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WO2024209036A1 - Génération de populations neuronales glutamatergiques de haute pureté par l'utilisation du facteur pro-neural ascl1 - Google Patents

Génération de populations neuronales glutamatergiques de haute pureté par l'utilisation du facteur pro-neural ascl1 Download PDF

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WO2024209036A1
WO2024209036A1 PCT/EP2024/059305 EP2024059305W WO2024209036A1 WO 2024209036 A1 WO2024209036 A1 WO 2024209036A1 EP 2024059305 W EP2024059305 W EP 2024059305W WO 2024209036 A1 WO2024209036 A1 WO 2024209036A1
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ascl1
neurons
polynucleotide
glutamatergic
cells
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Marcos Costa
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Institut Pasteur de Lille
Institut National de la Sante et de la Recherche Medicale INSERM
Centre Hospitalier Universitaire de Lille
Institut Pasteur
Universite de Lille
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Institut Pasteur de Lille
Institut National de la Sante et de la Recherche Medicale INSERM
Centre Hospitalier Universitaire de Lille
Institut Pasteur
Universite de Lille
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    • 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
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem 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
    • C12N2510/00—Genetically modified cells

Definitions

  • the present invention is in the field of medicine, in particular neurology.
  • hiPSCs human induced pluripotent stem cells
  • neural progenitors or fibroblasts using transcription factors
  • transcription factors have been indeed widely used to generate different populations of neurons, which have been used in neurodevelopment studies, disease modelling, drug screening, and neuronal replacement therapies.
  • transcription factor that would allow the differentiation towards glutamatergic neurons.
  • Glutamatergic neurons produce, accumulate and release in synapses the neurotransmitter glutamate, which is the main excitatory neurotransmitter in the mammalian central nervous system. It is involved in most of the brain's fundamental processes such as cognition, learning, memory, and sensory perception.
  • Asci 1 Mashl
  • Neurogenin2 Neurogenin2
  • Asci 1 Mashl
  • Neurogenin2 Neurogenin2
  • Asci 1 Mashl
  • Neurogenin2 Neurogenin2
  • Ascii and Neurog2 are respectively required to specify GAB Aergic and glutamatergic neurons in the forebrain and sympathetic and sensory neurons of the peripheral nervous system (Parras, Carlos M., et al.
  • the present invention is defined by the claims.
  • the present invention relates to methods for generating highly pure glutamatergic neuronal populations using the pro-neural factor ASCL1.
  • ASCL1 overexpression of ASCL1 induces the generation of a highly pure population of glutamatergic neurons. Said observation goes totally in the opposite direction of what has been previously taught, since ASLC1 was mainly described as inducing GABAergic neurons in the forebrain (Parras, Carlos M., et al. "Divergent functions of the proneural genes Mashl and Ngn2 in the specification of neuronal subtype identity. " Genes & development 16.3 (2002): 324-338).
  • the first object of the present invention relates to a method of generating a highly pure population of glutamatergic neurons comprising the steps consisting of i) expressing a polynucleotide encoding for the transcription factor ASCL1 in a population of human induced neural progenitor cells (hiNPCs) and ii) differentiation said population of cells into a highly pure population of glutamatergic neurons.
  • hiNPCs human induced neural progenitor cells
  • glutamatergic neuron has its general meaning in the art and refers to neurons that produce, accumulate in vesicles and release in synapses the neurotransmitter glutamate, which is the main excitatory neurotransmitter in the mammalian central nervous system.
  • the glutamatergic neuron is involved in most of the brain’s fundamental processes such as cognition, learning, memory, and sensory perception. Dysregulation of glutamatergic neurotransmission is associated with many neurological disorders including epilepsy, schizophrenia, Alzheimer’s disease, Huntington’s disease, Parkinson’s disease, multiple sclerosis, amyotrophic lateral sclerosis, and stroke.
  • the glutamatergic neuron is characterized by the expression of the Vesicular Glutamate Transporter 1 (VGLUT1) and 2 (VGLUT2).
  • the term "population” refers to a population of cells, wherein the majority (e.g., at least about 50%, preferably at least about 60%, more preferably at least about 70%, and even more preferably at least about 80%) of the total number of cells have the specified characteristics of the cells of interest and express the markers of interest (e.g. a population of glutamatergic neurons comprises at least about 50%, preferably at least about 60%, more preferably at least about 70%, and even more preferably at least about 80% of cells which have the markers of glutamatergic neurons).
  • the expression “highly pure population of cells” indicates that the population of cells contains at least 90% of the desired cells. Accordingly, a “highly pure population of glutamatergic neurons” means that the population contain 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% of glutamatergic neurons.
  • hiNPC human induced neural progenitor cell
  • iPSC induced pluripotent stem cell
  • iPSC a type of pluripotent stem cell artificially derived from a non-pluripotent cell — typically an adult somatic cell — by inducing a “forced” expression of specific genes.
  • Induced pluripotent stem cells are similar to natural pluripotent stem cells, such as embryonic stem (ES) cells, in many aspects, such as the expression of certain stem cell genes and proteins, chromatin methylation patterns, doubling time, embryoid body formation, teratoma formation, viable chimera formation, and potency and differentiability.
  • Different protocols can be used to obtain hiNPC.
  • most useful approaches rely solely on small molecule to produce highly proliferative hiNPC populations (Chambers, Stuart M., et al. "Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. " Nature biotechnology 27.3 (2009): 275-280 f.
  • hiNPCs A key characteristic of these hiNPCs is their capability of robust and homogenous expansion. At the same time, hiNPCs should be able to efficiently differentiate into neuronal and glial cell types (Reinhardt, Peter, et al. "Derivation and expansion using only small molecules of human neural progenitors for neurodegenerative disease modeling. " PloS one 8.3 (2013): e59252.).
  • ASCL1 has its general meaning in the art and refers to the Achaete- scute homolog 1 protein encoded by ASCL1 gene. The term is also known as ASH-1, Class A basic helix-loop-helix protein 46 or bHLHa46. ASCL1 is a transcription factor that plays a key role in neuronal differentiation.
  • An exemplary amino acid sequence for ASCL1 is shown as SEQ ID NO: !. SEQ ID NO : 1 >sp
  • polynucleotide refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, or analogues thereof.
  • a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogues, and may be interrupted by non-nucleotide components. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer.
  • the term polynucleotide, as used herein, refers interchangeably to double- and singlestranded molecules. Unless otherwise specified or required, any embodiment of the invention described herein that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the doublestranded form.
  • the population of hiNPCs is engineered to express a polynucleotide that encodes for a polypeptide comprising an amino acid sequence having at least 90% of identity with the amino acid sequence as set forth in SEQ ID NO: 1.
  • engineered refers to an aspect of having been manipulated and altered by the hand of man.
  • engineered cell refers to a cell that has been subjected to a manipulation, so that its genetic, epigenetic, and/or phenotypic identity is altered relative to an appropriate reference cell such as otherwise identical cell that has not been so manipulated.
  • the manipulation is or comprises a genetic manipulation.
  • a genetic manipulation is or comprises one or more of (i) introduction of a polynucleotide not present in the cell prior to the manipulation (i.e., of a heterologous polynucleotide); (ii) removal of a polynucleotide, or portion thereof, present in the cell prior to the manipulation; and/or (iii) alteration (e.g., by sequence substitution) of a polynucleotide, or portion thereof, present in the cell prior to the manipulation.
  • an engineered cell is one that has been manipulated so that it contains and/or expresses a particular agent of interest (e.g., a protein, a polynucleotide, and/or a particular form thereof) in an altered amount and/or according to altered timing relative to such an appropriate reference cell.
  • a particular agent of interest e.g., a protein, a polynucleotide, and/or a particular form thereof
  • reference to an “engineered cell” herein may, in some embodiments, encompass both the particular cell to which the manipulation was applied and also any progeny of such cell.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described below.
  • the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (Needleman, Saul B. & Wunsch, Christian D. (1970). "A general method applicable to the search for similarities in the amino acid sequence of two proteins". Journal of Molecular Biology. 48 (3): 443-53.).
  • the percent identity between two nucleotide or amino acid sequences may also be determined using for example algorithms such as EMBOSS Needle (pair wise alignment; available at www.ebi.ac.uk).
  • EMBOSS Needle may be used with a BLOSUM62 matrix, a “gap open penalty” of 10, a “gap extend penalty” of 0.5, a false “end gap penalty”, an “end gap open penalty” of 10 and an “end gap extend penalty” of 0.5.
  • the “percent identity” is a function of the number of matching positions divided by the number of positions compared and multiplied by 100. For instance, if 6 out of 10 sequence positions are identical between the two compared sequences after alignment, then the identity is 60%.
  • % identity is typically determined over the whole length of the query sequence on which the analysis is performed.
  • Two molecules having the same primary amino acid sequence or nucleic acid sequence are identical irrespective of any chemical and/or biological modification.
  • a first amino acid sequence having at least 90% of identity with a second amino acid sequence means that the first sequence has 90; 91; 92; 93; 94; 95; 96; 97; 98; 99 or 100% of identity with the second amino acid sequence.
  • a polynucleotide can be introduced into the hiNPCs as naked DNA or in a suitable vector.
  • Naked DNA generally refers to the DNA contained in a plasmid expression vector in proper orientation for expression.
  • Physical methods for introducing a polynucleotide construct into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, nucleofection, and the like.
  • Other means can be used including colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • the polynucleotide is introduced into the population of hiNPCs by a viral vector that is an adeno-associated virus (AAV), a retrovirus, lentivirus, bovine papilloma virus, an adenovirus vector, a vaccinia virus, a polyoma virus, or an infective virus.
  • the vector is a retroviral. Retroviruses may be chosen as gene delivery vectors due to their ability to integrate their genes into the host genome, transferring a large amount of foreign genetic material, infecting a broad spectrum of species and cell types and for being packaged in special cell- lines.
  • a retroviral vector In order to construct a retroviral vector, the polynucleotide of interest is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication defective.
  • a packaging cell line is constructed containing the gag, pol, and/or env genes but without the LTR and/or packaging components.
  • the packaging sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture media.
  • Retroviral vectors are able to infect a broad variety of cell types.
  • Lentiviruses are complex retroviruses, which, in addition to the common retroviral genes gag, pol, and env, contain other genes with regulatory or structural function. The higher complexity enables the virus to modulate its life cycle, as in the course of latent infection.
  • Some examples of lentivirus include the Human Immunodeficiency Viruses (HIV 1, HIV 2) and the Simian Immunodeficiency Virus (SIV).
  • Lentiviral vectors have been generated by multiply attenuating the HIV virulence genes, for example, the genes env, vif, vpr, vpu and nef are deleted making the vector biologically safe.
  • Lentiviral vectors are known in the art, see, e.g. U.S. Pat. Nos. 6,013,516 and 5,994,136, both of which are incorporated herein by reference.
  • the vectors are plasmid-based or virus-based and are configured to carry the essential sequences for incorporating foreign polynucleotide, for selection and for transfer of the polynucleotide into a host cell.
  • the gag, pol and env genes of the vectors of interest also are known in the art.
  • the relevant genes are cloned into the selected vector and then used to transform the target cell of interest.
  • Recombinant lentivirus capable of infecting a non-dividing cell wherein a suitable host cell is transfected with two or more vectors carrying the packaging functions, namely gag, pol and env, as well as rev and tat is described in U.S. Pat. No. 5,994,136, incorporated herein by reference.
  • This describes a first vector that can provide a polynucleotide encoding a viral gag and a pol gene and another vector that can provide a polynucleotide encoding a viral env to produce a packaging cell.
  • Introducing a vector providing a heterologous gene into that packaging cell yields a producer cell which releases infectious viral particles carrying the foreign gene of interest.
  • the env preferably is an amphotropic envelope protein which allows transduction of cells of human and other species.
  • control sequences' refers collectively to promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, enhancers, and the like, which collectively provide for the replication, transcription and translation of a coding sequence in a recipient cell. Not all these control sequences need always be present so long as the selected coding sequence is capable of being replicated, transcribed and translated in an appropriate host cell.
  • polynucleotide sequence is a "promoter" sequence, which is used herein in its ordinary sense to refer to a nucleotide region comprising a DNA regulatory sequence, wherein the regulatory sequence is derived from a gene which is capable of binding RNA polymerase and initiating transcription of a downstream (3'-direction) coding sequence.
  • Transcription promoters can include "inducible promoters” (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), “repressible promoters” (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), and “constitutive promoters”.
  • polynucleotides of the present invention may be operably linked to strong promoters, such as retroviral long terminal repeats (LTRs), cytomegalovirus (CMV), murine stem cell virus (MSCV) U3, phosphoglycerate kinase (PGK), P-actin, ubiquitin, and a simian virus 40 (SV40)/CD43 composite promoter, elongation factor (EF)-la and the spleen focus-forming virus (SFFV) promoter.
  • strong promoters such as retroviral long terminal repeats (LTRs), cytomegalovirus (CMV), murine stem cell virus (MSCV) U3, phosphoglycerate kinase (PGK), P-actin, ubiquitin, and a simian virus 40 (SV40)/CD43 composite promoter, elongation factor (EF)-la and the spleen focus-forming virus (SFFV) promoter.
  • the polynucleotide sequence that encodes for the ASCL1 polypeptide is placed under the control of an inducible promoter.
  • inducible promoters include, for example, promoters that respond to heavy metals, to thermal shocks, to hormones, promoters that respond to chemical agents, such as glucose, lactose, galactose or antibiotic (e.g., tetracycline or doxycycline).
  • a tetracycline-inducible promoter is an example of an inducible promoter that responds to an antibiotic.
  • the tetracycline-inducible promoter comprises a minimal promoter linked operably to one or more tetracycline operator(s) (i.e. TetO).
  • Tetracycline analogue includes any compound that displays structural homologies with tetracycline and can activate a tetracycline-inducible promoter.
  • exemplary tetracycline analogues include, for example, doxycycline, chlorotetracycline and anhydrotetracycline.
  • the sequence of the polynucleotides is codon optimized for expression in a mammalian cell.
  • Codon optimization refers to the discovery that the frequency of occurrence of synonymous codons (i.e., codons that code for the same amino acid) in coding DNA is biased in different species. Such codon degeneracy allows an identical polypeptide to be encoded by a variety of nucleotide sequences.
  • a variety of codon optimization methods is known in the art, and include, e.g., methods disclosed in at least U.S. Pat. Nos. 5,786,464 and 6,114,148.
  • hiNPCs are cultured in an appropriate culture medium for allowing the differentiation into glutamatergic neurons.
  • the terms “culture,” “culturing,” “grow,” “growing,” “maintain,” “maintaining,” “expand,” “expanding,” etc. when referring to cell culture itself or the process of culturing, can be used interchangeably to mean that a cell is maintained outside the body (e.g., ex vivo) under conditions suitable for survival. Cultured cells are allowed to survive, and culturing can result in cell growth, differentiation, or division. The term does not imply that all cells in the culture survive or grow or divide, as some may naturally senesce, etc. Cells are typically cultured in media, which can be changed during the culture.
  • the term "culture medium” refers to a chemical composition that supports the growth and/or differentiation of a cell, suitably of a mammalian cell.
  • Typical culture media include suitable nutrients (e.g. sugars, amino acids, proteins, and the like) to support the growth and/or differentiation of a cell.
  • the culture medium is a neuronal medium.
  • neuronal medium has its general meaning in the art and refers to a medium that supports the culture of neurons.
  • the neuronal medium includes one or more ingredients selected from: a cell culture medium containing growth-promoting factors and/or a nutrient mixture (e.g., DMEM/F12, MEM/D-valine, neurobasal medium etc., including mixtures thereof); media supplements containing hormones, proteins, vitamins and/or amino acids (e.g., N2 supplement, B27 supplement, non-essential amino acids (NEAA), L-glutamine, Glutamax, BSA, insulin, all trans retinoic acid, etc.
  • a cell culture medium containing growth-promoting factors and/or a nutrient mixture e.g., DMEM/F12, MEM/D-valine, neurobasal medium etc., including mixtures thereof
  • media supplements containing hormones, proteins, vitamins and/or amino acids (e.g., N2 supplement, B27 supplement, non-essential amino acids (NEAA), L-glutamine, Glutamax, BSA, insulin, all trans retinoic acid, etc.
  • ingredients may also include one or more of beta- mercaptoethanol, transferrin; sodium selenite; and cAMP. Suitable concentrations of each of these ingredients are known to those of skill in the art and/or may be empirically determined.
  • the cells are cultured in the BrainPhysTM Neuronal Medium that is based on the formulation published by Cedric Bardy and Fred H. Gage (C Bardy et al. Proc Natl Acad Sci USA, 2015).
  • the culture medium when the polynucleotide that encodes for the ASCL1 polypeptide is placed under the control of an inducible promoter that is responder to a chemical agent, the culture medium is thus supplemented with an amount of said chemical agent.
  • the culture medium when the polynucleotide is placed under the control of a tetracycline-inducible promoter, then the culture medium is supplemented with a tetracycline analogue (for example, doxycycline, chlorotetracycline and anhydrotetracycline).
  • the cells are cultured in appropriate culture system such as plates or dishes. Culture conditions may vary, but standard tissue culture conditions are preferably used. Typically, cells are incubated in 5% CO2 incubators at 37°C in the appropriate culture medium.
  • the hiNPCs are cultured with feeder cells.
  • the neural differentiation may be performed in the absence or presence of co-cultured astrocytes.
  • the differentiating includes culturing the hiNPC on a surface coated with feeder cells or polyomithine/ laminin.
  • the duration of the culturing step is in the range of about 5 to 25 days, more preferably about 14 to 35 days (2-5 weeks).
  • the hiNPCs differentiate effectively into mature and synaptically active glutamatergic neurons within 3 to 5 weeks, although this may vary depending on the cell line and the reprogramming protocol.
  • the method of the present invention is particularly suitable for the preparation of large amounts of glutamatergic neurons that can be subsequently used e.g. for research or therapeutics applications.
  • the glutamatergic neurons can be used in neurodevelopment studies, disease modelling, drug screening, and/or neuronal replacement therapies.
  • FIGURES are a diagrammatic representation of FIGURES.
  • FIG. 1 Comparison of ours and previously described models to generate hiNs.
  • ASCLl-hiNs ASCLl-induced human neurons
  • HiNPCs are first virally transduced with the FUW-M2rtTA (Addgene #20342) lentiviral construct and a passage later, the Tet-O-FUW-Ascl 1 (Addgene #27150) lentiviral construct was transduced. These cells are maintained in neural progenitor medium (NPM; StemCell Technologies, Inc) and expanded prior to differentiation. For differentiation in human induced neurons (hiNs), hiNPCs are plated onto PLO/laminin-coated imaging plates at density 50,000 cells/cm 2 in NPM.
  • NPM neural progenitor medium
  • BP BrainPhys medium
  • 2 pg/mL doxycycline Sigma-Aldrich
  • 1 pg/mL puromycin Sigma-Aldrich
  • 2- 3 days depending on the efficiency of antibiotic selection
  • 50,000 human cortical astrocytes were added in each well with BrainPhys (StemCell Technologies) containing doxycycline.
  • 2 pM of Ara-C Cytosine P-D-arabinofuranoside
  • HA Human cortical astrocytes
  • ScienCell # 1800 Human cortical astrocytes
  • astrocyte medium ScienCell # 1801
  • astrocyte growth supplement AGS, ScienCell #1852
  • fetal bovine serum ScienCell # 1800
  • This culture system was characterized at 4weeks using snRNA-seq showing that 70% of cells expressed the pan-neuronal markers SOX11, SNAP25, DCX and RBFOX3. Of those, more than 90% of cells express genes common to human forebrain glutamatergic neurons, and less than 5% of GABAergic neurons. Therefore, this method allows the generation of highly pure glutamatergic populations in a relatively short period of time from hNPCs.
  • ASCLl-hiNs were composed of glutamatergic neurons (-92%) with a small proportion of GABAergic neurons (-2%) or of cells co-expressing low levels of markers of both neuronal subtypes (-6%) (Figure 1).

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Abstract

Les neurones glutamatergiques produisent, accumulent et libèrent dans les synapses le neurotransmetteur glutamate, principal neurotransmetteur excitateur du système nerveux central des mammifères. Lesdits neurones sont impliqués dans la plupart des processus fondamentaux du cerveau tels que la cognition, l'apprentissage, la mémoire et la perception sensorielle. Il existe un intérêt pour l'identification de facteurs de transcription qui permettraient la différenciation vers les neurones glutamatergiques. Les inventeurs montrent de manière surprenante que la surexpression d'ASCL1 induit la génération d'une population de neurones glutamatergiques de haute pureté. Cette observation va totalement dans le sens contraire de ce que l'on connaissait jusqu'à présent, puisque l'ASLC1 était principalement décrit comme induisant des neurones GABAergiques dans le cerveau antérieur. Par conséquent, la présente invention concerne des procédés de génération de populations neuronales glutamatergiques de haute pureté par l'utilisation du facteur pro-neural ASCL1.
PCT/EP2024/059305 2023-04-07 2024-04-05 Génération de populations neuronales glutamatergiques de haute pureté par l'utilisation du facteur pro-neural ascl1 Pending WO2024209036A1 (fr)

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