring after the age of 50; when it is seen in young adults, it is called young onset PD (YOPD).
  • YOPD young onset PD
  • Treatments are effective at improving the early motor symptoms of the disease. This is typically with the medications L-DOPA and dopamine agonists. As the disease progresses and dopaminergic neurons continue to be lost, these drugs eventually become ineffective at treating the symptoms and at the same time produce a complication marked by involuntary writhing movements. Surgery and deep brain stimulation have been used to reduce motor symptoms as a last resort in severe cases where drugs are ineffective. Research directions include investigations into new animal models of the disease and of the potential usefulness of gene therapy, stem cell transplants and neuroprotective agents. In 2013 PD resulted in 103,000 deaths up from 44,000 deaths in 1990.
  • AD Alzheimer's disease
  • AD also known as Alzheimer disease, or just Alzheimer's
  • a chronic neurodegenerative disease that usually starts slowly and gets worse over time.
  • the most common early symptom is difficulty in remembering recent events (short- term memory loss).
  • symptoms can include problems with language, disorientation (including easily getting lost), mood swings, loss of motivation, loss of self-care, and behavioural issues.
  • the speed of progression can vary, the average life expectancy following diagnosis is three to nine years. No treatments stop or reverse its progression, though some may temporarily improve symptoms.
  • AD Alzheimer's disease
  • the mammalian cerebral cortex is tangentially subdivided into different functional areas, which are deputed to several higher functions, such as the processing of sensory and motor inputs as well as to the elaboration of thoughts and motor plans. All neocortical areas are radially organized into six neuronal layers that, despite similarities in their laminar organization, show remarkable diversities in features and functions. This is mainly due to differences in the molecular identity, morphology and long-range connectivity of residing neurons (Greig et al., 2013; Molyneaux et al., 2007).
  • Glutamatergic neurons constitute the major neuronal population of the neocortex and can be roughly subdivided into three main classes: corticothalamic (CT), intra-telencephalic (IT) and pyramidal tract (PT) projection neurons (Harris and Shepherd, 2015; Shepherd, 2013).
  • IT-type neurons project to the ipsi- and/or contralateral regions of the telencephalon (i.e. cortex and/or striatum).
  • PT-type neurons project ipsilaterally to different telencephalic and subcerebral targets, such as the striatum, the brainstem and the spinal cord
  • CT-type neurons mainly project to the ipsilateral reticular and thalamic nuclei.
  • Fezf2 FEZ Family Zinc Finger 2 protein
  • CPN corticofugal projection neurons
  • SCPNs subcerebral projection neurons
  • Andres de la Rossa et al. (2013) and Rouaux and Arlotta (2013) describe Fezf2 as an ideal molecular tool whose expression is necessary and sufficient to generate layer V neurons during corticogenesis.
  • Rouaux and Arlotta suggest the existence of a critical period of nuclear plasticity allowing reprogramming, while Andres de la Rossa et al. specify that the ability of Fezf2 to reprogram layer IV neurons is largely limited to the first postnatal week.
  • the present description relates to the combined use of Fezf2 and Lmo4 for differentiating a cell or for reprogramming a cell exhibiting a particular phenotype into a reprogrammed cell exhibiting a different phenotype.
  • Fezf2 and Lmo4 are expressed in vivo, in vitro or ex vivo either simultaneously or sequentially in order to allow the cell differentiation or cell reprogramming.
  • a nucleic acid sequence coding for Fezf2 and Lmo4 as well as a combination of a first nucleic acid sequence coding for Fezf2 and of a second nucleic acid sequence coding for Lmo4 are in particular herein described for use for differentiating a cell or for reprogramming a cell exhibiting a particular phenotype into a reprogrammed cell exhibiting a different phenotype, as well as a vector comprising such nucleic acid sequence(s), a cell comprising such a vector and a composition comprising said nucleic acid sequence(s), vector and/or cell.
  • the present invention allows the differentiation or reprogramming of a cell into a subcortical neuron whose axon projects to the striatum, to the thalamus, to the brainstem and/or to the spinal cord (once implanted in vivo).
  • a differentiated or reprogrammed cell is typically comparable to, and behaves like, a projection neuron naturally located in layer V or layer VI of the cerebral cortex.
  • Inventors herein disclose a in vivo, in vitro or ex vivo method for differentiating a cell or for reprogramming a cell exhibiting a particular phenotype into a reprogrammed cell exhibiting a different phenotype, wherein the method comprises a step of simultaneously or sequentially delivering Fezf2 and Lmo4, in particular Fezf2 and Lmo4 nucleic acid sequences, to the cell to be differentiated or reprogrammed. They further disclose the thereby obtained differentiated or reprogrammed cell and population of cells.
  • compositions in particular a pharmaceutical composition comprising a differentiated or reprogrammed cell or a population comprising such cells, preferably together with a pharmaceutically acceptable carrier or support, typically together with a cell preservation medium.
  • the differentiated or reprogrammed cell or the cell population herein described can be used as a cellular model, in particular for studying a neurodegenerative disease or disorder, a cerebral stroke or a spinal cord injury; for evaluating the therapeutic properties of a compound; or for screening for compounds useful in the prevention or treatment of a herein mentioned disease, disorder, stroke or injury.
  • the present invention also provides a kit for differentiating or reprogramming a cell exhibiting a particular phenotype into a reprogrammed cell exhibiting a different phenotype, preferably into a subcortical projection neuron whose axon projects to the striatum, to the thalamus, to the brainstem and/or to the spinal cord once implanted in vivo, wherein the kit comprises a nucleic acid sequence or combination of nucleic acid sequences, a vector and/or a composition as herein described.
  • This kit preferably comprises at least a nucleic acid sequence coding for Fezf2 and Lmo4, or a combination of a first nucleic acid sequence coding for Fezf2 and of a second nucleic acid sequence coding for Lmo4, optionally together with a nucleic acid sequence coding for the neurogenic factor Neurogenin 2 (Ngn2) and/or with a nucleic acid sequence coding for the neurogenic factor Ascll, and optionally together with a preservation medium and/or any appropriate pharmaceutical carrier or support.
  • Ngn2 neurogenic factor Neurogenin 2
  • Ascll neurogenic factor Ascll
  • the description also provides a therapeutic kit comprising a differentiated or reprogrammed cell or cell population as herein described together with a preservation medium, the cells or cell population being optionally associated to a pharmaceutical support.
  • Fig. 1 overexpressing the transcriptional adapter Lmo4 (Fig. 1) by in utero electroporation (Fig. 2) in the lower layers of the mouse somatosensory cortex induces the expression of Ctip2, an important transcription factor involved in cortico-spinal motor neuron differentiation (Arlotta et ah, 2005; Chen et ah, 2008; Srivatsa et ah, 2014), in approximately 30% of the electroporated cells (Fig. 3A-C).
  • Lmo4 de-represses Ctip2 transcription by preventing the interaction of Satb2, a transcription factor promoting callosal neuron specification (Alcamo et ah, 2008; Britanova et ah, 2008; Srivatsa et ah, 2014), with the histone deacetylase 1 (Hdacl) on the locus of Ctip2 (Fig. 4A-C).
  • Satb2 a transcription factor promoting callosal neuron specification
  • Hdacl histone deacetylase 1
  • Lmo4 overexpression induces Ctip2 expression in lower layers, but not in upper layers of the cortex (Fig. 3B-D), where Ctip2 is normally not present. This implies that Lmo4 must work together with a transcriptional activator that efficiently induces Ctip2 in the lower layers of the cortex.
  • the observed increase in Ctip2 expression does not merely represent the sum of the separate effects of Lmo4 and Fezf2 expression, but rather indicates an unexpected synergic action of these two factors.
  • the co-electroporation of Fezf2 and Lmo4 at embryonic stage 14.5 (or E14.5), which is the stage at which upper layer neurons (and callosal neurons among them) are produced changes the connectivity of upper layer cells from a callosal to a corticospinal one (Fig. 5).
  • the present invention relates to compositions and methods for obtaining a neuronal cell, in particular a subcortical projection neuron, from a cell to be differentiated or reprogrammed as herein defined.
  • the present invention in particular relates to a method for differentiating a cell or for reprogramming a cell exhibiting a particular phenotype into a reprogrammed cell exhibiting a different phenotype, wherein the method is performed in vitro, ex vivo or in vivo and comprises a step of simultaneously or separately delivering Fezf2 and Lmo4, typically Fezf2 and Lmo4 nucleic acid sequences, to the cell to be differentiated or reprogrammed, preferably thanks to a nucleic acid delivery vector as herein described.
  • nucleic acid sequence coding at least for Fezf2 and/or Lmo4 for use in vivo, in vitro or ex vivo for differentiating a cell or for reprogramming a cell exhibiting a particular phenotype into a reprogrammed cell exhibiting a different phenotype.
  • nucleic acid designates a DNA or a RNA molecule. It is typically a nucleic acid construct encoding for one or several, typically one, for example two, three or four proteins of interest, preferably selected from Fezf2, Lmo4, Neurogenin 2 (Ngn2) and Ascll .
  • a preferred nucleic acid construct codes for one protein of interest, either Fezf2 or Lmo4.
  • a single nucleic acid construct codes for several proteins of interest simultaneously, typically for Fezf2, Lmo4, Ngn2 and/or Ascll .
  • the nucleic acid construct codes simultaneously for Fezf2 and Lmo4.
  • the nucleic acid construct codes simultaneously for Fezf2, Lmo4 and Ngn2.
  • the nucleic acid construct codes simultaneously for Fezf2, Lmo4, Ngn2 andAscll .
  • nucleic acids each coding for at least one of the herein described proteins of interest for use in vitro or ex vivo for differentiating a cell or for reprogramming a cell exhibiting a particular phenotype into a reprogrammed cell exhibiting a different phenotype.
  • Such a combination typically involves a first nucleic acid sequence coding for Fezf2 and a second nucleic acid sequence coding for Lmo4.
  • Another combination of interest involves three or four nucleic acid sequences respectively coding for Fezf2, Lmo4, Ngn2 and Ascll .
  • nucleic acid sequence(s) coding for the protein(s) of interest is (are) recombinantly expressed via one or more vectors introduced into the cell to be differentiated or reprogrammed according to methods well known by the skilled person.
  • the present description indeed provides a vector comprising a nucleic acid sequence, typically a nucleic acid construct, as herein defined.
  • a vector is herein also identified as “expression vectors”, “nucleic acid delivery vector” or “gene delivery vector”.
  • the vectors used in the present invention may be any viral or non viral vectors suitable for introducing nucleic acids into a cell in vivo, ex vivo or in vitro.
  • Non-viral vectors include plasmids which can be used together with liposomes, electrically charged lipids (cytofectins), DNA-protein complexes and biopolymers.
  • the vectors are viral vectors, preferably retroviruses including lentiviruses, but also adenoviruses, herpes viruses, adeno-associated viruses (AAV), poxviridae, baculovirus, vaccinia or Epstein-Barr viruses. Most preferred vectors are lentiviruses.
  • Retroviral or AAV vectors may be produced following conventional techniques as described for instance in WO 92/07943, WO 90/02806, US 5,278,056; WO 97/09441 ; WO 97/06272, WO 96/39530, WO 95/34671, WO 96/22378.
  • the nucleic acid delivery vector provides a regulated expression of the nucleic acid or transgene and thus of the protein of interest.
  • expression may be constitutive and/or ubiquitous, preferred vectors provide for tissue-specific or inducible gene expression.
  • Tissue- specific expression can be achieved with regulated or tissue-specific promoters, such as NSE (neuron- specific enolase), etc.
  • regulated expression may be obtained using silencer elements that control expression from ubiquitous promoters.
  • a neuron-restrictive silencer element may be used in front of a ubiquitous promoter in the vectors according to the present invention, thereby limiting nucleic acid or transgene expression to the neurons and essentially avoiding expression in muscle cells.
  • a ubiquitous promoter in the vectors according to the present invention, thereby limiting nucleic acid or transgene expression to the neurons and essentially avoiding expression in muscle cells.
  • suitable, constitutive or tissue-specific promoters are described in WO 99/41396 and FR 2 774 698 and includes, for instance, viral promoters (RSV; LTR; CMV) or other promoters active in mammalian cells (actin, fibrin, PGK, enolase, etc.).
  • the in vivo, in vitro or ex vivo delivery step of Fezf2 and Lmo4 can be performed thanks to a nucleic acid delivery vector as explained previously.
  • administration may be performed into the motor and sensorymotor cortices (or in injured regions of these areas).
  • the vector is typically administered through a single or through several intracerebral injections of vectors by stereotaxis.
  • the vector(s) can also be directly administered in one or several part of the spinal cord, for example in area(s) where a loss of neurons has been detected.
  • Lmo4 is preferably administered or allowed to be expressed first, i.e. before Fezf2, in the cell to be differentiated or reprogrammed, for instance between 30 minutes and 1 week, typically between 6 hours and 1 day, prior to Fezf2.
  • Lmo4 and Fezf2 are preferably administered simultaneously, preferably in a 1 : 1 ratio.
  • Ngn2 nucleic acid is typically administered before Lmo4/Fezf2 nucleic acids to the cell to be differentiated or reprogrammed, for example from 1 hour to one week, typically between 6 hours and 1 day, for example 12 hours, before Lmo4 and Fezf2.
  • the nucleic acid sequence coding Ascll is typically administered before Lmo4/Fezf2 nucleic acids to the cell to be differentiated or reprogrammed, for example from 1 hour to one week, typically between 6 hours and 1 day, for example 12 hours, before Lmo4 and Fezf2.
  • the administration of nucleic acids is typically repeated one or more times per administration step occurring during the reprogramming/differentiation process, typically during a treatment or preventive method as herein described.
  • the administration step can be performed once or several times per day.
  • the administration step is preferably performed once per day.
  • each step of the reprogramming/differentiation process is performed only once.
  • the cell to be differentiated or reprogrammed is an animal cell, typically a mammalian cell, preferably a human cell.
  • the cell to be reprogrammed can be an immature or a mature cell.
  • the immature cell is typically selected from a stem cell, a progenitor cell, a precursor cell and an induced pluripotent stem cell (iPS).
  • the immature cell can have an embryonic or neonatal origin or can be derived from a mature animal whatever its age.
  • the immature cell is typically a neural (or neuronal) cell.
  • the cell to be differentiated is typically an immature cell as defined herein above.
  • stem cell refers to a cell that, by successive divisions can give rise to specialized cells.
  • neural stem cell and “neuronal stem cell”, as used herein, refers to a self-renewing, multipotent cell that generates the main phenotypes of the nervous system. Neural stem cells primarily differentiate into neurons, astrocytes and oligodendrocytes. Neural stem cells are identified by the expression of nestin, musashil, Sox2, CD133, vimentin and/or Notch markers.
  • Neural stem cells may be isolated from various areas of the adult brain, including, without limitation, the adult striatal tissue, the subventricular zone (SVZ) of the brain, the cerebellum and non-neurogenic areas, such as the spinal cord.
  • Said neural stem cells may be derived from a pluripotent stem cell selected from a non- human embryonic stem cell (ES) and an induced pluripotent stem cell (iPS). Therefore, in an embodiment the neural stem cell is derived from a pluripotent stem cell selected from a non-human embryonic stem cell (ES) and an induced pluripotent stem cell (iPS).
  • pluripotent stem cell refers to a stem cell that has the potential to differentiate into any of the three germ layers: endoderm (interior stomach lining, gastrointestinal tract, the lungs), mesoderm (muscle, bone, blood, urogenital), or ectoderm (epidermal tissues and nervous system). Pluripotent stem cells can give rise to any fetal or adult cell type but they cannot give rise to an entire organism.
  • a “pluripotent stem cell” may be identified by the expression of one or more of the cell markers Klf4, Sox2, Oct4, cMyc, Nanog and SSEA1.
  • Assays to assess the pluripotentiality of a cell are known in the art.
  • pluripotent stem cell includes non-human embryonic stem cells (ES) as well as induced pluripotent stem cells (iPS).
  • the pluripotent stem cell is selected from a non-human embryonic stem cell (ES) and an induced pluripotent stem cell (iPS).
  • embryonic stem cell refers to a pluripotent stem cell derived from the epiblast tissue of the inner cell mass (ICM) of a blastocyst or earlier morula stage embryos.
  • ICM inner cell mass
  • human embryonic stem cells are not encompassed by the term “embryonic stem cell”.
  • a “embryonic stem cell” is defined by the expression of several transcription factors and cell surface proteins including, without limitation, one or more of Oct4, Nanog, Sox2 and/or SSEA1 markers.
  • Embryonic stem cells are obtained from the inner cell mass of blastocysts by methods well-known by the person skilled in the art. Embryonic stem cells require different environments in order to maintain an undifferentiated state.
  • mouse ES cells are grown on a layer of gelatin as an extracellular matrix for support and require the presence of leukemia inhibitory factor (LIF). Without optimal culture conditions or genetic manipulation embryonic stem cells will rapidly differentiate. Culture conditions for ES cells are known in the art (Matise et al., 2000).
  • induced pluripotent stem cell refers to a pluripotent cell artificially derived from a non-pluripotent cell, typically an adult somatic cell, by inducing a forced expression of certain genes.
  • a "induced pluripotent stem cell” is defined by the expression of several transcription factors including one or more of Klf4, Sox2, Oct4 and cMyc.
  • iPS cells are typically derived by transfection of certain stem cell-associated genes into non-pluripotent cells, such as adult fibroblasts (Dimos et al., 2008). Transfection is typically achieved through viral vectors, such as retroviruses, and transfected genes include Oct-3/4 (Pou5fl) and Sox2.
  • Additional genes include certain members of the Klf family (Klfl, Klf2, Klf4 and Klf5), the Myc family (c-myc, L-myc, N-myc), Nanog and LIN28 have been identified to increase the induction efficiency.
  • Klfl, Klf2, Klf4 and Klf5 the Myc family (c-myc, L-myc, N-myc), Nanog and LIN28 have been identified to increase the induction efficiency.
  • Klfl, Klf2, Klf4 and Klf5 the Myc family (c-myc, L-myc, N-myc), Nanog and LIN28 have been identified to increase the induction efficiency.
  • Klfl, Klf2, Klf4 and Klf5 the Myc family (c-myc, L-myc, N-myc), Nanog and LIN28 have been identified to increase the induction efficiency.
  • the mature cell can be a neuronal cell or a non neuronal cell.
  • a non neuronal cell When selected from a non neuronal cell it is preferably selected from an astrocyte, a fibroblast (for example a dermal fibroblast) and a pericyte.
  • a fibroblast for example a dermal fibroblast
  • the mature cell When selected from a neuronal cell the mature cell is typically a neuronal cell, which is not a subcortical neuron, in particular, which is not a subcortical projection neuron.
  • the neuronal cell is typically a cortical neuronal cell, i.e. a cell whose cellular body is located in the cerebral cortex.
  • the neuronal cell can be selected for example from a cortical ipsilateral or contralateral projecting neuron, a corticostriatal cell, a corticothalamic cell, a corticobrainstem cell and a corticospinal cell.
  • the neuronal cell which is not a subcortical projection neuron can be selected for example from a callosal cell, a backward or forward (ipsilateral) projecting neuron, and an anterior commissural projecting neuron.
  • the cellular body of the selected cortical neuronal cell can be located in a cortex area distinct from the motor cortex.
  • the neuronal cell can also be a cell whose cellular body is not located in the cerebral cortex but is located in other regions of the central nervous system, such as in the striatum, thalamus, hippocampus, amygdala, brainstem and spinal cord, or in the peripheral nervous system such as, for example, sensory and motor neurons constituting the cervical spinal nerves, brachial and lumbosacral plexi.
  • the cell to be reprogrammed is a typical mature cell as defined herein above.
  • the selected cell is to be advantageously differentiated or reprogrammed into a subcortical projection neuron, preferably into a subcortical neuron whose axon projects to the striatum, to the thalamus, to the brainstem and/or to the spinal cord once implanted in vivo.
  • the selected cell can also be advantageously differentiated or reprogrammed into a subcerebral projection neuron whose axon typically projects to the thalamus, to the brainstem and/or to the spinal cord once implanted in vivo.
  • the differentiated or reprogrammed cell does not project to the contralateral hemisphere.
  • Fezf2 and Lmo4 are preferably expressed in combination with Ascll or Ngn2, and even more preferably in combination with both Ascll and Ngn2 in order to optimize the non-neuronal cell differentiation or reprogramming into a subcortical projection neuron as defined previously.
  • the differentiated or reprogrammed cell obtained thanks to the present invention is typically morphologically comparable to, and functionally behave like, a projection neuron naturally located in layer V or layer VI of the cerebral cortex.
  • the differentiated or reprogrammed cell typically expresses at least Ctip2, optionally together with at least Crym, Sox5 and/or bHLHB5 (Arlotta et al., 2005; Lodato et al., 2014). This expression can be determined by real-time qPCR and immunofluorescence.
  • the differentiated or reprogrammed cell can also exhibit a characteristic deep layer morphology, e.g. one apical long dendrite towards the pia, basal dendrites into deep layer V and a long axon projecting subcortically (Ideguchi et al., 2010; Gaspard et al., 2008; Eiraku et al., 2008).
  • a characteristic deep layer morphology e.g. one apical long dendrite towards the pia, basal dendrites into deep layer V and a long axon projecting subcortically
  • the differentiated or reprogrammed cell further functionally behaves like a normal projection neuron in terms of connectivity, morphology, gene expression and electrical activity.
  • a differentiated or reprogrammed cell according to the invention can be at least distinguished by the expression of a reporter gene, such as GFP, from a normal projection neuron.
  • the cell to be differentiated or reprogrammed is herein identified as the "manipulated cell" once said cell has been exposed to the combined action of the proteins of interest, i.e. of at least Fezf2 and Lmo4.
  • RNA or mRNA The nucleic acid (RNA or mRNA) or protein of interest expression levels or alternatively, the level of activity of said nucleic acid or protein of interest can be measured in the manipulated cell. Methods to assess such an expression are well known in the art.
  • the expression level of a gene can be measured by determining the protein level encoded by said gene. Practically, any conventional method can be used within the context of the present invention to quantify the levels of proteins of interest such as Western blot or immunohistochemistry.
  • any conventional method can be used within the context of the present invention to quantify the levels of proteins of interest such as Western blot or immunohistochemistry.
  • an increased expression or activity of said protein of interest with respect to the basal level can be measured once said cell has been manipulated.
  • the expression increase of a given protein (or corresponding encoding nucleic acid or gene) with respect to the expression basal level is of at least 20%, at least 25%>, at least 30%, at least 35%, at least 40%, or at least 45%, preferably at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, or more.
  • the in vivo, in vitro or ex vivo delivery step of Fezf2, Lmo4, or any other nucleic acids of interest as herein described can also be efficiently performed by using a number of techniques, such as by electroporation, iontophoresis, or sonophoresis, by using pneumatically delivered biologically active agent-coated particles such as gold particles used in a "gene gun” as well known by the skilled person.
  • a particle bombardment device, or "gene gun” a motive force is generated to accelerate coated high-density particles (such as gold or tungsten) to a high velocity that allows penetration of the neural tissues or cells.
  • Electroporation means are responsible for, or increase, permeability of a cell membrane and/or at least a portion of a targeted tissue to a biologically active agent such as a nucleic acid.
  • a brief electric impulse with a given field strength is used to allow transport or migration of nucleic acids through the tissue or across cell membranes into cells, by an electrophoretic effect.
  • the technique of electroporation is well known to those of ordinary skill in the art.
  • This method works on the principle that cells act as an electrical capacitor generally unable to pass current. Subjecting the cells to an electric field creates transient permeable structures or micropores in the cell membrane. The pores are large enough to allow the pharmaceuticals and/or nucleic acid to gain access to the cells. As a result of the "pores" briefly formed in the cell membrane, the biologically active molecules initially enter the cytoplasm or the nucleus in which they can already exert their function to be studied if necessary. With time, the pores in the cell membrane close and the cell once again becomes impermeable. In addition to the pore effect, the polyanionic, electrically charged nucleotide is also driven into tissue and cells by the electrophoretic effect of the applied electrical pulses.
  • the electrical field is constituted of one or more electrical pulse(s), the field intensity of which is between about 1 and 60 Volts/cm, preferably between 1 and 50 Volts/cm, even more preferably between about 30 and 40 Volts/cm.
  • a particularly preferred electrical field intensity usable in the present invention is an intensity of 35 Volts/cm.
  • the total duration of application of the electric field may be between 1 and 100 milliseconds, preferably between 20 and 80 milliseconds, more preferably between 30 and 60 milliseconds for example 35, 40, 45, 50, 55, 60 or 65 milliseconds. In a preferred embodiment, the total duration of application of the electric field is between 40 milliseconds and 60 milliseconds, for example 45, 50 or 65 milliseconds, and is even more preferably of 50 milliseconds.
  • Electric pulses applied may be between for example 1 and 10. Their frequency may be comprised between 10 and 100 hertz, for example between 40 and 70 hertz. A preferred frequency is 60 hertz. It is preferably a regular frequency.
  • Electric pulses may also be delivered in an irregular manner relative to each other, the function describing the intensity of the electric field as a function of the time for one pulse being preferably variable.
  • the delivered electric field may be for example the combination of at least a first electric field greater than 40 Volts/cm of less than 50 millisecond and one or more electric pulses of less than 35 Volts/cm and about 50 milliseconds.
  • the delivered electric field may further be for example the combination of at least a first electric field greater than 35 Volts/cm of less than 50 millisecond and one or more electric pulses of less than 35 Volts/cm and about 50 millisecond.
  • Electric pulses may be unipolar or bipolar wave pulses. They may be selected for example from square wave pulses, exponentially decreasing wave pulses, oscillating unipolar wave pulses of limited duration, oscillating bipolar wave pulses of limited duration, or other wave forms. Preferentially, electric pulses comprise square wave pulses or oscillating bipolar wave pulses.
  • the administration comprises an electroporation step implying the application, to the tissue(s), of an electric field comprising 4 unipolar square wave pulses, of frequency of 5 Hz, the intensity of each pulse being of 35 Volts/cm for a total duration of application of the electric field of 50 ms per pulse.
  • Electroporation is typically carried out by applying voltage pulses between a pair of electrodes that are applied to the tissue surface.
  • the voltage must be applied in proportion to the distance between the electrodes and the age of the sample.
  • the setting will be between 37V and 40V, at El 3.5 it will be between 33V and 37V, at E12.5 it will be between 30V and 35V, at El 1.5 it will be between 28V and 33V, etc. This depends on the dimensions of the embryos and the thickness of the cerebral cortex at different stages of development.
  • the present description also provides a cell comprising a vector as herein described, for example a differentiated or reprogrammed cell as herein defined or a cell which has been manipulated and is in the process of being differentiated or reprogrammed as herein taught.
  • the invention also relates to the differentiated or reprogrammed cells obtained thanks to the method of the invention, and to the cell population comprising such a differentiated or reprogrammed cell, as well as to their uses.
  • the herein described products are suitable for therapeutic, experimental or various other applications.
  • the differentiated or reprogrammed cell or cell population or the pharmaceutical composition herein described can be used as a medicament.
  • Such a medicament is useful in the context of a neural cell therapy, said therapy being based on the idea that neurological function lost to injury or neurodegeneration can be improved by introducing new cells that can form appropriate connections and replace the function of lost neurons.
  • nucleic acids, vectors, cells comprising such a nucleic acid and/or vectors, as well as the differentiated or reprogrammed cell or population of cells herein described are typically for use for preventing or treating a neurodegenerative disease or disorder, preferably a motor neurodegenerative disease or disorder such as ALS, Parkinson's disease or Alzheimer disease, or cerebral stroke; or for treating a spinal cord injury, in particular a lesion of the pyramidal tract.
  • a neurodegenerative disease or disorder preferably a motor neurodegenerative disease or disorder such as ALS, Parkinson's disease or Alzheimer disease, or cerebral stroke
  • spinal cord injury in particular a lesion of the pyramidal tract.
  • the products of interest herein described can of course also be used for preparing a composition, typically a pharmaceutical composition, for preventing or treating a disease or disorder as herein mentioned.
  • compositions in particular a pharmaceutical composition, comprising a nucleic acid, a vector and/or a differentiated or reprogrammed cell as herein described, or a population of cells comprising such differentiated or reprogrammed cells, preferably together with a pharmaceutically acceptable carrier or support.
  • a pharmaceutical composition comprising a nucleic acid, a vector and/or a differentiated or reprogrammed cell as herein described, or a population of cells comprising such differentiated or reprogrammed cells, preferably together with a pharmaceutically acceptable carrier or support.
  • the composition comprises several components (products of interest) said components can be separately formulated and combined prior to use.
  • pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia, or European Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • carrier or “support” refer to a diluent, adjuvant, excipient, or vehicle with which the nucleic acid or vector as herein described is administered, or a biodegradable or non-degradable porous or non- porous matrix or scaffold that can be used for transplantation of the differentiated or reprogrammed cell or cell population as herein described into a subject in need thereof.
  • biodegradable or non-degradable porous or non- porous matrix that can be used for transplantation of the differentiated or reprogrammed cell or cell population include for example synthetic and natural materials in the form of foams, sponges, gels, hydrogels, textiles, patches and nonwoven structures.
  • the incorporation of the cells of the present invention into a matrix or scaffold can be achieved by the simple depositing of cells onto the scaffold. Cells can enter into the scaffold by simple diffusion (J. Pediatr. Surg. 23 (1 Pt 2): 3-9 (1988)).
  • simple diffusion J. Pediatr. Surg. 23 (1 Pt 2): 3-9 (1988)
  • compositions can also contain minor amounts of pH buffering agents.
  • suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences” by E.W. Martin.
  • Such compositions will contain a prophylactically or therapeutically effective amount of the therapeutic agent of the invention preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the subject.
  • the formulation should suit the mode of administration.
  • the pharmaceutical compositions are sterile and in suitable form for administration to a subject, in particular to a human being.
  • the subject is preferably an animal subject, more preferably a mammalian subject, and most preferably a human being whatever its gender, age or race, typically a subject suffering of a neurodegenerative disease or disorder, or of a lesion at the level of the central nervous system, or at risk of suffering of such a neurodegenerative disease, disorder or lesion.
  • the respective nucleic acid sequences of Fezf2, Lmo4, Neurogenin 2 (Ngn2) and Ascll preferably comprise or consist in SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO: 5 and SEQ ID NO: 7, or any other sequence (functionally equivalent) having at least 50%, preferably at least 60%, 70%, or 80%, even more preferably at least 90%, 95%, 97%, 98%, 99% homology with each of said SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO: 5 and SEQ ID NO: 7, and their respective amino acid sequences preferably comprise or consist in SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO: 6 and SEQ ID NO: 8, or any other sequence (functionally equivalent) having at least 50%>, preferably at least 60%, 70%, or 80%, even more preferably at least 90%, 95%, 97%, 98%, 99% homology with each of said SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO: 6 and
  • the respective best sequences to be selected comprise or consist in the orthologous sequences corresponding to said animal species or any other sequence (functionally equivalent) having at least 50%>, preferably at least 60%>, 70%>, or 80%, even more preferably at least 90%, 95%, 97%, 98%, 99% homology with each of said sequences.
  • the pharmaceutical composition of the invention may be in a variety of forms. These include, for example, solid, semi-solid, and liquid dosage forms, such as lyophilized preparations, liquids solutions or suspensions, injectable and infusible solutions, etc.
  • the preferred form depends on the product to be administered, on the intended mode of administration and on the therapeutic application.
  • Nucleic acids can for example be formulated in liquid suspension, with isotonic solutions or excipients.
  • Viral vectors can be purified and injected as a suspension in any suitable composition or buffer, comprising pharmaceutically acceptable carrier or support as herein above defined such as PBS, salts, isotonic solution, stabilizing agents, etc.
  • suitable composition or buffer comprising pharmaceutically acceptable carrier or support as herein above defined such as PBS, salts, isotonic solution, stabilizing agents, etc.
  • Other delivery systems can include time-release, delayed release or sustained release delivery systems. Such systems can avoid repeated administrations of the product of interest, increasing convenience to the subject and the physician. Many types of release delivery systems are available and known to those of ordinary skill in the art.
  • Use of a long-term sustained release implant may be desirable. Long-term release, are used herein, means that the implant is constructed and arranged to delivery therapeutic levels of the active ingredient for at least 30 days, and preferably 60 days. Long-term sustained release implants such as biocompatible degradable polymeric support or capsule are well-known to those of ordinary skill in the art.
  • the product(s) may be injected at various doses, selected and/or adjusted by the skilled person.
  • the doses of product to be administered may be adjusted by the skilled person depending on the nature of the product to be administered (nucleic acid, vector, cell or cell population), on the nature of the protein of interest to be expressed (Fezf2, Lmo4, Ngn2 and/or Ascll for example), on the route of administration, on the target site, on the subject to be treated, as well as upon any other factors well known in the medical art.
  • the specific therapeutically effective dose level for any particular patient will depend upon a variety of factors/conditions including the age, body weight, general health, sex and diet of the patient. Selection comprises determination of dose effective to produce differentiation or reprogramming without essentially causing toxicity. For example, it is well known within the skill of the art to start doses of the product at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. It will be understood that the total daily usage of the product of the present invention will be decided by the attending physician within the scope of sound medical judgment. Doses indicated herein below are examples which can be easily adapted by the skilled person/physician.
  • the product of interest typically Fezf2, Lmo4, Ngn2 or Ascll nucleic acids may be injected via transfection at doses varying for example between about 1 and 10 ⁇ g each 6 cm plates for instance, preferably between about 2 and 8 ⁇ g, more preferably between about 3 and 7.5 ⁇ g, even more preferably between about 3.5 and 7 ⁇ g, for example about 4 ⁇ g, 4.5 ⁇ g, 5 ⁇ g, 5.5 ⁇ g, or 6 ⁇ g.
  • the product of interest typically Fezf2, Lmo4, Ngn2 or Ascll nucleic acids may be injected via electroporation at doses varying for example between about 0.1-10 ⁇ g of nucleic acid, preferably between about 0.2 - 5 ⁇ g, more preferably between about 0.3 - 2.5 ⁇ g, even more preferably between about 0.5 and 2 ⁇ g, for example about 1 ⁇ g or 1.5 ⁇ g.
  • a viral vector typically between about 10 4 -10 12 pfu/ml of viral vector, preferably between about 10 5 -10 12 pfu/ml, and even more preferably about nx lO 11 pfu/ml (n being an integer between 1 and 9) are administered per step of the differentiating/reprogramming process.
  • the administration of the cells or cell population of the invention, or the pharmaceutical composition comprising same, to the subject in need thereof can be carried out by conventional means.
  • said cells or cell population is administered to the subject by a method which involves transferring the cells to the desired tissue, in vitro, ex vivo or in vivo, to the animal tissue directly.
  • the herein described method for differentiating a cell or for reprogramming a cell is typically applicable in vivo in a mammalian subject, particularly in a human being, more particularly in a subject suffering of a degenerative disease or disorder such as Amyotrophic Lateral Sclerosis (ALS), Parkinson's disease or Alzheimer's disease, even after the onset of the first clinical signs of disease allowing diagnosis, or in a subject suffering of a spinal cord injury, in particular a lesion of the pyramidal tract, or of a cerebral stroke.
  • a degenerative disease or disorder such as Amyotrophic Lateral Sclerosis (ALS), Parkinson's disease or Alzheimer's disease
  • the method of the invention for differentiating a cell or for reprogramming a cell is also applicable in vitro or ex vivo on cells from a subject suffering of such a degenerative disease or disorder or suffering of a spinal cord injury or of a cerebral stroke, these cells being for use for reimplantation in said subject once adequately differentiated or reprogrammed.
  • the cells can be transferred to or implanted in the desired tissue by any appropriate method which generally will vary according to the type of tissue, optionally using any appropriate support.
  • the cells can be maintained in vitro on this support prior to implantation into the subject.
  • the support containing the cells can be directly implanted in the subject without additional in vitro culturing.
  • the support can optionally be incorporated with at least one pharmaceutical agent that facilitates the survival, differentiation and function of the transplanted cells.
  • Cells can also be directly seeded onto the desired site within the tissue to establish a population, etc. Cells can be transferred to sites in devices such as catheters, trocars, cannula, etc. (Kondziolka et al., 2004; Lee et al., 2015; Moro et al., 2012; Potts et al., 2013; Stone et al., 2013).
  • Cells' (re)implantation is typically an intracerebral or intraspinal implantation and is preferably performed via cellular injection or thanks to a cellular support (Stone et al., 2013).
  • Culture conditions of the differentiated or reprogrammed cells or of the cell population can be optimized by the person skilled in the art to achieve effective differentiation or reprogramming by, e.g., monitoring the percentage of cells that have differentiated or reprogrammed into desired subcortical projection neurons.
  • One or more pharmaceutical agents known in the art for example growth factors such as insulin- like growth hormone(s) and/or a human growth hormone(s) (Aberg et al., 2006; Luciani et al., 2012; Wang, 2009), antioxidants such as melatonin (Kostoglou-Athanassiou, 2013) or anti- inflammatory agents such as 5-aminosalicylic acid, glucocorticoid(s) or immunomodulatory(s) (Salas et al., 2009), or stem cell conditioned media (Stone et al., 2013) may be added in an amount sufficient to facilitate the differentiation or reprogramming as well as the survival and function of the differentiated or reprogrammed cells over a time period of about one to four weeks.
  • Such pharmaceutical agents can be administered before, simultaneously with, or after the administration of the cells.
  • the differentiated or reprogrammed cells of the present invention are fully differentiated or reprogrammed, prior to transplantation into a subject.
  • the cells of the present invention can be transplanted into a subject in a partially differentiated state. Further differentiation may take place in the subject, optionally with the help of one or more pharmaceutical agents as mentioned herein above.
  • the source of tissue from which the cells to be differentiated or reprogrammed of the present invention are isolated can be autologous in relation to the subject undergoing the therapeutic treatment.
  • the source may be allogeneic, or xenogeneic.
  • Cells to be administered to a subject can also be genetically modified to enhance proliferation and/or differentiation or prevent or lessen the risk of immune rejection.
  • the differentiated or reprogrammed cells obtained in accordance with the present invention can be used to modulate the recipient's immune response, prior to transplantation of differentiated cells prepared in accordance with the present invention. See, for example, U.S. Patent 6,328,960, U.S. Patent 6,281,012.
  • the amount of cells used for implantation depends on a number of factors including the patient's condition and response to the therapy, and can be determined by one skilled in the art.
  • the pharmaceutical composition of the invention can be used in a combination therapy.
  • the combination therapy is administered to a subject in need of treatment, such as a patient in need of repair or regeneration of a tissue, typically of a neuronal tissue.
  • the combination therapy is used in conjunction with other types of treatments to repair or regenerate neuronal tissue such as, for example, with a biomaterial scaffold, or together with the injection of neurotrophin(s), Chondroitinase ABC and/or MAI molecule(s) inhibitor(s) and/or antibody(ies) (McCreedy and Sakiyama-Elbert, 2012).
  • the combination therapies of the invention can be used prior to, concurrently or subsequent to the administration of the cells of the invention.
  • the invention relates to a method for the treatment of a subject suffering of a neurodegenerative disease or disorder, spinal cord injury or stroke as herein described, or for the prevention thereof in a subject at risk of developing such a disease or disorder, spinal cord injury or stroke, that comprises the administration to said subject of a product as herein described, for example of a nucleic acid, vector, differentiated or reprogrammed cell, cell population or pharmaceutical composition of the invention, optionally together with at least one pharmaceutical agent that facilitates the survival and function of the differentiated or reprogrammed cells.
  • a product for example of a nucleic acid, vector, differentiated or reprogrammed cell, cell population or pharmaceutical composition of the invention, optionally together with at least one pharmaceutical agent that facilitates the survival and function of the differentiated or reprogrammed cells.
  • compositions may be administered to a subject at risk of developing a particular disease, or to a subject reporting one or more of the physiological symptoms of a disease, even though a diagnosis of this disease may not have been made.
  • the present invention may also be used for experimental research, clinical research or other purposes (animal studies, etc.).
  • the differentiated or reprogrammed cell or cell population according to the invention can for example be used for evaluating the properties, typically the prophylactic and/or therapeutic properties, of a biological, pharmacological or chemical compound, or for screening for compounds useful in the treatment of a neurodegenerative disease or disorder, of a spinal cord injury or of a cerebral stroke.
  • the differentiated or reprogrammed cell or cell population of the invention can also advantageously be used as a cellular model for studying a neurodegenerative disease or disorder or a spinal cord injury, or for screening for compounds useful in the treatment of such a disease, disorder or injury.
  • a cellular model comprising differentiated or reprogrammed cells of the invention or populations of cells is also encompassed by the present invention.
  • Such a model can be used for evaluating the therapeutic properties of a compound or for screening for compounds useful in the prevention or treatment of a neurodegenerative disease or disorder, of a spinal cord injury, or of a cerebral stroke.
  • a method for screening for drugs for use for treating a neurodegenerative disease or disorder, a spinal cord injury or a cerebral stroke comprises the steps of testing compound(s) for its/their ability to further improve cell differentiation or reprogrammation when combined to Fezf2 and/or Lmo4, preferably when combined to both Fezf2 and Lmo4, to prolong the differentiated or reprogrammed cell survival, or to protect reprogrammed cells from stress-induced cell damages, such as hypoxia.
  • the screening methods involve assaying for compounds which enhance the expression or activity of Fezf2 and/or Lmo4 or which similarly allows the cell differentiation or reprogramming (as herein defined). Such methods are adaptable to automated, high throughput screening of compounds. Examples of such methods are described in US patent 5, 429, 921.
  • the candidate pharmacological agents can be derived from, for example, combinatorial peptide libraries, combinatorial chemical compound libraries, and natural products libraries. Convenient reagents for such assays are known in the art.
  • a plurality of assay mixtures are run in parallel with different agent concentrations to obtain a different response to the various concentrations.
  • one of these concentrations serves as a negative control, i.e., at zero concentration of agent or at a concentration of agent below the limits of assay detection.
  • the present invention also provides a kit for differentiating or reprogramming a cell exhibiting a particular phenotype into a reprogrammed cell exhibiting a different phenotype, preferably into a subcortical projection neuron whose axon projects to the striatum, to the thalamus, to the brainstem and/or to the spinal cord once implanted in vivo, wherein the kit comprises a nucleic acid sequence or combination of nucleic acid sequences, a vector and/or a composition as herein described.
  • This kit preferably comprises at least a nucleic acid sequence coding for Fezf2 and Lmo4, or a combination of a first nucleic acid sequence coding for Fezf2 and of a second nucleic acid sequence coding for Lmo4, optionally together with a nucleic acid sequence coding for Neurogenin 2 (Ngn2) and/or with a nucleic acid sequence coding for Ascll, and optionally together with a preservation medium and/or any appropriate pharmaceutical carrier or support as herein described.
  • Ngn2 Neurogenin 2
  • Ascll Ascll
  • This kit can comprise as many distinct (optionally sterile) containers as there are nucleic acid sequences of interest in the kit.
  • a therapeutic kit comprising differentiated or reprogrammed cells or a cell population according to the invention together with a preservation medium, the cells or cell population being optionally associated to a pharmaceutical support as herein described.
  • kit can further comprise means for administering said differentiated or reprogrammed cells or cell population to a subject, in particular a human being, at least one pharmaceutical agent that facilitates the survival, differentiation and function of the transplanted cells, and optionally an instruction manual including information for the administration of the effective amount the composition for treating and/or preventing a neurodegenerative disease or disorder or a cerebral stroke, or for treating a spinal cord injury.
  • FIGURES Figure 1 Whole-mount in situ hybridization for Lmo4 on P0 brain ⁇ up left panel) and immunofluorescence for Lmo4 on SI coronal sections ⁇ up right panel). ⁇ Bottom panels) High magnification views of frontal motor (F/M; left panel) and prospective somatosensory (pS; right panel) cortices coronal sections immuno-labelled for Lmo4. Scale bars: 1mm (upper panels) and ⁇ (bottom panels).
  • FIG. 2 (A) Schematic representation of the vector used to overexpress the Lmo4 cDNA.
  • B Coronal section of the P0 somatosensory cortex from the control (not electroporated) hemisphere.
  • C ⁇ Top right panels) Coronal sections of Cdk5r-Lmo4-IRES-GFP electroporated El 3.5 brains immunolabeled at P0 for Lmo4 and GFP. ⁇ Bottom right panels) High magnification views of boxes depicted in upper right panels. Note that the majority of GFP+ cells co-express Lmo4, indicating that the Lmo4-expressing vector is properly working.
  • C-D Quantification of Ctip2+/GFP+ cells on the total number of GFP+ in lower (C) and upper (D) layers of electroporated brains. Data are represented as means ⁇ SEM. (*) p ⁇ 0.05, (**) p ⁇ 0.01, (***) p ⁇ 0.001. Note that the co-electroporation of Fezf2 and Lmo4 increases the percentage of Ctip2+ neurons in lower layers from 7.57 ⁇ 4.6% (on the total of GFP electroporated, control, cells) to more than 81.05+3.29%, while only 31.26+3.79% or 49.52 ⁇ 5.78% of single Lmo4- or Fezf2-electroporated cells, respectively, show an induction of Ctip2 expression (C).
  • FIG. 4 (A) On the left, western blot performed on nuclear extracts from controls and COUP-TFI CKO PI cortices (in which there is an excess of Lmo4 (REF Alfano 2014 paper), immunoprecipitated with antibodies specific for Lmo4 (IP Lmo4), Satb2 (IP Satb2), or an unrelated epitope (IP control). IP fractions were analyzed using an antibody against Hdacl, whereas the corresponding input fractions were analyzed with an antibody specific for ⁇ -actin. On the right, ratio between Hdacl -LM04 and Hdacl -Satb2 complexes immunoprecipitated from control and COUP-TFI CKO extracts.
  • FIG. 5 (A) Schematic representation of the in utero electroporation timeline. To study the connectivity of Lmo4/Fezf2 overexpressing cells mice were electroporated at E14.5 and collected at P14. (B) Schematic representation of the connectivity of callosal and subcerebral projection neuron. (C) Details of immunofluorescences for Ctip2 and GFP performed on coronal (1,2) and sagittal (3) sections.
  • Figure 6 (A) Details of immunofluorescences for Ctip2, Satb2, Cuxl, Er81 performed on P14 cortices electroporated at E14.5 with either Cdk5r-Gfp or Cdk5r-Lmo4/Cdk5r-Fezf2 expressing plasmids. (B) Quantification of cells expressing the different markers on the total of Cdk5r-Gfp or Cdk5r-Lmo4/Cdk5r-Fezf2 electroporated cells.
  • electroporated GFP+ neurons express high levels of Ctip2 (tot 0% Cdk5r-GFP versus 80% Cdk5r- Lmo4/Cdk5r-Fezf2- expressing plasmids), lower levels of Satb2 (tot 100% Cdk5r-GFP versus 70% Cdk5r-Lmo4/Cdk5r-Fezf2- expressing plasmids), no Cuxl (tot 100%) Cdk5r-GFP versus 0% Cdk5r- Lmo4/Cdk5r-Fezf2- expressing plasmids) and Er81 (tot 0% Cdk5r-GFP versus 38% Cdk5r- Lmo4/Cdk5r-Fezf2- expressing plasmids), indicating that they have lost a upper layer identity and acquired a subcerebral one.
  • Figure 7 Astrocyte cultures transduced with lentiviral vectors expressing Fezf2, Lmo4 and Ngn2 (FLN).
  • A 15 days after transduction, reprogrammed cells express Tuj l and 80% of them express also Ctip2.
  • B After 30 days, branching is more complex and double Tuj l/Ctip2+ neurons appear more mature.
  • C The addition of small molecules increases the % of Ctip2+ reprogrammed neurons from 40 to 80%.
  • Figure 8 (A) Astrocyte cultures transduced with lentiviral vectors expressing Fezf2, Lmo4 and Ngn2 and immunolabeled for Tujl/Lmo4 and Tuj l/Sox5 after 30 days in culture. (B) Quantitative RT-PCR also shows high induction of Bhlhb5 transcript in infected/reprogrammed neurons (2 independent experiments) compared to non-infected neurons. The expression of these markers confirms that reprogrammed Tujl+ neurons are committed to a corticofugal projection neuron fate.
  • FIG. 9 Astrocytes transduced with lentiviral vectors expressing Fezf2 and Ngn2, Fezf2 and Lmo4 or an aspecific vector as control and immunolabeled for Tuj l and Ctip2 after 15 days in culture. Note that even if astrocytes do express Tuj l becoming neurons, they do not express Ctip2 and thus do not acquire a subcereberal fate, as is the case when Ngn2, Fezf2 and Lmo4 are transduced together (Fig. 7) ⁇ EXPERIMENTAL SECTION
  • EXAMPLE 1 - Lmo4 and Fezf2 work in synergy for reprogramming neurons. Materials and methods Animals
  • mice at P0, P7 and P21 were intracardially perfused with paraformaldehyde (PFA) 4%.
  • Embryonic and postnatal brain samples were fixed either for 2h (for immunohistochemistry) or over-night (for in situ hybridization) at 4°C in PFA 4%.
  • Samples for whole mount in situ hybridizations were, then, equilibrated to 30% sucrose, and cut on a Leica cryostat, or gradually dehydrated to 96% ethanol.
  • Samples to be used for floating immunofluorescences and cholera toxin- injected brains were embedded in 4% agarose after fixation and then cut on a Leica vibratome at 200 ⁇ .
  • Floating immunofluorescence was performed on vibratome sections, which were blocked with 10%) goat serum, 3%> bovin serum albumin (BSA), and 0.3%> triton X-100 over night at 4°C.
  • the following secondary antibodies were used: goat anti-rabbit FC (488, 594, 633), goat anti-rat FC (488, 594, 633), goat anti-mouse FC (488, 594, 633; dil.
  • CI 1 :300, Life Technologies), goat anti-rabbit FC 350 (1 :150, Life Technologies).
  • Slides were mounted with the following mounting solution: 80% glycerol, 2% N-propyl gallate, ⁇ g/ml Hoechst (Invitrogen).
  • ISH In Situ Hybridization
  • the Lmo4 ORF was amplified using available cDNA by the use of the following primers: Mlul- Lmo4.fw (5'GGACGCGTTGAGAGCAGCTC3') and MluI-Lmo4.rev
  • Cdk5r-IRES-EGFP In utero electroporations of Cdk5r-IRES-EGFP , Cdk5r-Lmo4-IRES-EGFP and/or Cdk5r-Fezf2-IRES- EGFP were performed as previously described (Alfano et al., 2011). Briefly, after performance of a 3cm laparotomy on deeply anesthetized female mice and once extroflected uteri, the DNA mix (lmg/ml) was injected into the lateral ventricle of E13.5 (or E14.5) embryos using a Femtojet microinjector (Eppendorf).
  • the electroporations were performed using a Tweezertrode electrode (diameter 7 mm; BTX) connected to a NEPA21 electroporator (NEPAGENE, Japan) with the following parameters: four 37V pulses (for E13.5; 40V for E14.5), P(on) 50 mseconds, P(off) 1 second, 5% decay rate. Then uteri were reallocated in the abdominal cavity and both peritoneum and abdominal skin were sewn with surgical sutures (B. Braun Surgical).
  • the bound fraction was separated by pulse centrifugation and pelleted beads and input were re-suspended in lx Nupage loading buffer (Invitrogen). Samples were loaded on a 10% SDS polyacrylamide gel and subjected to standard SDS-PAGE electrophoresis on Mini-Protean tetra cell (Biorad). Then, immunocomplexes were transferred to Hybond-P membrane (Amersham) by the use of a Trans-Blot SD Semi-Dry Transfer Cell (Biorad). Immunoblotting was performed with the following antibodies: rabbit anti-Hdacl (1 :500, Millipore) and rabbit anti- -actin (1 :500, Abeam).
  • Chromatin-immunoprecipitation (ChIP) assay on genomic DNA from controls and COUP-TFI ⁇ /jfmxCrei cort j ces was erformed as described in Kuo and Allis (1999).
  • Neocortices were dissected from 7 controls and COUP-TFI fl/fl EmxCrel pi pupSj and diced in ice cold Hanks Buffered Saline Solution. Proteins were crosslinked to DNA by adding 1% formaldehyde to the solution. The tissue was than disrupted by homogenization in lysis buffer (20mM HEPES pH7.4, ImM EDTA, 150mM NaCl, 1%SDS, 125mM Glycine, PMSF 0.2 mg/ml).
  • Nuclei were collected by centrifugation, resuspended in sonication buffer (20mM HEPES pH7.4, ImM EDTA, 150mM NaCl, 0.4% SDS, PMSF 0.2mg/ml) and disrupted by 6 pulses of 10 ⁇ amplitude in a Soniprepl50 Sonicator (Sanyo). Before immunoprecipitation, samples were pre-cleared lh in 50%> ProteinA-Sepharose slurry and then incubated ON at 4°C with 3 ⁇ g of the following antibodies: rabbit anti-Hdacl (Millipore), rabbit anti- H4K12 (Abeam), and a control antibody (rabbit anti-GFP, Molecular probe).
  • Lmo4 and Fezf2 can work in synergy to promote corticospinal projection neuron specification and to efficiently reprogram upper layer neurons from a callosal to a subcortical cell fate as described below.
  • Lmo4 overexpression induces Ctip2 de-repression in lower layers of the cerebral cortex.
  • the transcriptional adapter Lmo4 is mainly expressed at perinatal stages in postmitotic neurons of the cerebral cortex and is scarcely expressed at P0 in cortical somatosensory areas of mice (Fig. l).
  • Lmo4 sequence in a plasmidic vector under the control of the Cdk5r promoter (which is exclusively active in postmitotic cells of the cortex) it was possible to induce Lmo4 expression only in postmitotic neurons of the somatosensory cortex (Fig.2).
  • Lmo4 overexpression in the lower layers of the mouse somatosensory cortex increased the expression of Ctip2, an important transcription factor involved in cortico-spinal motor neuron differentiation (Arlotta et al, 2005; Chen et al, 2008; Srivatsa et al, 2014), in approximately 30% of electroporated cells (Fig. 3A-C).
  • the inventors found that after co-immunoprecipitating Hdacl -Lmo4 and Hdacl -Satb2 complexes with specific antibodies against Lmo4 and Satb2, the Lmo4-Hdacl interaction increased while Satb2-Hdacl complexes strongly decreased in protein extracts of COUP-TFI conditional knock-out mice compared to control ones (Fig.4A). Moreover, by Chromatin Immuno-precipitation (ChIP) the inventors found a decrease in the amount of Hdacl bound to Ctip2 locus in COUP-TFI mutant mice.
  • ChIP Chromatin Immuno-precipitation
  • Lmo4 is able to de-repress Ctip2 expression by preventing the interaction of Satb2, a transcription factor promoting callosal neuron specification (Alcamo et ah, 2008; Britanova et ah, 2008; Srivatsa et al, 2014), with the histone deacetylase 1 (Hdacl) on the locus of Ctip2 (Fig. 4C).
  • Satb2 a transcription factor promoting callosal neuron specification
  • Hdacl histone deacetylase 1
  • Lmo4 overexpression induces Ctip2 expression in lower layers, but not in upper layers of the cortex (Fig.3B-D), where Ctip2 is normally not present. This implies that Lmo4 works together with a transcriptional activator to efficiently induce Ctip2 in lower layers of the cortex.
  • inventors co-electroporated the Lmo4-expressing vector together with a Fezf2-expressing one (Rouaux and Arlotta, 2013) at E13.5 and calculated the percentage of Ctip+ neurons among electroporated (GFP+) cells. The presence of both vectors induced the expression of Ctip2 in over 80% of electroporated cells in lower layers and in more than 65% in the upper layers (Fig. 3B-D).
  • lentiviral vectors for the cortical transcription factors were cloned into lentiviral vectors under the control of the tetracycline operator.
  • Replication-incompetent, Vesicular stomatitis Indiana virus Protein G (VSVG)-coated lentiviral particles were packaged in 293T cells.
  • Mouse astrocytes were infected in DMEM 10%> FBS media. 16-20 h after infection cells were switched into fresh mouse embryonic fibroblast (MEF) media containing doxycycline (2 mg.ml _1 ; Sigma).
  • MEF mouse embryonic fibroblast
  • neuronal inducing media DMEM/F12 (Invitrogen), 25 ⁇ g.m ⁇ 1 insulin (Sigma), 50 ⁇ g.m ⁇ 1 transferrin (Sigma), 30 nM sodium selenite, 20 nM progesterone (Sigma), 100 nM putrescine (Sigma) and penicillin/streptomycin (Sigma)
  • the medium was changed every 2-3 days for a further 45-55 days.
  • RNA isolation and real-time RT-PCR were as follows: mouse anti- ill-tubulin (1 :500, Covance), rabbit anti-Ctip2 (1 :500, Abeam), rabbit anti-Sox5 (1 :500, Gentaur) and rat anti-Lmo4 (1 :400, kind gift from J. Visvader).
  • qRT-PCR Quantitative RT-PCR
  • the thermal profile consisted of 2 minutes at 50°C and 10 minutes at 95°C, followed by 40 cycles of 15 seconds each at 95°C and 1 minute at 60°C.
  • mRNA levels were calculated according to the threshold cycle numbers within a linear range of amplification of 20 to 32 cycles. Data were standardized versus the housekeeping gene 18S, which was used as an internal standard and amplified for every sample in parallel assays.
  • the reprogramming efficiency of the Fezf2/Lmo4 cocktail was subsequently tested in vitro in non- neuronal cells, such as postnatal astrocytes, in collaboration with the group of Dr. V. Broccoli lab (San Raffaele Institute, Milan, Italy), an expert in the field of direct lineage reprogramming (Broccoli et al, 2014; Caiazzo et al, 2011 ; Caiazzo et al, 2015; Colasante et al, 2015).
  • the Broccoli team has transduced newborn mouse astrocytes with inducible lentivirus expressing Lmo4 and Fezf2 together with Ngn2 (which induces a generic glutamatergic neuron identity (Heinrich et al, 2010; Heinrich et al, 2011).
  • the cultures were left for 15 and 30 days after induction and tested for their morphology as well as their capacity to express Tuj 1 (a neuronal marker) and Ctip2 (a cortical subcerebral projection neuron marker).
  • Tuj 1 a neuronal marker
  • Ctip2 a cortical subcerebral projection neuron marker
  • Astrocytes have normally a stellate-like shape and never express Tuj l or Ctip2 (Heinrich et al, 2011).
  • reprogrammed astrocytes acquire a pyramidal like structure and express the neuronal marker Tuj l ; 40% of these Tuj l+ neurons also express Ctip2 (Fig.7A).
  • reprogrammed Tuj l/Ctip2+ neurons acquire an even more complex and branched structure (Fig.7B).
  • small molecules Zhang et al, 2015 increases the reprogramming induction of astrocytes into neurons expressing Tuj l and Ctip2 from 40% to 80%> (Fig. 7C).
  • Reprogrammed Tuj l+ neurons also express Lmo4, known to induce a corticopontine phenotype (Cederquist et al., 2013), the general corticofugal marker Sox5 (Lai et al, 2008) and the transcription factor Bhlhb5, normally expressed by layers II to V cortical neurons and known to be involved in proper development of corticospinal motor neurons (Joshi et al, 2008) (Fig. 8A-C).
  • COUP-TFI promotes radial migration and proper morphology of callosal projection neurons by repressing Rnd2 expression. Development 138, 4685-4697.
  • COUP-TFI is required for the formation of commissural projections in the forebrain by regulating axonal growth. Development 133, 4151-4162.
  • Satb2 is a postmitotic determinant for upper- layer neuron specification in the neocortex. Neuron 57, 378-392.
  • Glial cells generate neurons: the role of the transcription factor Pax6. Nat Neurosci 5, 308-315.
  • Bhlhb5 regulates the postmitotic acquisition of area identities in layers II-V of the developing neocortex. Neuron 60, 258-272.
  • SOX5 controls the sequential generation of distinct corticofugal neuron subtypes. Neuron 57, 232-247.

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Abstract

La présente invention concerne le domaine de la médecine humaine et vétérinaire et plus particulièrement la thérapie génique et cellulaire. Les inventeurs identifient pour la première fois des molécules actives, Fezf2 et Lmo4, dont l'expression combinée oriente la différenciation d'une cellule et permet la reprogrammation d'une cellule présentant un phénotype particulier en une cellule reprogrammée présentant un phénotype différent. La présente invention concerne en particulier des produits, des utilisations et des procédés d'obtention d'un neurone de projection sous-corticale dont l'axone se projette vers le striatum, le thalamus, le tronc cérébral et/ou la moelle épinière après son implantation in vivo, à partir d'une cellule mature ou immature. L'invention concerne également les cellules reprogrammées ou différenciées ainsi obtenues, la population de cellules comprenant lesdites cellules différenciées et/ou reprogrammées, ainsi que les compositions et les kits comprenant lesdites cellules ou une population de cellules, et des utilisations correspondantes.
PCT/EP2016/077029 2015-11-09 2016-11-09 Différentiation ou reprogrammation cellulaire à l'aide de fezf2 et de lmo4 Ceased WO2017081033A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022052964A1 (fr) * 2020-09-08 2022-03-17 纽伦捷生物医药科技（苏州）有限公司 Fragment fonctionnel pour la reprogrammation, composition et application associée
WO2025146440A1 (fr) 2024-01-02 2025-07-10 Leibniz-Institut Für Immuntherapie (Lit) Utilisation de lmo4 pour renforcer la pluripotence et l'efficacité antitumorale de lymphocytes t

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EP2733205A1 (fr) * 2012-11-20 2014-05-21 Centro De Investigacíon Biomédica En Red De Enfermedades Neurodegenerativas Ciberned Neurones moteurs supérieurs cortico-spinaux, procédés et compositions permettant de différencier des cellules souches neurales par modulation de la signalisation des récepteurs cannabinoïdes CB1 et leurs utilisations

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EP2733205A1 (fr) * 2012-11-20 2014-05-21 Centro De Investigacíon Biomédica En Red De Enfermedades Neurodegenerativas Ciberned Neurones moteurs supérieurs cortico-spinaux, procédés et compositions permettant de différencier des cellules souches neurales par modulation de la signalisation des récepteurs cannabinoïdes CB1 et leurs utilisations

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022052964A1 (fr) * 2020-09-08 2022-03-17 纽伦捷生物医药科技（苏州）有限公司 Fragment fonctionnel pour la reprogrammation, composition et application associée
WO2025146440A1 (fr) 2024-01-02 2025-07-10 Leibniz-Institut Für Immuntherapie (Lit) Utilisation de lmo4 pour renforcer la pluripotence et l'efficacité antitumorale de lymphocytes t

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