Method of generating glutamatergic neurons

Abstract

The invention relates to methods of generating glutamatergic neurons using forward programming.

Description

[0001] BIT-C-P3888PCT

[0002] 1

[0003] METHOD OF GENERATING GLUTAMATERGIC NEURONS

[0004] FIELD OF THE INVENTION

[0005] The invention relates to methods of generating glutamatergic neurons by overexpressing one or more polypeptides that have the activity of combinations of transcription factors and / or combinations of transcription factors themselves, i.e. through forward programming.

[0006] BACKGROUND OF THE INVENTION

[0007] Glutamate is the most common neurotransmitter in the central nervous system. Almost 40% of all neurons are classified as glutamatergic. Most glutamatergic neurons are located in the frontal cortex. Because of its extensive projection circuits, glutamate is involved in learning, memory formation and storage, and synaptic plasticity. The ability to induce long-term potentiation or long-term depression in the hippocampus by glutamatergic receptors, both ionotropic and metabotropic, determines synaptic plasticity. Synaptic plasticity is often calciumdependent and requires alteration of the actin cytoskeleton in dendrites and local mRNA translation of synaptic proteins.

[0008] Neurons in the aging brain are particularly susceptible to excitotoxicity. Malfunction of the glutamatergic system, which may readily lead to excitotoxicity, can be particularly dangerous for the nervous system. Glutamate plays a pivotal role in the etiology of neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease, because of its abundance in brain tissue and, in part, because it is at the crossroads of multiple metabolic pathways. It has been shown that if the balance of glutamate turnover is disrupted, the perturbation of glutamate neurotransmission has severe consequences, leading to the onset of neurodegenerative diseases. Understanding the role of the glutamatergic system in pathophysiology may allow the development of improved therapeutics for neurodegenerative disorders.

[0009] Methods for differentiating stem cells into glutamatergic neurons generally include treating stem cells with differentiation-inducing materials and culturing them for a long time. However, these differentiation-inducing materials can be expensive and the efficiency of cell differentiation is low. By contrast, forward programming strategies provide mature human cell types with unprecedented speed and efficiency. Forward programming involves directly converting pluripotent stem cells, including human pluripotent stem cells (hPSCs), to mature cell types through the forced expression of polypeptides having the activity of key lineage transcription factors and / or the key lineage transcription factors themselves, in order to convert the stem cell into a particular mature cell type.BIT-C-P3888PCT

[0010] 2

[0011] There is a need in the art to provide methods for generating glutamatergic neurons, particularly human glutamatergic neurons, suitable for use as potential therapeutic agents, in research and in tissue engineering.

[0012] SUMMARY OF THE INVENTION

[0013] According to a first aspect of the invention, there is provided a method of generating glutamatergic neurons comprising expressing one or more polypeptides having the activity of two or more transcription factors and / or increasing the expression of two or more transcription factors, the transcription factors comprising NGN2 and one or more transcription factors selected from the group consisting of: TBR1, NFIA, BCL11B, MEF2C, ZBTB18, POU4F2, POU4F1, FOXP1, NR4A2, TLE4, PAX6, FOXG1, FEZF2, EMX1 and variants thereof in a cell population, preferably a human pluripotent stem cell population, and culturing the cell population to obtain glutamatergic neurons.

[0014] According to a further aspect of the invention, there is provided a method for the production of glutamatergic neurons from a source cell, preferably a human cell, comprising the steps of:

[0015] a) insertion (preferably targeted insertion) of a gene encoding a transcriptional regulator protein into a first genomic safe harbour site of the source cell; and

[0016] b) insertion (preferably targeted insertion) of at least one nucleotide sequence encoding one or more polypeptides having the activity of two or more transcription factors and / or encoding two or more transcription factors, the transcription factors comprising NGN2 and one or more transcription factors selected from the group consisting of: TBR1, NFIA, BCL11B, MEF2C, ZBTB18, POU4F2, POU4F1, FOXP1, NR4A2, TLE4, PAX6, FOXG1, FEZF2, EMX1 and variants thereof, operably linked to an inducible promoter into a second genomic safe harbour site of the source cell, wherein said inducible promoter is regulated by the transcriptional regulator protein; and

[0017] c) culturing the source cell(s) comprising the insertions to obtain glutamatergic neurons.

[0018] According to a further aspect of the invention, there is provided a composition comprising one or more vectors, preferably one vector, encoding polypeptides having the activity of two or more transcription factors, wherein the transcription factors comprise a combination of NGN2 and one or more transcription factors selected from the group consisting of: TBR1, NFIA, BCL11B, MEF2C, ZBTB18, POU4F2, POU4F1, FOXP1, NR4A2, TLE4, PAX6, FOXG1, FEZF2 and EMX1.BIT-C-P3888PCT

[0019] 3

[0020] According to a further aspect of the invention, there is provided a use of one or more polypeptides having the activity of two or more transcription factors and / or two or more transcription factors, wherein the transcription factors comprise the combination of NGN2 and one or more transcription factors selected from the group consisting of: TBR1, NFIA, BCL11B, MEF2C, ZBTB18, POU4F2, POU4F1, FOXP1, NR4A2, TLE4, PAX6, FOXG1, FEZF2, EMX1 and variants thereof, to generate glutamatergic neurons, preferably human glutamatergic neurons.

[0021] According to a further aspect of the invention, there is provided a cell obtainable by any one of the methods defined herein.

[0022] According to a further aspect of the invention, there is provided a cell, preferably a human cell, comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of two or more transcription factors and / or encoding two or more transcription factors, wherein the transcription factors comprise the combination of NGN2 and one or more transcription factors selected from the group consisting of: TBR1, NFIA, BCL11B, MEF2C, ZBTB18, POU4F2, POU4F1, FOXP1, NR4A2, TLE4, PAX6, FOXG1, FEZF2, EMX1 and variants thereof.

[0023] According to a further aspect of the invention, there is provided a cell, preferably a human cell, as defined herein, for use in therapy, in vitro diagnostics or drug screening.

[0024] According to a further aspect of the invention, there is provided a kit for differentiating a cell, preferably a human cell, into a glutamatergic neuron comprising:

[0025] (i) a source cell and an agent that activates or increases the expression or amount of at least one or more transcription factors; and / or

[0026] (ii) one or more expression cassette(s) comprising nucleotide sequences encoding one or more polypeptides having the activity of two or more transcription factors and / or encoding two or more transcription factors,

[0027] wherein the transcription factors comprise the combination of NGN2 and one or more transcription factors selected from the group consisting of: TBR1, NFIA, BCL11B, MEF2C, ZBTB18, POU4F2, POU4F1, FOXP1, NR4A2, TLE4, PAX6, FOXG1, FEZF2, EMX1 and variants thereof.

[0028] According to a further aspect of the invention, there is provided a use of a kit as defined herein, for differentiating a cell into a glutamatergic neuron.BIT-C-P3888PCT

[0029] 4

[0030] According to a further aspect of the invention, there is provided a method of drug screening comprising contacting a glutamatergic neuron generated using the method or a glutamatergic neuron as defined herein, with the drug and observing a change in the glutamatergic neuron induced by the drug.

[0031] According to a further aspect of the invention, there is provided a method of treating a subject having or at risk of a disease or disorder comprising administering to the subject a therapeutically effective amount of glutamatergic neurons generated using the method or glutamatergic neurons as defined herein.

[0032] In one embodiment the transcription factors comprise NGN2 and two or more additional transcription factors selected from the list consisting of: TBR1, NFIA, BCL11B, MEF2C, ZBTB18, POU4F2, POU4F1, FOXP1, NR4A2, TLE4, PAX6, FOXG1, FEZF2 and EMX1. In one embodiment the transcription factors comprise NGN2 and one or more additional transcription factors selected from the list consisting of: TBR1, NFIA, BCL11B, MEF2C, ZBTB18, POU4F2, POU4F1, FOXP1, NR4A2, TLE4, PAX6 and FOXG1. In one embodiment the transcription factors comprise NGN2 and one or more additional transcription factors selected from the list consisting of: TBR1, NFIA, BCL11B, ZBTB18, FOXP1, NR4A2, TLE4, FOXG1, FEZF2 and EMX1. In one embodiment the transcription factors comprise NGN2 and one or more additional transcription factors selected from the list consisting of: TBR1, NFIA, BCL11B, MEF2C, ZBTB18, POU4F2, POU4F1, FOXP1, NR4A2, TLE4, PAX6 and FEZF2. In one embodiment the transcription factors comprise NGN2 and one or more additional transcription factors selected from the list consisting of: TBR1, NFIA, BCL11B, ZBTB18, FOXP1, NR4A2, TLE4 and FEZF2. In one embodiment the transcription factors comprise NGN2 and one or more additional transcription factors selected from the list consisting of: TBR1, NFIA, POLI4F2 and POLI4F1. In one embodiment the transcription factors comprise NGN2 and one or more additional transcription factors selected from the list consisting of: NFIA, POLI4F2 and POLI4F1. In one embodiment the transcription factors comprise NGN2 and one or more additional transcription factors selected from the list consisting of: TBR1 and NFIA. In one embodiment the transcription factors comprise NGN2 and TBR1. In one embodiment the transcription factors comprise NGN2 and NFIA. In one embodiment the transcription factors comprise one of the following combinations: (i) NGN2, TBR1 and EMX1; (ii) NGN2, TBR1 and FOXG1; (iii) NGN2, BCL11B and EMX1; (iv) NGN2, BCL11B and FOXG1; (v) NGN2, FEZF2 and EMX1; or (vi) NGN2, FEZF2 and FOXG1.BIT-C-P3888PCT

[0033] 5

[0034] In one embodiment the transcription factors comprise NGN2 and one or more additional transcription factors selected from the list consisting of: NFIA, BCL11B, FOXP1, FOXG1, POU4F2, ZBTB18 and EMX1 (such as (i) NFIA, BCL11B, FOXP1 and POU4F2, (ii) NFIA, BCL11B and FOXP1 or (iii) NFIA and POLI4F2). Such transcription factors are particularly beneficial for the reprogramming to deep layer glutamatergic neurons.

[0035] In one embodiment the transcription factors comprise NGN2 and one or more additional transcription factors selected from the list consisting of: NR4A2, TLE4, PAX6, MEF2C, POLI4F1 and TBR1 (such as TBR1 and POLI4F1). Such transcription factors are particularly beneficial for the reprogramming to upper-layer glutamatergic neurons.

[0036] BRIEF DESCRIPTION OF THE FIGURES

[0037] Figure 1. Neuronal subtype gene signature ratios for each transcription factor (TF) (in addition to NGN2) compared against NGN2 alone as control (Flue).

[0038] Figure 2. Deep and upper layer gene signature ratios for each TF (in addition to NGN2) ordered by its ratio for deep layer gene signature (high to low) compared against NGN2 alone (Flue).

[0039] Figure 3. Mean expression values of deep layer (black) and upper layer (grey) gene signatures for each TF (in addition to NGN2) compared against NGN2 alone (FLuc).

[0040] DETAILED DESCRIPTION

[0041] The present invention provides methods for producing glutamatergic neurons from source cells, preferably human cells, by expressing one or more polypeptides having the activity of one or more transcription factors and / or increasing the expression of a combination of transcription factors which the present inventors have identified as inducing cell differentiation into glutamatergic neurons.

[0042] Definitions

[0043] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. As used herein, the following terms have the meanings ascribed to them below.

[0044] References to “transcription factor” as used herein, refer to proteins that are involved in gene regulation in both prokaryotic and eukaryotic organisms. In one embodiment, transcriptionBIT-C-P3888PCT

[0045] 6

[0046] factors can have a positive effect on gene expression and, thus, may be referred to as an “activator” or a “transcriptional activation factor”. In another embodiment, a transcription factor can negatively affect gene expression and, thus, may be referred to as “repressors” or a “transcription repression factor”. Activators and repressors are generally used terms and their functions may be discerned by those skilled in the art.

[0047] The term “increasing the expression of” or “increasing the amount of’ with respect to increasing an amount, level or expression of a transcription factor, refers to increasing the quantity of the transcription factor in a cell of interest {e.g., a source cell). In some embodiments, the amount of transcription factor is increased in a cell {e.g., via an expression cassette directing expression of a polynucleotide encoding one or more transcription factors) when the quantity of transcription factor is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more relative to a control e.g., a source cell without said expression cassette(s) or a control cell where the baseline expression is zero or negligible). In some of the embodiments, increasing the expression comprises “overexpressing” the transcription factor, i.e., increasing the expression of the transcription factor above the endogenous expression level of the transcription factor in the cell.

[0048] Methods of the invention may be used in a “cell population”, i.e., a collection of cells which may be differentiated into the desired cell type. Said cell population may comprise “source cells”, also referred to as “starting cells”, i.e., a cell type prior to differentiation into the desired cell type.

[0049] References herein to “pluripotent”’ refer to cells which have the potential to differentiate into all types of cell found in an organism. One form of pluripotent stem cell, known as induced pluripotent stem cells, are of particular interest to the present invention. “Induced pluripotent stem cells” (iPSCs) are cells that have been reprogrammed to an embryonic stem cell-like state by being forced to express genes and factors important for maintaining the defining properties of embryonic stem cells. In 2006, it was shown that overexpression of four specific transcription factors could convert adult cells into pluripotent stem cells. Oct-3 / 4 and certain members of the SOX gene family have been identified as potentially crucial transcriptional regulators involved in the induction process. Additional genes including certain members of the Klf family, the Myc family, Nanog, and Lin28, may increase the induction efficiency. Examples of the genes which may be used as reprogramming factors to generate iPSCs include Oct3 / 4, SOX2, SOX1, SOX3, SOX15, SOX17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15-2, Tell , beta-catenin, Lin28b, Sall4, Esrrb, Tbx3 and Glisl,BIT-C-P3888PCT

[0050] 7

[0051] GATA3, GATA6 and these reprogramming factors may be used singly, or in combination of two or more kinds thereof. In particular, the reprogramming factors may comprise at least the Yamanaka factors, i.e., Oct3 / 4, SOX2, Klf4 and c-Myc. These reprogramming factors may also be used in combination with the transcription factors of interest in the present invention.

[0052] References herein to “somatic” refer to any type of cell that makes up the body of an organism, excluding germ cells. Somatic cells therefore include, for example, skin, heart, muscle, bone or blood cells and their stem cells. Somatic cells may also be referred to as differentiated cells. In one embodiment, the somatic cell may be an adult cell or a cell derived from an adult which displays one or more detectable characteristics of an adult or non-embryonic cell.

[0053] Methods of the invention (e.g. forward programming of iPSCs) are for use in generating “glutamatergic neurons”. The term “glutamatergic neuron” includes glutamatergic neuron-like cells that exhibit some but not all characteristics of adult glutamatergic neurons, as well as mature, fully functional and / or metabolically active adult glutamatergic neurons. The glutamatergic neurons produced by this method may be at least as functional as the glutamatergic neurons produced by directed differentiation to date.

[0054] References herein to “culturing” include the addition of cells {e.g., the cell population, i.e., the source cells), to media comprising growth factors and / or essential nutrients. It will be appreciated that such culture conditions may be adapted according to the cells or cell population to be generated according to methods of the invention.

[0055] References to a “variant” when referring to a polypeptide could be, for example, an amino acid sequence at least 80%, 85%, 90%, 95%, 98%, or 99% identical to the original, full-length polypeptide. When referring to a nucleic acid sequence, the term “variant” could be, for example, a nucleic acid sequence at least 80%, 85%, 90%, 95%, 98%, or 99% identical to the original, full-length nucleic acid sequence. The variant could be a fragment of full-length polypeptide, in particular a functional fragment of the polypeptide. The fragment may be at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, or 99% as long as the full-length wild type polypeptide or a domain thereof having an activity of interest such as the ability to differentiate a source cell into a glutamatergic neuron. Variations known in the art to eliminate or substantially reduce the activity of the protein are preferably avoided. In some embodiments, the variant lacks an N- and / or C-terminal portion of the full-length polypeptide, e.g., up to 10, 20, or 50 amino acids from either terminus is lacking. In some embodiments, a functional variant or fragment has at least 50%, 60%, 70%, 80%, 90%, 95% or more of the activity of theBIT-C-P3888PCT

[0056] 8

[0057] full-length wild type polypeptide. One of skill in the art will be aware of, or will readily be able to ascertain, whether a particular polypeptide variant or fragment is functional using assays known in the art. For example, the ability of a variant of a transcription factor to generate glutamatergic neurons can be assessed using the assays as described herein. In particular, the variant may be a biologically active variant. A "biologically active variant" includes any variant of a molecule having substantially, at least in part, the same functional and / or biological properties of said molecule, such as binding properties, and / or the same structural features, such as binding domain. It also refers to a molecule that exhibits the functional features as the transcription factors disclosed herein.

[0058] In one embodiment, the variant is an isoform of the listed transcription factor. Many transcription factors have one or more isoforms which result, for example, from alternative splicing or from a shifted transcription initiation. Based on the different transcript variants (i.e. mRNA), different polypeptides are generated. It is possible that different transcript variants have different translation initiation sites.

[0059] A “promoter” is a nucleotide sequence which is recognised by proteins involved in initiating and regulating transcription of a polynucleotide sequence. An “inducible promoter” is a nucleotide sequence where expression of a genetic sequence operably linked to the promoter is controlled by an analyte, co-factor, regulatory protein, etc. It is intended that the term “promoter” or “control element” includes full-length promoter regions and functional {e.g., controls and / or affects transcription or translation) segments of these regions.

[0060] The term “operably linked” refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function. Thus, a given promoter operably linked to a genetic sequence is capable of effecting the expression of that sequence when the regulatory factors are present. The promoter need not be contiguous with the sequence, so long as it functions to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between the promoter sequence and the genetic sequence and the promoter sequence can still be considered “operably linked” to the genetic sequence. Thus, the term “operably linked” is intended to encompass any spacing or orientation of the promoter element and the genetic sequence in the inducible cassette which allows for initiation of transcription of the inducible cassette upon recognition of the promoter element by a transcription complex.BIT-C-P3888PCT

[0061] 9

[0062] The term “vector”, as used herein, is intended to refer to a nucleic acid molecule which is used as a vehicle to carry genetic material into a cell. One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop or circle into which additional DNA segments may be ligated. Another type of vector is an infectious but non-pathogenic viral vector, wherein additional DNA segments may be ligated to certain viral genetic elements. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian and yeast vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply, “expression vectors”). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, lentiviral vectors, adenoviruses, Sendai viruses and adeno-associated viruses), which serve equivalent functions, and also bacteriophage and phagemid systems. Another type of vector includes synthetic and in vitro transcribed RNA molecules, e.g., mRNA and stabilised RNA, to carry coding genetic information to the cells. This also includes synthetic-self- replicating RNA vectors.

[0063] References to “subject”, “patient” or “individual” refer to a subject, in particular a mammalian subject, to be treated. Mammalian subjects include humans, non-human primates, farm animals (such as cows), sports animals, or pet animals, such as dogs, cats, guinea pigs, rabbits, rats or mice. In some embodiments, the subject is a human. In alternative embodiments, the subject is a non-human mammal, such as a mouse.

[0064] The term "sufficient amount" means an amount sufficient to produce a desired effect. The term "therapeutically effective amount" is an amount that is effective to ameliorate a symptom of a disease or disorder. A therapeutically effective amount can be a "prophylactically effective amount" as prophylaxis can be considered therapy.

[0065] As used herein, the term “about” when used herein includes up to and including 10% greater and up to and including 10% lower than the value specified, suitably up to and including 5% greater and up to and including 5% lower than the value specified, especially the value specified. The term “between” includes the values of the specified boundaries.BIT-C-P3888PCT

[0066] 10

[0067] It will be understood that any method as described herein may have one or more, or all, steps performed in vitro, ex vivo or in vivo. Preferably, the methods described herein do not modify the germ line genetic identity of a human being.

[0068] Transcription factor activity

[0069] The method described herein may comprise increasing the expression (in particular, the protein expression) of a sufficient number of polypeptides having the activity of the transcription factors (or the transcription factors themselves) capable of causing forward programming of a cell population to glutamatergic neurons, therefore differentiating the cell population into glutamatergic neurons. In the context of the present invention, these factors may also be referred to as “forward programming factors”. As described herein, the expression of an exogenous or endogenous (in particular an exogenous) transcription factor may be increased.

[0070] According to an aspect of the invention, there is provided a method of generating glutamatergic neurons comprising expressing one or more polypeptides having the activity of two or more transcription factors and / or increasing the expression of two or more transcription factors, the transcription factors comprising NGN2 and one or more transcription factors selected from the group consisting of: TBR1, NFIA, BCL11B, MEF2C, ZBTB18, POU4F2, POU4F1, FOXP1, NR4A2, TLE4, PAX6, FOXG1, FEZF2, EMX1, and variants thereof, in a cell population, preferably a human cell population, and culturing the cell population to obtain glutamatergic neurons.

[0071] References to “NGN2” relate to Neurogenin 2. It is also known as NEUROG2, BHLHa8, Math4A and Atoh4. In one embodiment, the NGN2 transcription factor is human NGN2. Wild type human NGN2 is identified by UniProt ID: Q9H2A3 and is encoded by the NGN2 gene which is identified by Ensembl Gene ID: ENSG00000178403. NGN2 plays a role in the differentiation and survival of midbrain dopaminergic neurons.

[0072] References herein to “TBR1” relate to T-box Brain Transcription Factor 1. TBR1 is also known as T-box brain protein and T-brain. In one embodiment the TBR1 is human TBR1. Wild type human TBR1 is identified by UniProt ID: Q16650, and is encoded by the TBR1 gene, identified by Ensembl Gene ID: ENSG00000136535. TBR1 plays an important role in neuronal migration and axonal projection. TBR1 is associated with intellectual development, and its absence can lead to autism and speech delay.BIT-C-P3888PCT

[0073] 11

[0074] References herein to “NFIA” relate to nuclear factor 1 A-type. NFIA is also known as NF1a, NF1-A and NFI-A. In one embodiment the NFIA is human NFIA. Wild type human NFIA is identified by UniProt ID: Q12857, and is encoded by the NFIA gene, identified by Ensembl Gene ID: ENSG00000162599. Diseases associated with NFIA include bilateral polymicrogyria.

[0075] References herein to “BCL11B” relates to BCL11 Transcription Factor B. In one embodiment, the BCL11 B is human BCL11 B. Wild type human BCL11 B is identified by UniProt ID: Q9C0K0, and is encoded by the BCL11B gene, identified by Ensembl Gene ID: ENSG00000127152. BCL11B encodes a C2H2-type zinc finger protein and is closely related to BCL11A, a gene whose translocation may be associated with B-cell malignancies.

[0076] References herein to “MEF2C” relate to Myocyte Enhancer Factor 20. In one embodiment, the MEF2C is human MEF2C. Wild type human MEF2C is identified by UniProt ID: Q06413, and is encoded by the MEF2C gene, identified by Ensembl Gene ID: ENSG00000081189. MEF2C binds specifically to the MEF2 element present in the regulatory regions of many musclespecific genes.

[0077] References herein to “ZBTB18” relate to Zinc Finger And BTB Domain Containing 18. In one embodiment, the ZBTB18 is human ZBTB18. Wild type human ZBTB18 is identified by UniProt ID: ZBTB18, and is encoded by the ZBTB18 gene, identified by Ensembl Gene ID: ENSG00000179456. ZBTB18 is a zinc finger protein involved in myogenesis by directly repressing the expression of ID2 and ID3, 2 inhibitors of skeletal myogenesis. ZBTB18 is also involved in controlling cell division of progenitor cells and regulating the survival of postmitotic cortical neurons.

[0078] References herein to “POU4F2” and “P0U4F1” relate to homeoboxes 2 and 1 of POU class 4. POU4F2 and P0U4F1 are involved in the development of the sensory nervous system. In one embodiment, POU4F2 and / or P0U4F1 are human POU4F2 and / or P0U4F1. Wild type human POU4F2 is identified by UniProt ID: Q12837 and is encoded by the POU4F2 gene which is identified by Ensembl Gene ID: ENSG00000151615. Wild type human P0U4F1 is identified by UniProt ID: Q01851 and is encoded by the P0U4F1 gene which is identified by Ensembl Gene ID: ENSG00000152192.

[0079] References herein to “FOXP1” relate to the P1 paralog of the Forkhead Box or winged-helix family of transcription factors. FOXP1 acts as a transcriptional repressors in order to regulate immune cell development. In one embodiment, FOXP1 is human FOXP1. Wild type humanBIT-C-P3888PCT

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[0081] F0XP1 is identified by UniProt ID: Q9H334 and is encoded by the FOXP1 gene which is identified by Ensembl Gene ID: ENSG00000114861.

[0082] References herein to “NR4A2” relate to Nuclear Receptor Subfamily 4 Group A Member 2. In one embodiment, the NR4A2 is human NR4A2. Wild type human NR4A2 is identified by UniProt ID: P43354, and is encoded by the NR4A2 gene, identified by Ensembl Gene ID: ENSG00000153234. NR4A2 is a member of the steroid-thyroid hormone-retinoid receptor superfamily. It is important for the differentiation and maintenance of meso-diencephalic dopaminergic neurons during development.

[0083] References herein to “TLE4” relate to TLE Family Member 4, Transcriptional Corepressor. In one embodiment, the TLE4 is human TLE4. Wild type human TLE4 is identified by UniProt ID: Q04727, and is encoded by the TLE4 gene, identified by Ensembl Gene ID: ENSG00000106829. TLE4 is a transcriptional corepressor that inhibits the transcriptional activation mediated by PAX5, and by CTNNB1 and TCF family members in Wnt signaling.

[0084] References herein to “PAX6” relate to Paired Box 6. PAX6 is also known as Aniridia Type II Protein. In one embodiment, the PAX6 is human PAX6. Wild type human PAX6 is identified by UniProt ID: P26367, and is encoded by the PAX6 gene, identified by Ensembl Gene ID: ENSG00000007372. PAX6 has important functions in the development of the eye, nose, central nervous system and pancreas. PAX6 is required for the natural differentiation of pancreatic islet alpha cells.

[0085] References herein to “FOXG1” relate to Forkhead Box Protein G1. FOXG1 is also known as BF1, FKHL1, HBF-1 and HFK1. In one embodiment the FOXG1 is human FOXG1. Wild type human FOXG1 is identified by UniProt ID: P55316, and is encoded by the FOXG1 gene, identified by Ensembl Gene ID: ENSG00000176165. The absence of FOXG1 leads to FOXG1 syndrome characterized by microcephaly and brain malformations. It affects most aspects of development and can cause seizures. FOXG1 is a transcription repression factor that plays an important role in the establishment of the regional subdivision of the developing brain and in the development of the telencephalon.

[0086] References herein to “FEZF2” relate to Forebrain Embryonic Zinc Finger-Like Protein 2. FEZF2 is also known as FEZL, FKSG36, Zfp312, Znf312 and TOP. In one embodiment the FEZF2 is human FEZF2. Wild type human FEZF2 is identified by UniProt ID: Q8TBJ5, and is encoded by the FEZF2 gene, identified by Ensembl Gene ID: ENSG00000153266. DiseasesBIT-C-P3888PCT

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[0088] associated with FEZF2 include uterine inversion and ceftriaxone allergy. FEZF2 is required for the specification of corticospinal motor neurons and other subcerebral projection neurons.

[0089] References herein to “EMX1” relate to Empty Spiracles Homeobox 1. In one embodiment the EMX1 is human EMX1. Wild type human EMX1 is identified by UniProt ID: Q04741, and is encoded by the EMX1 gene, identified by Ensembl Gene ID: ENSG00000135638. EMX1 enables sequence-specific double-stranded DNA binding activity. EMX1 is involved in brain development, neuron differentiation and the regulation of transcription by RNA polymerase II.

[0090] According to an aspect of the invention, there is provided a method of generating glutamatergic neurons comprising expressing one or more polypeptides having the activity of two or more transcription factors and / or increasing the expression of two or more transcription factors, the transcription factors comprising NGN2 and one or more additional transcription factors selected from the list consisting of: TBR1, NFIA, BCL11B, MEF2C, ZBTB18, POU4F2, POLI4F1, FOXP1, NR4A2, TLE4, PAX6 and FEZF2 and variants thereof, in a cell population, preferably a human cell population, and culturing the cell population to obtain glutamatergic neurons.

[0091] In one embodiment, the transcription factors comprise the combination of NGN2, TBR1 and one or more additional transcription factors selected from the listed consisting of: NFIA, BCL11B, MEF2C, ZBTB18, POU4F2, POU4F1, FOXP1, NR4A2, TLE4, PAX6 and FEZF2. In one embodiment, the transcription factors comprise the combination of NGN2, NFIA and one or more additional transcription factors selected from the listed consisting of: TBR1, BCL11B, MEF2C, ZBTB18, POU4F2, POU4F1, FOXP1, NR4A2, TLE4, PAX6 and FEZF2. In one embodiment, the transcription factors comprise the combination of NGN2, BCL11B and one or more additional transcription factors selected from the listed consisting of: TBR1, NFIA, MEF2C, ZBTB18, POU4F2, POU4F1, FOXP1, NR4A2, TLE4, PAX6 and FEZF2. In one embodiment, the transcription factors comprise the combination of NGN2, MEF2C and one or more additional transcription factors selected from the listed consisting of: TBR1, NFIA, BCL11B, ZBTB18, POU4F2, POU4F1, FOXP1, NR4A2, TLE4, PAX6 and FEZF2. In one embodiment, the transcription factors comprise the combination of NGN2, ZBTB18 and one or more additional transcription factors selected from the listed consisting of: TBR1, NFIA, BCL11B, MEF2C, POU4F2, POU4F1, FOXP1, NR4A2, TLE4, PAX6 and FEZF2. In one embodiment, the transcription factors comprise the combination of NGN2, POLI4F2 and one or more additional transcription factors selected from the listed consisting of: TBR1, NFIA, BCL11B, MEF2C, ZBTB18, POU4F1, FOXP1, NR4A2, TLE4, PAX6 and FEZF2. In oneBIT-C-P3888PCT

[0092] 14

[0093] embodiment, the transcription factors comprise the combination of NGN2, POLI4F1 and one or more additional transcription factors selected from the listed consisting of: TBR1, NFIA, BCL11B, MEF2C, ZBTB18, POU4F2, FOXP1, NR4A2, TLE4, PAX6 and FEZF2. In one embodiment, the transcription factors comprise the combination of NGN2, FOXP1 and one or more additional transcription factors selected from the listed consisting of: TBR1, NFIA, BCL11B, MEF2C, ZBTB18, POU4F2, POU4F1, NR4A2, TLE4, PAX6 and FEZF2. In one embodiment, the transcription factors comprise the combination of NGN2, NR4A2 and one or more additional transcription factors selected from the listed consisting of: TBR1, NFIA, BCL11B, MEF2C, ZBTB18, POU4F2, POU4F1, FOXP1, TLE4, PAX6 and FEZF2. In one embodiment, the transcription factors comprise the combination of NGN2, TLE4 and one or more additional transcription factors selected from the listed consisting of: TBR1, NFIA, BCL11B, MEF2C, ZBTB18, POU4F2, POU4F1, FOXP1, NR4A2, PAX6 and FEZF2. In one embodiment, the transcription factors comprise the combination of NGN2, PAX6 and one or more additional transcription factors selected from the listed consisting of: TBR1, NFIA, BCL11B, MEF2C, ZBTB18, POU4F2, POU4F1, FOXP1, NR4A2, TLE4 and FEZF2. In one embodiment, the transcription factors comprise the combination of NGN2, FEZF2 and one or more additional transcription factors selected from the listed consisting of: TBR1, NFIA, BCL11B, MEF2C, ZBTB18, POU4F2, POU4F1, FOXP1, NR4A2, TLE4 and PAX6.

[0094] Once the activity of a transcription factor is appreciated, the endogenous transcription machinery can be modulated using not only the transcription factors themselves, but also polypeptides engineered to replicate the action of the transcription factor, such as synthetic transcription factors or artificial transcription factors. For example, CRISPR (clustered regularly interspaced palindromic repeats), TALE (transcriptional activator-like effector) or Zinc Finger technologies can be used to modulate the expression of endogenous cellular genes, to allow for faster and more efficient nuclear forward programming under conditions amenable for clinical and commercial applications. This is set out in, for example, US2016 / 362705, incorporated herein by reference.

[0095] Alternatively, with the development of highly accurate protein structure prediction with artificial intelligence tools such as AlphaFold, it is now straightforward for polypeptides to be developed that have very similar structure and / or activity to a transcription factor of interest whilst at the same time having an amino acid sequence that has very little resemblance to that of the transcription factor of interest. For example, large language models trained on biological diversity have been used to develop proteins only around 70% identical to CRISPR-Cas proteins that occur in nature and yet with comparable or improved biological activity andBIT-C-P3888PCT

[0096] 15

[0097] specificity (Ruffolo et al. (2024) bioRxiv, doi: https: / / doi.org / 10.1101 / 2024.04.22.590591). Such polypeptides are covered within the scope of the invention.

[0098] In some embodiments of the present invention, a polypeptide (in particular a single polypeptide) is engineered to mimic the activity of more than one transcription factor of interest. In a further embodiment, polypeptides having the activity of one or more transcription factors is expressed, in combination with increasing the expression of another transcription factor. For example, a polypeptide having the activity of NGN2 can be expressed in combination with increasing the expression of TBR1 (or vice versa).

[0099] Methods of the invention encompass the use of variants of the transcription factors of interest. References to the transcription factors also encompasses species variants, isoforms, homologues, allelic forms, mutant forms, and equivalents thereof, including conservative substitutions, additions, deletions therein not adversely affecting the structure and / or function. Changes in the nucleic acid sequence of the transcription factor gene can result in conservative changes or substitutions in the amino acid sequence. Therefore, the invention includes polypeptides having conservative changes or substitutions. The invention includes sequences where conservative substitutions are made that do not alter the activity of the transcription factor protein of interest.

[0100] Cell Types

[0101] The method may be used on any cell type, including stem cells. In the case of stem cells, the generation of glutamatergic neurons using the method may be referred to as “forward programming”, “direct programming” or “direct differentiation”, i.e. , the pluripotent stem cell is differentiated into a glutamatergic neuron. Furthermore, glutamatergic neuron forward programming may be used as generic terminology referring to the use of transcription factors to differentiate a source cell into glutamatergic neurons. Alternatively, the methods also cover transdifferentiation where a non-glutamatergic neuron (preferably a somatic cell) is converted into a glutamatergic neuron.

[0102] Sources of cells suitable for methods of the invention may include, for example, any stem cells or cells that are not glutamatergic neurons. For example, the stem cells may be pluripotent stem cells, for example induced pluripotent stem cells, embryonic stem cells or pluripotent stem cells derived by nuclear transfer or cell fusion. It may be preferred that the embryonic stem cell is derived without destruction of the embryo, particularly where the cells are human. In some embodiments, the stem cells are not derived from human or animal embryos, i.e., theBIT-C-P3888PCT

[0103] 16

[0104] invention does not extend to any methods which involve the destruction of human or animal embryos. The stem cells may also include multipotent stem cells, oligopotent stem cells, or unipotent stem cells. The stem cells may also include fetal stem cells or adult stem cells, such as hematopoietic stem cells, mesenchymal stem cells, neural stem cells, epithelial stem cells, skin stem cells. In certain aspects, the stem cells may be isolated from umbilical, placenta, amniotic fluid, chorion villi, blastocysts, bone marrow, adipose tissue, brain, peripheral blood, cord blood, menstrual blood, blood vessels, skeletal muscle, skin and liver.

[0105] In one embodiment, the cell population is of human origin. The source cell e.g., a cell that is not a glutamatergic neuron, may be of human origin. It is well known that, compared with nonhuman pluripotent cells, genome engineering in human pluripotent stem cells is challenging due to, for example, partially due to low transfection / transduction efficiency and high apoptosis under stresses such as low-density plating, drug-selection and sorting (Cerbini et al., PLOS ONE, 10(1), e0116032).

[0106] In one embodiment, the cell population is not an immortalized cell line. An immortalised cell line is a population of cells that can be grown and divided indefinitely in vitro, unlike normal cells with limited lifespans. These cells become immortalized either naturally (like cancer cells) or through intentional modification.

[0107] In one embodiment, the cell population is of animal origin. The source cell e.g., a cell that is not a glutamatergic neuron, may be of animal origin. In certain aspects, the cell is preferably one from a livestock animal. Livestock animals include, for example, pigs, cows, horses, buffalo, bison, goats, sheep, deer, reindeer, donkeys, bantengs, yaks, chickens, ducks and turkeys. In one embodiment, the cell population is not of mouse origin.

[0108] In one embodiment, the cell population comprises stem cells, e.g., induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs), haematopoietic stem cells, mesenchymal stem cells or neuronal stem cells. In a further embodiment, the cell population comprises pluripotent stem cells, e.g., iPSCs or ESCs.

[0109] In one embodiment, the source cell is a stem cell, e.g., an iPSC, an ESC, a haematopoietic stem cell, a mesenchymal stem cell or a neuronal stem cell. In a further embodiment, the source cell is a pluripotent stem cell, e.g., an iPSC or an ESC. In some embodiments, the source cell is an iPSC.BIT-C-P3888PCT

[0110] 17

[0111] Methods of preparing induced pluripotent stem cells are also known in the art. Induction of iPSCs typically require the expression of or exposure to at least one member from SOX family and at least one member from Oct family. SOX and Oct are thought to be central to the transcriptional regulatory hierarchy that specifies ES cell identity. For example, SOX may be S0X1, S0X2, S0X3, S0X15, or S0X18; Oct may be Oct-4. Additional factors may increase the reprogramming efficiency, like Nanog, Lin28, Klf4, orc-Myc; specific sets of reprogramming factors may be a set comprising S0X2, Oct-4, Nanog and, optionally, Lin-28; or comprising S0X2, Oct4, Klf and, optionally, c-Myc. In one method, iPSC may be generated by transfecting cells with transcription factors Oct4, S0X2, c-Myc and Klf4 using viral transduction. In an alternative method, iPSCs may be generated by transfecting cells with RNA encoding transcription factors inducing the development of stem cell characteristics, such as transcription factors selected from Oct4, S0X2, c-Myc and Klf4.

[0112] In one embodiment, the glutamatergic neurons are human glutamatergic neurons.

[0113] In one embodiment, the induced pluripotent stem cells are derived from somatic or germ cells of the patient. Such use of autologous cells would remove the need for matching cells to a recipient. Alternatively, commercially available iPSC may be used, such as those available from WICELL (WiCell Research Institute, Inc, Wisconsin, US). Alternatively, the cells may be a tissue-specific stem cell which may also be autologous or donated.

[0114] Delivery of transcription factors

[0115] It will be understood that methods for expressing polypeptides having transcription factor activity and / or increasing the expression of the transcription factors in the cells to be programmed into glutamatergic neurons may include any method known in the art, for example, by induction of expression of one or more expression cassettes previously introduced into the cells, or by introduction of nucleic acids (such as DNA or RNA), polypeptides, or small molecules to the cells to stimulate expression of the endogenous or exogenous transcription factors. Increasing the expression of certain endogenous but transcriptionally repressed genes may also reverse the silencing or inhibitory effect on the expression of these genes by regulating the upstream transcription factor expression or epigenetic modulation. Therefore, methods of the invention may involve culturing the cell population under conditions to artificially increase the expression level of one or more of the transcription factors described herein.

[0116] In one embodiment, expression of the polypeptides having transcription factor activity and / or the transcription factors themselves takes place at the same time. It is understood in the artBIT-C-P3888PCT

[0117] 18

[0118] that this is very different to what takes place in nature, where transcription factors involved in, for example, differentiating a pluripotent stem cell to a neural stem cells would be different to the transcription factors involved in differentiating a neural stem cell to a glutamatergic neuron. As discussed above, the mechanisms by which forward programming take place are very different to differentiation that takes place in nature or directed differentiation methods of the art.

[0119] “At the same time” is understood to mean that the induction of the polypeptides with transcription factor activity and / or the transcription factors themselves, the introduction of the nucleic acids, polypeptides, or small molecules to stimulate expression of the endogenous or exogenous transcription factors, or the artificial increase of said factors, takes place at the same time (i.e. simultaneously). Therefore, the method may comprise simultaneous expression of the one or more polypeptides having the activity of one or more transcription factors and / or simultaneously increasing the expression of one or more transcription factors. It will be appreciated that the downstream activities of the polypeptides and / or transcription factors may take place at different rates.

[0120] In one embodiment, the expression of the polypeptides having transcription factor activity and / or the transcription factors themselves is increased by contacting the cell population with the polypeptides and / or the transcription factors (i.e., the proteins encoding the transcription factors). Delivery of the transcription factors may occur using direct electroporation of transcription factor proteins to the cells.

[0121] In an alternative embodiment, the expression of the transcription factors is increased by introducing a promoter (e.g. a strong promoter) ahead of an endogenous gene encoding the transcription factor(s).

[0122] In a further alternative embodiment, the expression of the polypeptides having transcription factor activity and / or the transcription factors themselves is increased by contacting the cell population with one or more agents that activate or increase the expression amount of the (exogenous or endogenous) transcription factors. In the case of polypeptides having transcription factor activity or exogenous transcription factors, the agents may still be used after the genes for the polypeptides and / or transcription factors have been inserted into the cell.BIT-C-P3888PCT

[0123] 19

[0124] In one embodiment, the agent is selected from the group consisting of: a nucleic acid (i.e., polynucleotide, e.g., messenger RNA (mRNA), coding DNA sequence), a protein, an aptamer and small molecule, ribosome, RNAi agent, guide RNA (gRNA) and peptide nucleic acid (PNA) and analogues or variants thereof. In one embodiment, the agent is a transcriptional activation system (e.g., a gRNA for use in a gene activation system such as CRISPR / Cas or TALEN) for increasing the expression of the one or more endogenous transcription factors.

[0125] The method of inducing differentiation of the cell population (i.e., source cells), may comprise delivering to the cells a nucleic acid comprising an open reading frame encoding one or more of the polypeptides having transcription factor activity, one or more of the transcription factors themselves (e.g., in an expression cassette), the transcription factor protein, and / or an activator of transcription of the open reading frame encoding the polypeptide and / or transcription factor. This results in the amount of the transcription factor in the cells being increased, and the cells differentiate to form glutamatergic neurons. Said open reading frame may be part of a recombinant expression cassette.

[0126] In one embodiment, the nucleic acid comprises a recombinant or exogenous expression cassette comprising the one or more transcription factor sequences (or genes) (and / or sequences encoding one or more polypeptides having transcription factor activity) in a sufficient number to cause forward programming of source cells to glutamatergic neurons. Preferably the sequences would be present a single expression cassette.

[0127] The exogenous expression cassette may comprise an externally inducible transcriptional regulatory element for inducible expression of the one or more polypeptides having transcription factor activity and / or the transcription factors themselves, such as an inducible promoter, e.g., comprising a tetracycline response element or variant thereof. If a single expression cassette is used then induction from the externally inducible transcriptional regulatory element would lead to increased expression of all of the polypeptides and / or transcription factors at the same time.

[0128] If expression of the transcription factors is increased by introducing an exogenous sequence encoding the transcription factor (e.g., the transcription factor gene), then it would be understood that any suitable system for delivering the sequence may be used. The gene delivery system may be a transposon system; a viral gene delivery system; an episomal gene delivery system; or a homologous recombination system such as utilizing a zinc fingerBIT-C-P3888PCT

[0129] 20

[0130] nuclease, a transcription activator-like effector nuclease (TALENs), a meganuclease, or CRISPR / Cas, or the like.

[0131] Alternatively, introduction of a nucleic acid, such as DNA or RNA, into cells may use any suitable methods for nucleic acid delivery for transformation of a cell, as described herein or as would be known to one of ordinary skill in the art. Such methods include, but are not limited to, direct delivery of DNA such as by ex vivo transfection, by injection (including microinjection), by electroporation, by calcium phosphate precipitation, by using DEAE-dextran followed by polyethylene glycol, by direct sonic loading, by liposome mediated transfection, by receptor-mediated transfection, by microprojectile bombardment, by agitation with silicon carbide fibers, by Agrobacterium-mediated transformation, and any combination of such methods. Through the application of these techniques, cells may be stably or transiently transformed.

[0132] Further, the expression cassette (e.g., an inducible recombinant expression cassette) may include cleavable sequences. Such sequences are sequences that are recognised by an entity capable of specifically cutting DNA, and include restriction sites, which are the target sequences for restriction enzymes or sequences for recognition by other DNA cleaving entities, such as nucleases, recombinases, ribozymes or artificial constructs. At least one cleavable sequence may be included, but preferably two or more are present. These cleavable sequences may be at any suitable point in the cassette, such that a selected portion of the cassette, or the entire cassette, can be selectively removed if desired. The cleavable sites may thus flank the part / all of the genetic sequence that it may be desired to remove. The method may therefore also comprise removal of the expression cassette and / or the genetic material.

[0133] In an alternative embodiment, the cell population is contacted with one or more agents that has the same effect as activating or increasing the expression or amount of the transcription factors (i.e. an indirect method of increasing the expression transcription factor). In this aspect of the invention, the method comprises introducing an exogenous agent which mimics the effect of increasing the expression of the transcription factors described herein. For example, such a method may comprise introducing a protein (e.g. an engineered zinc finger nuclease) that has a DNA-binding activity analogous to the transcription factor.

[0134] It will be understood that a combination of one or more of the methods for expressing the polypeptides having transcription factor activity or increasing the expression of the transcription factors may be used where the combination overall results in activity necessary for the forward programming to glutamatergic neurons.BIT-C-P3888PCT

[0135] 21

[0136] Vectors

[0137] In one embodiment, the polypeptides having transcription factor activity or the transcription factors themselves (e.g., combinations of polypeptides and / or transcription factors) are introduced into the cell population using a vector. One of skill in the art would be well equipped to construct a vector through standard recombinant techniques. Vectors include but are not limited to plasmids, cosmids, viruses (bacteriophage, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs).

[0138] In one embodiment, a nucleic acid sequence encoding one or more transcription factors is introduced into a cell by a transposon system (i.e. involving a transposon plasmid). A transposon delivery system is comprised of two plasmids, one encoding the transposase and one encoding the transcription factor(s). The transposase protein mediates random integration of the transcripts encoded in the transposon plasmid into the genome. In one embodiment, the transposon system is selected from a PiggyBac or Sleeping Beauty transposon system. The transposon plasmid encodes a payload flanked by two ITRs (internal terminal repeats). The payload may comprise a Tet inducible promoter, the transcription factor(s), and optionally a selection marker, e.g. an antibiotic selection cassette under a constitutive promoter.

[0139] In one embodiment, the transposase and transposon plasmids are delivered by nucleofection or lipofection into the cells. The number of integration events, and therefore the number of copies of payload per cell can be in part controlled by adjusting the total and relative amounts of transposase and transposon plasmid DNA. This allows the combinatorial delivery of transcription factors at a single cell level.

[0140] In one embodiment, the vector is a viral vector. The viral gene delivery system may be an RNA-based or DNA-based viral vector. Viral vectors include retroviral vectors, lentiviral vectors (e.g., derived from HIV-1, HIV-2, SIV, BIV, FIV etc.), gammaretroviral vectors, adenoviral (Ad) vectors (including replication competent, replication deficient and gutless forms thereof), adeno-associated virus-derived (AAV) vectors, simian virus 40 (SV-40) vectors, bovine papilloma virus vectors, Epstein-Barr virus vectors, herpes virus vectors, vaccinia virus vectors, Harvey murine sarcoma virus vectors, murine mammary tumour virus vectors, Rous sarcoma virus vectors and Sendai virus vectors. In a further embodiment, the viral vector is selected from: a lentiviral vector, an adeno-associated virus vector or a Sendai virus vector. In a yet further embodiment, the viral vector is a lentiviral vector.BIT-C-P3888PCT

[0141] 22

[0142] Lentiviral vectors are well known in the art. Lentiviral vectors are complex retroviruses capable of integrating randomly into the host cell genome, which, in addition to the common retroviral genes gag, pol, and env, contain other genes with regulatory or structural function (e.g., accessory genes Vif, Nef, Vpu, Vpr). Lentiviral vectors have the advantage of being able to infect non-dividing cells and can be used for both in vivo and ex vivo gene transfer and expression of nucleic acid sequences. For example, recombinant lentiviral vector 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.

[0143] In one embodiment, the vector is a self-replicating RNA vector expression system. For example, the system may comprise self-replicating RNA vectors that remain ectopic to the host cell genome and encode the transcription factors that induce forward programming. Selfreplicating RNA vectors are known in the art and many are based on positive strand RNA viruses, such as alphaviruses.

[0144] In one embodiment, the viral vector is used at a high multiplicity of infection (MOI). A high MOI helps to ensure that more than one transcription factor is introduced into the source cell. In one embodiment, the MOI is greater than 0.5, such as 1.0 or above.

[0145] In one embodiment, a nucleic acid sequence encoding the one or more polypeptides having transcription factor activity and / or transcription factors is introduced into a cell by a plasmid. In one embodiment, at least one nucleic acid sequence encoding the polypeptides having transcription factor activity and / or the transcription factors is introduced into a cell on a single plasmid.

[0146] In one embodiment, the plasmid is episomal. Episomal vectors are able to introduce large fragments of DNA into a cell but are maintained extra-chromosomally, replicated once per cell cycle, partitioned to daughter cells efficiently, and elicit substantially no immune response. In alternative embodiments, an Epstein-Barr virus (EBV)-based episomal vector, a yeast-based vector, an adenovirus-based vector, a simian virus 40 (SV40)-based episomal vector, or a bovine papilloma virus (BPV)-based vector may be used.

[0147] As described herein, adding any one of the transcription factors TBR1, NFIA, BCL11B, MEF2C, ZBTB18, POU4F2, POU4F1, FOXP1 , NR4A2, TLE4, PAX6, FOXG1 , FEZF2 or EMX1 to NGN2 achieves stable forward programming to glutamatergic neurons in a human induced pluripotent stem cell line (cells that have not been immortalized). Therefore, the inventionBIT-C-P3888PCT

[0148] 23

[0149] includes vectors and compositions encoding these combinations of transcription factors (i.e. at least two transcription factors).

[0150] According to a further aspect of the invention, there is provided a vector (or construct) comprising a combination of polynucleotide sequences encoding polypeptides having the activity of NGN2 and additionally one or more polynucleotides encoding polypeptides having the activity of one or more (preferably one) transcription factor(s) selected from the group consisting of: TBR1, NFIA, BCL11B, MEF2C, ZBTB18, POU4F2, POU4F1, FOXP1, NR4A2, TLE4, PAX6, FOXG1, FEZF2, EMX1.

[0151] According to a further aspect of the invention, there is provided a composition comprising one or more vectors encoding polypeptides having the activity of two or more transcription factors, wherein the transcription factors comprise a combination of NGN2 and one or more transcription factors selected from the group consisting of: TBR1, NFIA, BCL11B, MEF2C, ZBTB18, POU4F2, POU4F1, FOXP1, NR4A2, TLE4, PAX6, FOXG1, FEZF2, EMX1. This aspect of the invention may include the individual transcription factors encoded by separate vectors (i.e. a composition comprising a combination of at least two vectors) and / or combinations of transcription factors encoded by one or more vectors (e.g. a composition comprising one vector encoding at least two transcription factors).

[0152] These vectors by their nature are distinguishable from endogenous genes within a cell. In one embodiment, the vectors are isolated vectors. In one embodiment, the vectors are exogenously inserted into a cell, such as a pluripotent stem cell, and so the compositions may be present within a cell. In one embodiment, the vectors are operably linked to inducible promoters.

[0153] Site-specific delivery

[0154] Any suitable technique for insertion of a nucleic acid sequence into a specific sequence may be used, and several are described in the art. Suitable techniques include any method which introduces a break at the desired location and permits recombination of the vector into the gap. Thus, a crucial first step for targeted site-specific genomic modification is the creation of a double-strand DNA break (DSB) at the genomic locus to be modified. Distinct cellular repair mechanisms can be exploited to repair the DSB and to introduce the desired sequence, and these are non-homologous end joining repair (NHEJ), which is more prone to error; and homologous recombination repair (HR).BIT-C-P3888PCT

[0155] 24

[0156] Several techniques exist to allow customized site-specific generation of DSB in the genome. Many of these involve the use of customized endonucleases, such as zinc finger nucleases, TALENs or the clustered regularly interspaced short palindromic repeats / CRISPR associated protein (CRISPR / Cas, e.g. CRISPR / Cas9) system.

[0157] Zinc finger nucleases are artificial enzymes which are generated by fusion of a zinc-finger DNA-binding domain to the nuclease domain of the restriction enzyme Fokl. The latter has a non-specific cleavage domain which must dimerise in order to cleave DNA. This means that two zinc finger nuclease monomers are required to allow dimerisation of the Fokl domains and to cleave the DNA. The DNA binding domain may be designed to target any genomic sequence of interest, is a tandem array of Cys2HiS2 zinc fingers, each of which recognises three contiguous nucleotides in the target sequence. The two binding sites are separated by 5-7bp to allow optimal dimerization of the Fokl domains. The enzyme thus is able to cleave DNA at a specific site, and target specificity is increased by ensuring that two proximal DNA-binding events must occur to achieve a double-strand break.

[0158] Transcription activator-like effector nucleases, or TALENs, are dimeric transcription factor / nucl eases. They are made by fusing a TAL effector DNA-binding domain to a DNA cleavage domain (a nuclease). Transcription activator-like effectors (TALEs) can be engineered to bind practically any desired DNA sequence, so when combined with a nuclease, DNA can be cut at specific locations. TAL effectors are proteins that are secreted by Xanthomonas bacteria, the DNA binding domain of which contains a repeated highly conserved 33-34 amino acid sequence with divergent 12th and 13th amino acids. These two positions are highly variable and show a strong correlation with specific nucleotide recognition. This straightforward relationship between amino acid sequence and DNA recognition has allowed for the engineering of specific DNA-binding domains by selecting a combination of repeat segments containing appropriate residues at the two variable positions. TALENs are thus built from arrays of 33 to 35 amino acid modules, each of which targets a single nucleotide. By selecting the array of modules, almost any sequence may be targeted. Again, the nuclease used may be Fokl or a derivative thereof.

[0159] Three types of CRISPR mechanisms have been identified, of which type II is the most studied. The CRISPR / Cas9 system (type II) utilises the Cas9 nuclease to make a double-stranded break in DNA at a site determined by a short guide RNA. The CRISPR / Cas system is a prokaryotic immune system that confers resistance to foreign genetic elements. CRISPR are segments of prokaryotic DNA containing short repetitions of base sequences. Each repetitionBIT-C-P3888PCT

[0160] 25

[0161] is followed by short segments of “protospacer DNA” from previous exposures to foreign genetic elements. CRISPR spacers recognize and cut the exogenous genetic elements using RNA interference. The CRISPR immune response occurs through two steps: CRISPR-RNA (crRNA) biogenesis and crRNA-guided interference. CrRNA molecules are composed of a variable sequence transcribed from the protospacer DNA and a CRISPR repeat. Each crRNA molecule then hybridizes with a second RNA, known as the trans-activating CRISPR RNA (tracrRNA) and together these two eventually form a complex with the nuclease Cas9. The protospacer DNA encoded section of the crRNA directs Cas9 to cleave complementary target DNA sequences, if they are adjacent to short sequences known as protospacer adjacent motifs (PAMs). This natural system has been engineered and exploited to introduce DSB breaks in specific sites in genomic DNA, amongst many other applications. In particular, the CRISPR type II system from Streptococcus pyogenes may be used. At its simplest, the CRISPR / Cas9 system comprises two components that are delivered to the cell to provide genome editing: the Cas9 nuclease itself and a gRNA. The gRNA is a fusion of a customised, site-specific crRNA (directed to the target sequence) and a standardised tracrRNA.

[0162] Once a DSB has been made, a donor template with homology to the targeted locus is supplied; the DSB may be repaired by the homology-directed repair (HDR) pathway allowing for precise insertions to be made.

[0163] Derivatives of this system are also possible. Mutant forms of Cas9 are available, such as Cas9D10A, with only nickase activity. This means it cleaves only one DNA strand and does not activate NHEJ. Instead, when provided with a homologous repair template, DNA repairs are conducted via the high-fidelity HDR pathway only. Cas9D10A may be used in paired Cas9 complexes designed to generate adjacent DNA nicks in conjunction with two sgRNAs complementary to the adjacent area on opposite strands of the target site, which may be particularly advantageous.

[0164] The elements for making the double-strand DNA break may be introduced in one or more vectors, such as plasmids, for expression in the cell.

[0165] Thus, any method of making specific, targeted double strand breaks in the genome in order to effect the insertion of a gene / inducible cassette may be used in the method of the invention. It may be preferred that the method for inserting the gene / inducible cassette utilises any one or more of zinc finger nucleases, TALENs and / or CRISPR / Cas9 systems or any derivative thereof.BIT-C-P3888PCT

[0166] 26

[0167] Once the DSB has been made by any appropriate means, the gene / inducible cassette for insertion may be supplied in any suitable fashion as described below. The gene / inducible cassette and associated genetic material form the donor DNA for repair of the DNA at the DSB and are inserted using standard cellular repair machinery / pathways. How the break is initiated will alter which pathway is used to repair the damage, as noted above.

[0168] Other methods in the art for site specific delivery include the use of homologous recombination (HR) and recombinase mediated cassette exchange (RMCE). DNA damage mediated site specific insertion methods (such as CRISPR / Cas) can also be used to perform site specific integration of DNA recognition sequences fatt' sites) which in turn mediate site specific insertion via the activity of tyrosine and serine recombinases or integrases. These sites (e.g. attP) once inserted into the genome, can mediate site specific HR and RMCE. Insertion of exogenous nucleic acid sequences occurs through homologous recombination between cognate attP and attB sites mediated by the expression of the appropriate and cognate recombinase (e.g. Flp, Cre) or integrase (PhiC31, Bxb1). Using targeting vectors, as described above, flanked by attB sites, site specific exogenous DNA insertion of transgenes can be achieved.

[0169] Controlled expression

[0170] In one embodiment, expression of the transcription factors is under inducible control. In this aspect of the invention, the transcription and translation (expression) of the transcription factors may be controlled within the cell. This permits overexpression of the transcription factor(s), if required.

[0171] In an alternative embodiment, expression of the polypeptides having transcription factor activity and / or the transcription factors themselves is under inducible control. In this aspect of the invention, the transcription and translation (expression) of the polypeptides having transcription factor activity and / or the transcription factors may be controlled within the cell. This permits overexpression of the transcription factor(s), if required, preferably in response to external stimuli.

[0172] An exogenous expression cassette carrying the polypeptides having transcription factor activity and / or the transcription factors themselves may comprise an externally inducible transcriptional regulatory element (i.e., an inducible promoter) for rapid induction of protein expression in response to external stimuli, i.e. inducible gene (or transgene) expression. TheBIT-C-P3888PCT

[0173] 27

[0174] presence or addition of the appropriate external stimuli (e.g. protein, compound, chemical, light or temperature change) to cell culture media modulates the controlled expression of the genetic sequence within the inducible expression cassette; and may be administered continuously or transiently to modulate transcription as required.

[0175] Expression of the transcription factors described herein may be increased using a dual cassette expression system, such as the system described in WO2018096343, which is incorporated herein by reference. In this instance, induced transgene over-expression is achieved by using the Tet-ON system components with transgene expression controlled by doxycycline. The components are split between two genetic safe harbour sites (GSH) to reduce the risk of epigenetic gene silencing. The components are (i) transcriptional activator protein (reverse tetracycline trans-activator (rtTA)), which in the presence of doxycycline binds (ii) tetracycline response element (TRE; multiple TetO repeat sequences & minimal Cytomegalovirus (CMV) promoter). TRE binding by rtTA trans-activates transgene expression. Trans-activatable coding sequences for transgenes may be of human origin.

[0176] Therefore, in one embodiment, a sequence encoding one or more (e.g., two or more or three or more) of the polypeptides having transcription factor activity and / or the transcription factors is introduced into the cell population, preferably human cell population, using a method comprising:

[0177] - insertion (preferably targeted insertion) of a coding sequence for a transcriptional regulator protein into a first genomic safe harbour site of a source cell present in the cell population; and

[0178] - insertion (preferably targeted insertion) of one or more inducible cassettes into one or more second genomic safe harbour sites of the source cell, wherein said one or more inducible cassettes comprises said sequence encoding the one or more polypeptides having the activity of one or more transcription factors and / or the transcription factors operably linked to an inducible promoter, and said promoter is regulated by the transcriptional regulator protein.

[0179] According to another aspect of the invention, there is provided a dual expression system comprising:

[0180] (a) a first expression cassette comprising a gene encoding a transcriptional regulator protein flanked by one or more homology arms targeting the first expression cassette; and (b) a second expression cassette comprising a sequence encoding one or more polypeptides having the activity of two or more transcription factors and / or two or more transcription factors operably linked to an inducible promoter, flanked by one or more homologyBIT-C-P3888PCT

[0181] 28

[0182] arms targeting the second expression cassette, wherein the inducible promoter is regulated by the transcriptional regulator protein of the first expression cassette, and wherein the two or more transcription factors comprise the combination of NGN2 and one or more transcription factors selected from the group consisting of: TBR1, NFIA, BCL11B, MEF2C, ZBTB18, POU4F2, POU4F1, FOXP1, NR4A2, TLE4, PAX6, FOXG1, FEZF2, EMX1 and variants thereof.

[0183] This embodiment of the invention provides a dual expression cassette system. The insertion of the gene encoding a transcriptional regulator protein provides the control mechanism for the expression of the inducible cassette which is operably linked to the inducible promoter.

[0184] In one embodiment, the first and / or second expression cassettes are inserted into genomic safe harbour sites (GSH sites). Preferably the first and second GSH site are different (i.e. are located at different positions in the genome). It will be understood that if more than one transcription factor is to be introduced into the cell using the dual expression system, then the transcription factors may be introduced into the second GSH site (such as within a multicistronic cassette at the same GSH site), or into multiple GSH sites (i.e. as separate cassettes across different GSH sites).

[0185] Alternatively, the dual expression cassette system utilises different alleles of the same GSH site. In this embodiment, the inducible cassette may be inserted into one allele of the GSH site and the system controlling the expression of the inducible cassette into the other allele of the GSH site (e.g. as described in DeKelver et al., 2010, Genome Res., 20, 1133-43 and Qian et al., 2014, Stem Cells, 32, 1230-8).

[0186] A GSH site is a locus within the genome wherein a gene or other genetic material may be inserted without any deleterious effects on the cell or on the inserted genetic material. Most beneficial is a GSH site in which expression of the inserted gene sequence is not perturbed by any read-through expression from neighbouring genes and expression of the inducible cassette minimizes interference with the endogenous transcription programme. More formal criteria have been proposed that assist in the determination of whether a particular locus is a GSH site in future (Papapetrou etal. (2011) Nature Biotechnology, 29(1): 73-8) These criteria include a site that is (i) 50 kb or more from the 5’ end of any gene, (ii) 300 kb or more from any gene related to cancer, (iii) 300 kb or more from any microRNA (miRNA), (iv) located outside a transcription unit and (v) located outside ultraconserved regions (UCR). It may not be necessary to satisfy all of these proposed criteria, since GSH sites already identified do notBIT-C-P3888PCT

[0187] 29

[0188] fulfil all of the criteria. It is thought that a suitable GSH site will satisfy at least 2, 3, 4 or all of these criteria. Any suitable GSH site may be used in the method of the invention, on the basis that the site allows insertion of genetic material without deleterious effects to the cell and permits transcription of the inserted genetic material. Those skilled in the art may use these simplified criteria to identify a suitable GSH site, and / or the more formal criteria set out above.

[0189] Insertion of the coding sequence for a transcriptional regulator protein and / or the inducible cassette may be carried out through direct delivery methods as described above. It is understood that although such direct delivery methods may lead to the random insertion of the genetic material, screening may be carried out in order to identify clones that show no deleterious effects, are able to express the genetic material and are able to be forward programmed or reprogrammed to glutamatergic neurons, and by doing so one is able to confirm that the transcriptional regulator protein I inducible cassette has been inserted into a GSH site.

[0190] In one embodiment the insertion of the transcriptional regulator protein or the inducible cassette is targeted. In a further embodiment, the insertion of the transcriptional regulator protein and the inducible cassette is targeted. “Targeted insertion”, as with site-specific delivery, is understood as the insertion of the genetic material into a pre-chosen GSH site. As discussed above, this can be carried out using techniques known in the art such as zinc finger nucleases, TALENs or the clustered regularly interspaced short palindromic repeats / CRISPR associated protein (CRISPR / Cas, e.g. CRISPR / Cas9) system.

[0191] In one embodiment, the first and second genetic safe harbour sites (GSH sites) are selected from (in particular any two) of the hROSA26 locus, the AAVS1 locus, the CLYBL gene, the CCR5 gene or the HPRT gene. Insertions specifically within genetic safe harbour sites is preferred over random genome integration, since this is expected to be a safer modification of the genome, and is less likely to lead to unwanted side effects such as silencing natural gene expression or random insertional mutagenesis.

[0192] The adeno-associated virus integration site 1 locus (AAVS1) is located within the protein phosphatase 1, regulatory subunit 12C (PPP1R12C) gene on human chromosome 19, which is expressed uniformly and ubiquitously in human tissues. AAVS1 has been shown to be a favourable environment for transcription, since it comprises an open chromatin structure and native chromosomal insulators that enable resistance of the inducible cassettes against silencing. There are no known adverse effects on the cell resulting from disruption of theBIT-C-P3888PCT

[0193] 30

[0194] PPP1R12C gene. Moreover, an inducible cassette inserted into this site remains transcriptionally active in many diverse cell types.

[0195] The human ROSA26 (hROSA26) site has been identified on the basis of sequence analogy with a GSH site from mice (ROSA26 - reverse oriented splice acceptor site #26). The hROSA26 locus is on chromosome 3 (3p25.3), and can be found within the Ensembl database (GenBank:CR624523). The integration site lies within the open reading frame (ORF) of the THUMPD3 long non-coding RNA (reverse strand). Since the hROSA26 site has an endogenous promoter, the inserted genetic material may take advantage of that endogenous promoter, or alternatively may be inserted operably linked to a promoter.

[0196] Intron 2 of the Citrate Lyase Beta-like (CLYBL) gene, on the long arm of Chromosome 13, was identified as a suitable GSH site since it is one of the identified integration hot-spots of the phage derived phiC31 integrase. Studies have demonstrated that randomly inserted inducible cassettes into this locus are stable and expressed. It has been shown that insertion of inducible cassettes at this GSH site does not perturb local gene expression (Cerbini et al. (2015) PLOS One, 10(1): e0116032). CLYBL thus provides a GSH site which may be suitable for use in the present invention.

[0197] CCR5, which is located on chromosome 3 (position 3p21.31) is a gene which codes for HIV-1 major co-receptor. Interest in the use of this site as a GSH site arises from the null mutation in this gene that appears to have no adverse effects, but predisposes to HIV-1 infection resistance. Zinc-finger nucleases that target the third exon have been developed, thus allowing for insertion of genetic material at this locus.

[0198] The hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene encodes a transferase enzyme that plays a central role in the generation of purine nucleotides through the purine salvage pathway.

[0199] Other GSH sites have been described in the art, such as in Sadelain et al. (2012) Nature Reviews 12:51-58 and in WO2021 / 152086, which are herein incorporated by reference.

[0200] GSH sites in other organisms have been identified and include ROSA26, HRPT and Hippl 1 (H11) loci in mice. Mammalian genomes may include GSH sites based upon pseudo attP sites. For such sites, hiC31 integrase, the Streptomyces phage-derived recombinase, has beenBIT-C-P3888PCT

[0201] 31

[0202] developed as a non-viral insertion tool, because it has the ability to integrate an inducible cassette-containing plasmid carrying an attB site into pseudo attP sites.

[0203] Technically, the insertions into the first and / or second GSH site may occur on one chromosome, or on both chromosomes. The GSH sites exist at the same genetic loci on both chromosomes of diploid organisms. Insertion within both chromosomes is advantageous since it may enable an increase in the level of transcription from the inserted genetic material within the inducible cassette, thus achieving particularly high levels of transcription.

[0204] Specific insertion of genetic material into the particular GSH site based upon customised sitespecific generation of DNA double-strand breaks at the GSH site may be achieved. The genetic material may then be introduced using any suitable mechanism, such as homologous recombination. Any method of making a specific DSB in the genome may be used, but preferred systems include CRISPR / Cas9 and modified versions thereof, zinc finger nucleases and the TALEN system, or via HR or ROME mediated integration or recombination.

[0205] One or more genetic sequences may be controllably transcribed from within the second and / or further GSH sites. Indeed, the inducible cassette may contain 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 genetic sequences (e.g., transcription factor sequences) which it is desired to insert into the GSH site and the transcription of which be controllably induced. Therefore, the transcription factors required by the present invention may be included within the same cassette introduced into the second genetic safe harbour site. For example, the three or more transcription factors may be included in, for example, three mono-cistronic constructs, one mono-cistronic and one bi-cistronic construct or one tri-cistronic construct. It will be understood that similar combinations of constructs may be used to achieve higher orders of transcription factor expression.

[0206] Alternatively, if a combination of transcription factors is used, the individual transcription factors may be introduced into separate GSH sites and / or under the control of the same, different or orthogonal inducible promoters. Therefore, in one embodiment, the transcription factors are introduced into separate GSH sites. For example, this may be achieved by utilising three or more different GSH sites for three or more transcription factors (i.e. , wherein the transcription factors are introduced as mono-cistronic cassettes). Alternatively, this may be achieved by utilising the fact that a GSH site exists at the same genetic loci on both chromosomes of diploid organisms, e.g., introducing one transcription factor into the GSH site on one chromosome and a different transcription factor into the same GSH site on the other chromosome. ThisBIT-C-P3888PCT

[0207] 32

[0208] embodiment is advantageous if different expression levels or timing of expression of the transcription factors is desired. In one embodiment, the method comprises targeted insertion of the transcription factors, each operably linked to an inducible promoter into a second, third and fourth genetic safe harbour site of the source cell. The inducible promoter may be the same of each transcription factor and therefore are all regulated by the transcriptional regulator protein.

[0209] A transcriptional regulator protein is a protein that binds to DNA, preferably sequence-specifically to a DNA site located in or near a promoter, and either facilitating the binding of the transcription machinery to the promoter, and thus transcription of the DNA sequence (a transcriptional activator) or blocks this process (a transcriptional repressor).

[0210] The DNA sequence that a transcriptional regulator protein binds to is called a transcription factor-binding site or response element, and these are found in or near the promoter of the regulated DNA sequence. Transcriptional activator proteins bind to the response element and promote gene expression. Such proteins are preferred in the methods of the present invention for controlling inducible cassette expression. Transcriptional repressor proteins bind to the response element and prevent gene expression.

[0211] Transcriptional regulator proteins may be activated or deactivated by a number of mechanisms including binding of a substance, interaction with other transcription factors (e.g., homo- or hetero-dimerization) or coregulatory proteins, phosphorylation, and / or methylation. The transcriptional regulator protein may be controlled by activation or deactivation.

[0212] If the transcriptional regulator protein is a transcriptional activator protein, it is preferred that the transcriptional activator protein requires activation. This activation may be through any suitable means, but it is preferred that the transcriptional regulator protein is activated through the addition to the cell of an exogenous substance. The supply of an exogenous substance to the cell can be controlled, and thus the activation of the transcriptional regulator protein can be controlled. Alternatively, an exogenous substance can be supplied in order to deactivate a transcriptional regulator protein, and then supply withdrawn in order to activate the transcriptional regulator protein.

[0213] If the transcriptional regulator protein is a transcriptional repressor protein, it is preferred that the transcriptional repressor protein requires deactivation. Thus, a substance is supplied toBIT-C-P3888PCT

[0214] 33

[0215] prevent the transcriptional repressor protein repressing transcription, and thus transcription is permitted.

[0216] Any suitable transcriptional regulator protein may be used, preferably one that may be activated or deactivated. It is preferred that an exogenous substance, light or change in temperature may be supplied to control the transcriptional regulator protein.

[0217] Tetracycline-Controlled Transcriptional Activation is a method of inducible gene expression where transcription is reversibly turned on or off in the presence of the antibiotic tetracycline or one of its derivatives (e.g., doxycycline which is more stable). In this system, the transcriptional activator protein is reverse tetracycline-controlled transactivator (rtTa, which may also be referred to as tetracycline - responsive transcriptional activator protein) or a derivative thereof. The rtTA protein is able to bind to DNA at specific TetO operator sequences. Several repeats of such TetO sequences are placed upstream of a minimal promoter (such as the CMV promoter), which together form a tetracycline response element (TRE). There are two forms of this system, depending on whether the addition of tetracycline or a derivative activates (Tet-On) or deactivates (Tet-Off) the rtTA protein.

[0218] In a Tet-Off system, tetracycline or a derivative thereof binds rtTA and deactivates the rtTA, rendering it incapable of binding to TRE sequences, thereby preventing transcription of TRE-controlled genes. This system was first described in Gossen et al. (1992) PNAS 89 (12): 5547-5551.

[0219] The Tet-On system is composed of two components; (1) the constitutively expressed reverse tetracycline-controlled transactivator (rtTa) and the rtTa-sensitive inducible promoter (Tet Responsive Element, TRE). This may be bound by tetracycline or its more stable derivatives, including doxycycline (dox), resulting in activation of rtTa, allowing it to bind to TRE sequences and inducing expression of TRE-controlled genes. The use of this may be preferred in the method of the invention.

[0220] Thus, the transcriptional regulator protein may thus be a reverse tetracycline-controlled transactivator (rtTa) protein, which can be activated or deactivated by the antibiotic tetracycline or one of its derivatives, which are supplied exogenously. If the transcriptional regulator protein is rtTA, then the inducible promoter inserted into the second GSH site includes the tetracycline response element (TRE). The exogenously supplied substance is the antibiotic tetracycline or one of its derivatives.BIT-C-P3888PCT

[0221] 34

[0222] Variants and modified rtTa proteins may also be used in the methods of the invention, these include Tet-On Advanced transactivator (also known as rtTA2S-M2) and Tet-On 3G (also known as rtTA-V16, derived from rtTA2S-S2).

[0223] The tetracycline response element (TRE) generally consists of 7 repeats of the 19bp bacterial TetO sequence separated by spacer sequences, together with a minimal promoter. Variants and modifications of the TRE sequence are possible, since the minimal promoter can be any suitable promoter. Preferably the minimal promoter shows no or minimal expression levels in the absence of rtTa binding. The inducible promoter inserted into the second GSH site may thus comprise a TRE.

[0224] A modified system based upon tetracycline control is the T-REX System (Thermo-Fisher Scientific), in which the transcriptional regulator protein is a transcriptional repressor protein, TetR. The components of this system include (i) an inducible promoter comprising a strong human cytomegalovirus immediate-early (CMV) promoter and two tetracycline operator 2 (TetO2) sites, and a Tet repressor (TetR). In the absence of tetracycline, the Tet repressor forms a homodimer that binds with extremely high affinity to each TetO2 sequence in the inducible promoter, and prevent transcription from the promoter. Once added, tetracycline binds with high affinity to each Tet repressor homodimer rendering it unable to bind to the Tet operator. The Tet repressor: tetracycline complex then dissociates from the Tet operator and allows induction of expression. In this instance, the transcriptional regulator protein is TetR and the inducible promoter comprises two TetO2 sites. The exogenously supplied substance is tetracycline or a derivative thereof.

[0225] The invention further relates to a codon-optimised tetR (OPTtetR). This may be used in any method described herein, or for any additional use where inducible promotion is desirable. This entity was generated using multiparameter-optimisation of the bacterial tetR cDNA sequence. OPTtetR allows a ten-fold increase in the tetR expression when compared to the standard sequence (STDtetR). Homozygous OPTtetR expression of tetR was sufficient to prevent shRNA leakiness whilst preserving knockdown induction in the Examples. The sequence for OPTtetR is included here, with the standard sequence shown as a comparison. Sequences with at least 75%, 80%, 85% or 90% homology for this sequence are hereby claimed, more particularly 91, 92, 93, 94, 95, 96, 97 or 99% homology. Residues shown to be changed between STDtetR and OPTtetR have been indicated in the sequences, and it is preferred that these residues are not changed in any derivative of OPTtetR since these are thought to beBIT-C-P3888PCT

[0226] 35

[0227] important for the improved properties. Any derivative would optionally retain these modifications at the indicated positions.

[0228] Other inducible expression systems are known and can be used in the method of the invention. These include the Complete Control Inducible system from Agilent Technologies. This is based upon the insect hormone ecdysone or its analogue ponasterone A (ponA) which can activate transcription in mammalian cells which are transfected with both the gene for the Drosophila melanogaster ecdysone receptor (EcR) and an inducible promoter comprising a binding site for the ecdysone receptor. The EcR is a member of the retinoid-X-receptor (RXR) family of nuclear receptors. In humans, EcR forms a heterodimer with RXR that binds to the ecdysoneresponsive element (EcRE). In the absence of PonA, transcription is repressed by the heterodimer.

[0229] Thus, the transcriptional regulator protein can be a repressor protein, such as an ecdysone receptor or a derivative thereof. Examples of the latter include the VgEcR synthetic receptor from Agilent technologies which is a fusion of EcR, the DNA binding domain of the glucocorticoid receptor and the transcriptional activation domain of Herpes Simplex Virus VP16. The inducible promoter comprises the EcRE sequence or modified versions thereof together with a minimal promoter. Modified versions include the E / GRE recognition sequence of Agilent Technologies, in which mutations to the sequence have been made. The E / GRE recognition sequence comprises inverted half-site recognition elements for the retinoid-X-receptor (RXR) and GR binding domains. In all permutations, the exogenously supplied substance is ponasterone A, which removes the repressive effect of EcR or derivatives thereof on the inducible promoter, and allows transcription to take place.

[0230] Alternatively, inducible systems may be based on the synthetic steroid mifepristone as the exogenously supplied substance. In this scenario, a hybrid transcriptional regulator protein is inserted, which is based upon a DNA binding domain from the yeast GAL4 protein, a truncated ligand binding domain (LBD) from the human progesterone receptor and an activation domain (AD) from the human NF-KB. This hybrid transcriptional regulator protein is available from Thermo-Fisher Scientific (Gene Switch™). Mifepristone activates the hybrid protein, and permits transcription from the inducible promoter which comprises GAL4 upstream activating sequences (UAS) and the adenovirus E1b TATA box. This system is described in Wang etal. (1994) PNAS 91: 8180-8184.BIT-C-P3888PCT

[0231] 36

[0232] The transcriptional regulator protein can thus be any suitable regulator protein, either an activator or repressor protein. Suitable transcriptional activator proteins are tetracyclineresponsive transcriptional activator protein or the Gene Switch hybrid transcriptional regulator protein. Suitable repressor proteins include the Tet-Off version of rtTA, TetR or EcR. The transcriptional regulator proteins may be modified or derivatised as required.

[0233] The inducible promoter can comprise elements which are suitable for binding or interacting with the transcriptional regulator protein. The interaction of the transcriptional regulator protein with the inducible promoter is preferably controlled by the exogenously supplied substance, light or temperature change.

[0234] The exogenously supplied substance can be any suitable substance that binds to or interacts with the transcriptional regulator protein. Suitable substances include tetracycline (or derivatives thereof, such as doxycycline), ponasterone A and mifepristone.

[0235] Alternatively, the cumate system may be used as the transcriptional regulator system. The cumate system is derived from the regulatory mechanisms of bacterial operons (cmt and cym) to regulate gene expression. Regulation is mediated by the binding of the repressor (CymR) to the operator site (CuO). Addition of cumate, a small molecule, removes the CymR repressor from the CuO operator site, thus allowing the expression of genetic material to take place.

[0236] Alternatively, the transcriptional regulator system may be based on the hepatitis C virus (HCV) NS3 protease domain. NS3 is a serine cis-protease that excises itself from the HCV polyprotein by cleaving recognition sites that flank it at either end. Because it is essential for HCV replication, numerous inhibitors targeting the viral protease have been developed, such as danoprevir and grazoprevir. The protease has been used as a ligand-inducible connection to control the association between modular DNA-binding and transcriptional activation domains. In one embodiment, the protease is inserted between minimal DNA-binding and transcriptional activator sequences. In this configuration, the viral protease would serve as a self-immolating connection, excising itself from the fusion construct and, in doing so, separating the DNA-binding and transcriptional activator elements. However, in the presence of an NS3 inhibitor, self-excision of the protease would be blocked, resulting in the preservation of full-length gene capable of activating the expression of targeted genes. These systems may be developed so that one NS3 protease regulates transcription, but may also involve the use of more than one NS3 protease, such as NS3 variants binding danoprevir and / or grazoprevir.BIT-C-P3888PCT

[0237] 37

[0238] As discussed above, the transcriptional regulator protein may be activated or deactivated by light. Such proteins are described in, for example, W02023 / 004031 (incorporated herein by reference). The protein may comprise a light-activatable domain that responds to light of a particular wavelength. In some cases, the light-activatable domain, upon stimulation with light of a particular wavelength or within a particular spectral range, dimerizes or oligomerizes (e.g., with another light activatable domain). In some cases, the light-activatable domain may form a homodimer or a heterodimer (e.g., may dimerize with a second, different light-activatable domain). In some cases, the light-activatable domain may exist in a (e.g., homo or hetero) dimer or (e.g., homo or hetero) oligomer (e.g., in the absence of light), and may dissociate into a monomeric form after exposure to light. The light-activatable domain may be derived from a natural source (e.g., a naturally occurring protein) or may be synthetically produced. The light-activatable domain may comprise or may be a functional domain or portion of a naturally occurring protein, such as, by way of example only, the PHR domain of Arabidopsis cryptochrome 2. The light-activatable domain may comprise an amino acid sequence identical to an amino acid sequence of a wildtype protein, or may comprise one or more variants (e.g., amino acid substitutions, deletions, insertions, etc.) relative to a wild-type protein. This domain may activate or deactivate the transcriptional regulator protein.

[0239] In various aspects, a combination of light-activatable domains (e.g., a first light activatable domain and a second light-activatable domain) may be used. In this scenario, the first light-activatable domain and the second light-activatable domain are binding partners, such that upon illumination with light at a particular wavelength or within a particular spectral range, the first and second light-activatable domains heterodimerize or heterooligomerize. This heterodimerization or heterooligomerization may activate or deactivate the transcriptional regulator protein.

[0240] In various aspects, the light-activatable domain comprises a Light-Oxygen-Voltage (LOV) photoreceptor domain, a LOV2 photoreceptor domain, a Cryptochrome (CRY) domain, Blue-light-using FAD (BLLIF) photoreceptor domain, a Phytochrome (PHY) domain, a CIBN (N-terminal domain of CIB1 (cryptochrome-interacting basic-helix-loop-helix protein 1)) domain, a PIF (phytochrome interacting factor) domain, a Dronpa domain, a LIVR8 photoreceptor domain, a COP1 domain, a BphP1 domain, a QPAS-1 domain, a cobalamin binding domain (CBD), ora combination thereof. In one example, the light-activatable domain is a LOV domain (e.g., such as a LOV domain derived from Vaucheria frigida Aureochrome 1).BIT-C-P3888PCT

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[0242] In some instances, a combination of light-activatable domains is used, wherein the first light-activatable domain is cryptochrome 2 (or a variant or a functional portion thereof) and the second-light activatable domain is CIBN (or a variant or a functional portion thereof). In some instances, a combination of light-activatable domains is used, wherein the first light-activatable domain is BphP1 (or a variant or a functional portion thereof) and the second-light activatable domain is QPAS1 (or a variant or a functional portion thereof).

[0243] The light-activatable domain may be fused with a domain that, upon activation, interacts with an inducible promoter to allow expression of the genetic material to take place. Alternatively the light-activatable domain may be fused with a domain that, upon activation, interacts with a recombinase that removes a blocker sequence, allowing expression of the genetic material to take place. The advantage of the combination with a recombinase is that only a short burst of light is necessary in order to switch on expression, rather than constant light exposure.

[0244] Alternatively the transcriptional regulator protein may be activated or deactivated by a change in temperature. In this scenario, the cells may be exposed to a period of temperature increase or temperature decrease in order to either activate or deactivate the transcriptional regulator protein. The extent of the temperature change and the length of time the cells are exposed to the temperature increase / decrease may be optimised so that cell viability is not detrimentally affected. It is known in the art that some heat shock proteins are expressed only when a temperature increase and / or decrease takes place, and this mechanism could be utilised in a transcriptional regulator protein.

[0245] It is preferred that the gene encoding the transcriptional regulator protein is operably linked to a constitutive promoter. Alternatively, the insertion site can be selected such that it already has a constitutive promoter that can also drive expression of the transcriptional regulator protein gene and any associated genetic material. Constitutive promoters ensure sustained and high-level gene expression. Commonly used constitutive promoters, including the human p-actin promoter (ACTB), cytomegalovirus (CMV), elongation factor-1a, (EF1a), phosphoglycerate kinase (PGK) and ubiquitin C (UbC). The CAG promoter is a strong synthetic promoter frequently used to drive high levels of gene expression and was constructed from the following sequences: (C) the cytomegalovirus (CMV) early enhancer element, (A) the promoter, the first exon and the first intron of chicken beta-actin gene, and (G) the splice acceptor of the rabbit beta-globin gene.BIT-C-P3888PCT

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[0247] According to a further aspect of the invention, there is provided a method for the production of glutamatergic neurons from a source cell, preferably a human source cell, comprising the steps of:

[0248] a) insertion (preferably targeted insertion) of a gene encoding a transcriptional regulator protein into the source cell; and

[0249] b) insertion (preferably targeted insertion) of at least one nucleotide sequence encoding one or more polypeptides having the activity of two or more transcription factors and / or two or more transcription factors, the transcription factors comprising the combination of NGN2 and one or more transcription factors selected from the group consisting of: TBR1, NFIA, BCL11B, MEF2C, ZBTB18, POU4F2, POU4F1, FOXP1, NR4A2, TLE4, PAX6, FOXG1, FEZF2, EMX1, and variants thereof, operably linked to an inducible promoter into the source cell, wherein said inducible promoter is regulated by the transcriptional regulator protein; and

[0250] c) culturing the source cell(s) comprising the insertions to obtain glutamatergic neurons.

[0251] It will be understood that these aspects of the invention may be used with any of the combinations of transcription factors described herein.

[0252] Obtaining glutamatergic neurons

[0253] In one embodiment, the method additionally comprises monitoring the cell population for at least one characteristic of a glutamatergic neuron. Cells may be monitored throughout culturing to identify expression of key lineage markers.

[0254] For example, monitoring may be through the use of engineered ‘reporter’ cell lines (i.e. endogenously tagged proteins or positive selection markers under the control of glutamatergic neurons specific promoters) or immunostaining and detected, using fluorescence microscopy or flow cytometry. Such material includes genes for markers or reporter molecules, such as genes that induce visually identifiable characteristics including fluorescent and luminescent proteins. Examples include the gene that encodes jellyfish green fluorescent protein (GFP), which causes cells that express it to glow green under blue / UV light, luciferase, which catalyses a reaction with luciferin to produce light, and the red fluorescent protein from the gene dsRed.

[0255] The cell may further comprise a positive selection marker and / or selectable reporter expression cassette, e.g., comprising a promoter specific to a glutamatergic neuron operably linked to a reporter gene.BIT-C-P3888PCT

[0256] 40

[0257] Selectable markers may include resistance genes to antibiotics or other drugs. Examples of drug resistance genes may include: a puromycin resistance gene, an ampicillin resistance gene, a neomycin resistance gene, a tetracycline resistance gene, a kanamycin resistance gene or a chloramphenicol resistance gene. Cells can be cultured on a medium containing the appropriate drug (i.e. , a selection medium) and only those cells which incorporate and express the drug resistance gene will survive. Therefore, by culturing cells using a selection medium, it is possible to select for cells comprising and expressing a drug resistance gene, positively enriching for a target cell population.

[0258] Examples of fluorescent protein genes which may be used as markers include: a green fluorescent protein (GFP) gene, yellow fluorescent protein (YFP) gene, red fluorescent protein (RFP) gene or aequorin gene. Cells expressing the fluorescent protein can be detected using a fluorescence microscope and fluorescence activated cell sorting (FACS) used to identify and select cell populations based on the expression of fluorescent proteins.

[0259] Fluorescent protein genes may be tagged with a nuclear localization signal peptide to confine expression of the fluorescent proteins to the nucleus. This may be helpful in cell types with a high lipid content which may not be suitable for FACS. This allows end-point fluorescence-activated cell sorting to be carried out on either whole cell populations, or purified nuclei which maintain an intact fluorescent signal.

[0260] Examples of chromogenic enzyme genes which may be used as markers, and known in the art, include but are not limited to: p-galactosidase gene, p-glucuronidase gene, alkaline phosphatase gene, or secreted alkaline phosphatase SEAP gene. Cells expressing these chromogenic enzyme genes can be detected by applying the appropriate chromogenic substrate (e.g., X-gal for p galactosidase) so that cells expressing the marker gene will produce a detectable colour (e.g., blue in a blue-white screen test).

[0261] The method may therefore comprise a selection or enrichment step for glutamatergic neurons provided from the methods described herein. In one embodiment, the method comprises the step of sorting the glutamatergic neurons using fluorescence activated cell sorting (FACS) or immunomagnetic sorting methods based on the expression of markers specific for glutamatergic neurons and / or absence of cell markers not specific for glutamatergic neurons. A labelled binding agent directed to target cell surface proteins may be used. Any binding agent capable of specific binding to a particular epitope may be used for this purpose, for example an antibody or a fragment thereof, a peptide or a synthetic binder such as a plastic antibody,BIT-C-P3888PCT

[0262] 41

[0263] or an aptamer or oligonucleotide, capable of specific binding to an epitope. The binding agent may be labelled with a detectable marker, such as a luminescent, fluorescent (e.g. fluorochrome), enzyme or radioactive marker; alternatively or additionally an affinity tag, e.g. a biotin, avidin, streptavidin or His (e.g. hexa-His) tag. In one embodiment, fluorochrome conjugated antibodies targeting cell surface proteins (e.g. glutamatergic neuron markers) may be used to sort target cells.

[0264] In another embodiment, glutamatergic neurons are enriched by drug-resistance selection from genetically engineered source cells expressing an antibiotic-resistance gene under the control of a promoter specific for glutamatergic neurons.

[0265] The method may generate cells (i.e. , differentiated cells) exhibiting at least one characteristic of a glutamatergic neurons. One or more characteristics may be used to select for the glutamatergic neurons generated by methods of the invention.

[0266] Characteristics include but are not limited to the detection or quantitation of expressed cell markers, enzymatic activity, and the characterization of morphological features and intercellular signaling. The biological function of a glutamatergic neuron may also be evaluated.

[0267] In one embodiment, the characteristic (i.e., of a glutamatergic neuron, in particular a human glutamatergic neuron) is selected from one or more of:

[0268] (i) expression of one or more cell markers, such as a glutamate transporter protein, VGLLIT1 (or the associated gene SLC17A7), VGLLIT2 (or the associated gene SLC17A6), POU3F2, SATB2, BCL11B, PCP4, FOXP2, FEZF2, TSHZ3, TLE4, TBR1, SV2A, SV2B, or a combination thereof;

[0269] (ii) glutamatergic neuron morphological features; and

[0270] (iii) show spontaneous electrophysiological properties that can be inhibited pharmacologically by known inhibitors of glutamatergic neurotransmission.

[0271] The detection of the one or more markers may be unrelated to the incorporation of exogenous genes expressing one or more transcription factors for reprogramming purposes, or the overexpression of endogenous transcription factors for reprogramming purposes. For example, where an inducible system is used for the expression of the reprogramming transcription factors, preferably the markers would still be detectable after the induced expression has been stopped. In other words, it is preferable that once reprogramming has taken place the markers (that may or may not be the same as the transcription factors usedBIT-C-P3888PCT

[0272] 42

[0273] for reprogramming) are expressed as a result of the successful reprogramming and are detectable even after the forced expression of reprogramming transcription factors has stopped.

[0274] The glutamatergic neuron markers may be markers obtained by transcriptome analysis. For example, single cell RNA sequencing has been used to provide detailed transcriptional profiles of human glutamatergic neurons obtained from primary human tissues. This information can be used to identify glutamatergic neurons generated by the methods described herein. Additional resources, such as Human Cell Atlas and CellTypist may also be used to identify markers of glutamatergic neurons.

[0275] The method may comprise assaying the differentiated cells obtained by the method described herein and determining a set of transcribed genes; comparing the set of transcribed genes of the differentiated cells to one or more reference sets of transcribed genes from one or more reference glutamatergic neurons; and identifying a match between the differentiated cells and a reference glutamatergic neuron.

[0276] The glutamatergic neurons may be deep layer glutamatergic neurons or upper layer glutamatergic neurons.

[0277] Deep layer glutamatergic neurons are neurons located in layers 5 and 6 of the neocortex of the brain. They form a major source of output from the neocortex. Deep layer glutamatergic neurons (particularly human deep layer glutamatergic neurons) may be identified through the detection of one or more markers selected from the list consisting of: BCL11 B, PCP4, FOXP2, FEZF2, TSHZ3, TLE4 and TBR1. Such neurons may also express one or more general glutamatergic neuron markers such as SLC17A7, SLC17A6, SV2A or SV2B. Transcription factors particularly useful for reprogramming to deep layer glutamatergic neurons include NGN2 and one or more additional transcription factors selected from the list consisting of: NFIA, BCL11B, FOXP1, FOXG1, POU4F2, ZBTB18 and EMX1.

[0278] Upper layer glutamatergic neurons are neurons located in layers 2 and 3 of the cerebral cortex. They play a key role in information processing and communication between different cortical regions by forming long-range connections with other brain areas. They are considered crucial for higher cognitive functions due to their extensive inter-hemispheric connections. Upper layer glutamatergic neurons (particularly human upper layer glutamatergic neurons) may be identified through the detection of POU3F2 and / or SATB2. Such neurons may also expressBIT-C-P3888PCT

[0279] 43

[0280] one or more general glutamatergic neuron markers such as SLC17A7, SLC17A6, SV2A or SV2B. Transcription factors particularly useful for reprogramming to deep layer glutamatergic neurons include NGN2 and one or more additional transcription factors selected from the list consisting of: NR4A2, TLE4, PAX6, MEF2C, POU4F1 and TBR1.

[0281] Cell culturing

[0282] In one embodiment, the method includes culturing the cell population for a sufficient time and under conditions to allow differentiation to a glutamatergic neuron. Generally, cells of the present invention are cultured in a culture medium, which is a nutrient-rich buffered solution capable of sustaining cell growth.

[0283] The cell culture medium may contain any of the following in an appropriate combination: salt(s), buffer(s), amino acids, glucose or other sugar(s), antibiotics, serum or serum replacement, and other components such as peptide growth factors, etc. Cell culture media ordinarily used for particular cell types are known to those skilled in the art. For example, the media may comprise Basal Medium (e.g. DMEM / F12, Neurobasal or STEMPRO-34) supplemented with GLUTAMAX, antibiotics (such as penicillin or streptomycin), B27 supplement and / or N2 supplement (all available from Thermo Fisher Scientific). The media may then be further supplemented at different time points during the culturing process. For example, one or more peptide hormones and / or cytokines can be at 2, 4 and / or 10 days during the culturing process.

[0284] In one embodiment, the culture media comprises one or more components selected from the group consisting of: growth factors (such as neurotrophin-3 (NT-3) and brain-derived neurotrophic factor (BDNF)), small-molecules (such as DAPT) and cell culture supplements (such as dibutyryl cyclic adenosine monophosphate (dbcAMP)).

[0285] Glutamatergic neurons may be obtained using methods of the invention at least about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 days after culturing, where the cells are expressing one or more relevant polypeptides having the activity of one or more transcription factors and / or have increased the expression of one or more of the relevant transcription factors discussed herein. In one embodiment, the method comprises culturing under suitable conditions for at least 14 days, such as at least 17 days or about 21 days. In further embodiments, method comprises culturing cells for a duration (e.g., at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 21 days, at least 28 days, or longer, e.g., from 5 days to 40BIT-C-P3888PCT

[0286] 44

[0287] days, from 7 days to 35 days, from 14 days to 28 days, or about 21 days) which is sufficient to generate glutamatergic neurons.

[0288] After culturing, the cell population may comprise two cell types. For example, such a cell population may have two cell types including the stem cells and glutamatergic neurons. In one embodiment, the cell population comprises up to 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 99.5% (or any intermediate ranges) of glutamatergic neurons in the resulting cell population.

[0289] The invention may be used to prepare purer populations of glutamatergic neurons, in particular a cell population with less sensory neurons (e.g. cholinergic, dopaminergic GABAergic, serotonergic or noradrenergic neuronal cells). As described herein, the invention produced a cell population with a decrease in sensory neuronal cells compared to a cell population using NGN2 alone. Therefore, after culturing, the cell population may comprise only one ora majority cell type, such as only one neuronal cell type. For example, in one embodiment, the cell population comprises 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 99.5% (or any intermediate ranges) glutamatergic neurons in the resulting cell population.

[0290] Culturing the cells may either help to induce cells to commit to a more mature phenotype, preferentially promote survival of the mature cells, or have a combination of both these effects.

[0291] According to a further aspect of the invention, there is provided a cell obtainable by any one of the methods defined herein.

[0292] According to a further aspect of the invention, there is provided a cell, preferably a human cell, comprising one or more exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of two or more transcription factors and / or encoding two or more transcription factors, wherein the transcription factors comprise the combination of NGN2 and one or more transcription factors selected from the group consisting of: TBR1, NFIA, BCL11B, MEF2C, ZBTB18, POU4F2, POU4F1, FOXP1, NR4A2, TLE4, PAX6, FOXG1, FEZF2, EMX1 and variants thereof.

[0293] As described herein, the exogenous expression cassettes encoding the one, two or three or more polypeptides having transcription factor activity and / or transcription factors may be integrated into the genome of the cell. In a further embodiment, exogenous expression cassettes encoding the two or more polypeptides having transcription factor activity and / orBIT-C-P3888PCT

[0294] 45

[0295] transcription factors are integrated into a (specific) target site in the genome of the cell. Alternatively, exogenous expression cassettes encoding the two or more polypeptides having transcription factor activity and / or transcription factors are integrated into a non-specific target site in the genome of the cell.

[0296] Cell compositions

[0297] According to a further aspect, there is provided a pharmaceutical composition comprising the glutamatergic neurons produced by the method as described herein and a pharmaceutically acceptable carrier.

[0298] Pharmaceutical compositions may include glutamatergic neurons as described herein in combination with one or more pharmaceutically or physiologically acceptable carrier, diluents, or excipients. Such compositions may include buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminium hydroxide); and preservatives. Cryopreservation solutions which may be used in the pharmaceutical compositions of the invention include, for example, DMSO.

[0299] For purposes of manufacture, distribution, and use, the glutamatergic neurons described herein may be supplied in the form of a cell culture or suspension in an isotonic excipient or culture medium, optionally frozen to facilitate transportation or storage.

[0300] Uses of glutamatergic neurons

[0301] The cells produced according to any of the methods of the invention have applications in basic and medical research, diagnostic and therapeutic methods. The cells may be used in vitro to study cellular development, provide test systems for new drugs, enable screening methods to be developed, scrutinise therapeutic regimens, provide diagnostic tests and the like. These uses form part of the present invention. Alternatively, the cells may be transplanted into a human or animal patient for diagnostic or therapeutic purposes. The use of the cells in therapy is also included in the present invention.

[0302] According to one aspect of the invention, there is provided a glutamatergic neuron as defined herein, for use in in vitro diagnostics or drug screening.BIT-C-P3888PCT

[0303] 46

[0304] Glutamatergic neurons generated by methods of the invention may find particular use in drug screening. Therefore, in one embodiment, the method additionally comprises contacting the glutamatergic neurons with a test substance and observing a change (e.g., an effect) in the glutamatergic neurons induced by the test substance. The change or effect may be observed using methods known in the art, for example using pharmacological or toxicological assays. In one aspect, the cells may be used in a method of assessing a test substance (e.g., a drug, such as a compound), comprising assaying a pharmacological or toxicological property of the test substance on the glutamatergic neurons provided by the methods described herein. The method may comprise: a) contacting the glutamatergic neurons described herein with the test substance; and b) assaying an effect of the test substance on the glutamatergic neurons.

[0305] Assessment of the activity of a candidate molecule may involve combining the glutamatergic neurons described herein with the candidate molecule, determining any change in the morphology, phenotype, or metabolic activity of the glutamatergic neurons that is attributable to the molecule (i.e. , compared with a control, such as untreated cells or cells treated with an inert compound), and then correlating the effect of the molecule with the observed change. The screening may be done either because the candidate molecule is designed to have a pharmacological effect on glutamatergic neurons, or because the molecule is designed to have effects elsewhere but there is a need to determine if it has any unintended side effects.

[0306] Cytotoxicity can be determined in the first instance by the effect on cell viability, survival, morphology, and leakage of enzymes into the culture medium. More detailed analysis may be conducted to determine whether a test substance affects cell function without causing toxicity.

[0307] Alternatively, the cells can be used to assess changes in gene expression patterns caused by a potential drug candidate. In this embodiment, the changes in gene expression pattern from addition of the candidate drug can be compared with the gene expression pattern caused by a control drug with a known effect on glutamatergic neurons.

[0308] Therefore, according to a further aspect, there is provided a method for drug screening (e.g., evaluating drug reactivity), comprising a step of using the glutamatergic neurons produced by the method as described herein. According to a further aspect of the invention, there is provided a method of drug screening comprising contacting a glutamatergic neuron generated using the method as defined herein, or a glutamatergic neuron as defined herein, with the drug and observing a change in the glutamatergic neuron induced by the drug.BIT-C-P3888PCT

[0309] 47

[0310] According to a further aspect of the invention, there is provided the glutamatergic neuron as defined herein for use in therapy.

[0311] In one embodiment, the method additionally comprises transplanting the glutamatergic neurons into a patient. In this aspect of the invention, the cells used to generate the glutamatergic neurons may be autologous (i.e. , mature cells removed, modified and returned to the same individual) or from a donor (i.e., allogeneic, including a stem cell line). Forward programming of cells into glutamatergic neurons is amenable to the production of autologous and allogeneic glutamatergic neurons.

[0312] Therefore, according to a further aspect of the invention there is provided a method of treating a subject having or at risk of a disease or disorder comprising administering to the subject a therapeutically effective amount of glutamatergic neurons generated using the method as defined herein, or glutamatergic neurons as defined herein. For example, glutamatergic neurons may help promote arousal and recovery of consciousness in patients with traumatic brain injury.

[0313] Glutamatergic neurons of the invention may find use in the development for therapies and the treatment of disorders of glutamatergic neuron development and function (e.g. in motor neuron diseases and neurodegenerative diseases).

[0314] In a different aspect, the cells may be used in tissue engineering. Tissue engineering requires the generation of tissue which could be used to replace tissues or even whole organs of a human or animal. Methods of tissue engineering are known to those skilled in the art, but include the use of a scaffold (an extracellular matrix) upon which the cells are applied in order to generate tissues / organs. These methods can be used to generate an “artificial” tissue or organ. Methods of generating tissues may include additive manufacturing, otherwise known as three-dimensional (3D) printing, which can involve directly printing cells to make tissues. The present invention thus provides a method for generating tissues using the cells produced as described in any aspect of the invention.

[0315] Forward programming kits

[0316] According to a further aspect of the invention, there is provided a kit for differentiating a cell, preferably a human cell, into a glutamatergic neuron comprising:

[0317] (i) a source cell and an agent that activates or increases the expression or amount of at least one or more transcription factors; and / orBIT-C-P3888PCT

[0318] 48

[0319] (ii) one or more expression cassette(s) comprising nucleotide sequences encoding one or more polypeptides having the activity of two or more transcription factors and / or encoding two or more transcription factors,

[0320] wherein the transcription factors comprise the combination of NGN2 and one or more transcription factors selected from the group consisting of: TBR1, NFIA, BCL11B, MEF2C, ZBTB18, POU4F2, POU4F1, FOXP1, NR4A2, TLE4, PAX6, FOXG1, FEZF2, EMX1, and variants thereof.

[0321] In one embodiment, the expression cassette comprises an inducible expression construct comprising a sequence encoding one or more polypeptides having transcription factor activity and / or the transcription factors themselves.

[0322] As described herein, combinations of polypeptides having transcription factor activity and / or the transcription factors described herein are of particular use in the present invention. If a combination of polypeptides and / or transcription factors is required, these may be encoded on the same or on different expression cassettes. Therefore, in one embodiment, the kit comprises an expression cassette (preferably an inducible expression cassette) encoding two or more polypeptides and / or transcription factors, such as three, four, five, six, seven or eight transcription factors. Preferably, the kit comprises an expression cassette encoding three or more, more preferably four or more, polypeptides and / or transcription factors.

[0323] According to a further aspect, there is provided a use of a kit as defined herein, for differentiating a cell into a glutamatergic neuron.

[0324] The kit may include one or more articles and / or reagents for performance of the method. For example, one or more transcription factor genes, derivatives, variants or fragments thereof, for use in the methods described herein may be provided in isolated form and may be part of a kit, e.g., in a suitable container such as a vial in which the contents are protected from the external environment.

[0325] In one embodiment, the kit additionally comprises at least one source cell, such as a pluripotent stem cell (such as an induced pluripotent stem cell) or a non-pluripotent cell that is not a glutamatergic neuron.BIT-C-P3888PCT

[0326] 49

[0327] In one embodiment, the kit additionally comprises a medium for culturing the cell and instructions for preparing the enhanced potency cells or reprogrammed pluripotent cells in accordance with the method defined herein.

[0328] It will be understood that all embodiments described herein may be applied to all aspects of the invention.

[0329] Other features and advantages of the present invention will be apparent from the description provided herein. It should be understood, however, that the description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications will become apparent to those skilled in the art. The invention will now be described using the following, non-limiting examples:

[0330] EXAMPLES

[0331] Methods

[0332] Plasmid library encoding the transcription factors and dual-harbour mediated delivery Transcription factors (TFs) and Luciferase (FLuc) controls were cloned separately into vectors for genomic integration mediated by CRISPR-CAS9, targeting the mutated allele in AAVS1. TF expression was controlled by a promoter activated by a reverse tetracycline-controlled transactivator (rtTA). The gene for the rtTA was inserted into the hROSA locus and was operably linked to a constitutive promoter. Vectors encoded a puromycin-resistance cassette for the selection of cells with successful genomic integration. Each TF expression cassette encodes a unique DNA barcode. We designed primers for the concurrent amplification of TF and cell barcodes in the 10x Genomics workflow, allowing us to assign TF combinations to single-cell transcriptome profiles.

[0333] Vectors were pooled in equimolar ratios to ensure even representation of TFs followed by retransformation in bacteria and large-scale DNA preparation. A mix of TF library and CAS9 was nucleofected into iPSCs. Cell culture scale and nucleofection parameters were optimised to ensure an adequate coverage of each TF.

[0334] Screening outline

[0335] Three replicates of a screening experiment were performed using the same pooled plasmid library. Prior to nucleofection, iPSCs were expanded in GIBCO ESSENTIAL 8 Medium on standard tissue culture plates coated with Vitronectin (Life Technologies). FollowingBIT-C-P3888PCT

[0336] 50

[0337] nucleofection, cells were cultured as above and selected for successful genomic integration by adding puromycin to the culture media. Following selection, iPSCs were plated for reprogramming and cultured for up to 14 days in media promoting maintenance and survival of glutamatergic neurons, in presence of doxycycline. At all stages, enough cells were maintained in culture to ensure adequate coverage for each combination of TFs.

[0338] After reprogramming, cells were harvested and analysed by scRNA-seq using 10x Genomics.

[0339] Single-cell transcriptome analysis and TF barcode capture

[0340] Non-sorted cells were purified and analysed by scRNA-seq using the 10x Genomics Chromium Single Cell 3’ (v3) kit following manufacturer’s instructions for 10,000 cells per replicate. Libraries were then sequenced to obtain data for -10,000 cells per replicate. After the cDNA amplification step, gene expression libraries were created and sequenced on a NovaSeq aiming for at least 50,000 reads per cell, as per the Chromium Single Cell 3’ v3 protocol. In addition to the gene expression libraries, the matched cDNA was used as a template for further targeted amplification of the TF barcodes, which were sequenced on a NovaSeq.

[0341] Data Analysis

[0342] Single-cell gene expression data was processed using the 10x Genomics pipeline and TF barcode reads processed through a custom workflow. 10x cell barcodes were used to assign exogenous transcription factor (eTF) barcodes to single cells and their corresponding marker gene expression profiles. The data were further analysed in Seurat v4 (Stuart et al., Cell, 2019) and visualised on uniform manifold approximation and projection (LIMAP) plots (Becht, Nature Biotech, 2019). Then, for each eTF the expression profile for different neuronal subtypes was determined, i.e. glutamatergic, cholinergic, sensory, GABAergic, dopaminergic, serotonergic and noradrenergic, and for cortical layers, i.e deep and upper layer. A gene signature for each neuronal subtype cortical layer was created based on existing knowledge in literature (Table 1). Then, for each eTF, the total mean expression for all genes in a given signature was determined. Gene signature ratios for neuronal subtypes and cortical layers were determined by dividing the mean expression value for each signature by the sum of mean expression values for all subtypes or layers, respectively.

[0343] Table 1: Marker genes associated with neuronal subtype

[0344]

[0345] BIT-C-P3888PCT

[0346] 51

[0347]

[0348] Results

[0349] As shown in Figure 1, forward programming iPSCs with NGN2 alone (“Flue”) led to glutamatergic neuronal cells, but also a noticeable amount of undesirable sensory neuronal cells. The addition of any one of ZBTB18, TBR1, TLE4, NFIA, BCL11B, FOXP1, NR4A2, EMX1, MEF2C, POLI4F2, POLI4F1 or PAX6 led to either an increase in glutamatergic neuronal cells or a decrease in sensory neuronal cells. The amount of cholinergic, dopaminergicBIT-C-P3888PCT

[0350] 52

[0351] GABAergic, serotonergic or noradrenergic forward programming was minimal across all TF combinations.

[0352] Figure 2 shows that NFIA, BCL11B, FOXP1, FOXG1, POU4F2, ZBTB18 and EMX1 (when combined with NGN2) improve forward programming to deep layer glutamatergic neurons specifically and NR4A2, TLE4, PAX6, MEF2C, POLI4F1 and TBR1 (when combined with NGN2) improve forward programming to upper layer glutamatergic neurons specifically when compared with NGN2 alone (Flue).

[0353] Figure 3 shows thatTBRI, POU4F1, NR4A2, FOXP1. TLE4, PAX6, ZBTB18, BCL11B, NFIA and MEF2C (when combined with NGN2) all increase the expression of glutamatergic neuron layers in general (both deep layer and upper layer combined) compared with NGN2 alone (FLuc). All TFs apart from PAX6 lead to an improvement in deep layer glutamatergic neuron expression (when combined with NGN2), and all TFs apart from FOXP1 and NFIA lead to an improvement in upper layer glutamatergic neuron expression (when combined with NGN2).

Claims

BIT-C-P3888PCT53CLAIMS1. A method of generating glutamatergic neurons comprising expressing one or more polypeptides having the activity of two or more transcription factors and / or increasing the expression of two or more transcription factors, the transcription factors comprising NGN2 and one or more transcription factors selected from the group consisting of: TBR1, NFIA, BCL11B, MEF2C, ZBTB18, POU4F2, POU4F1, FOXP1, NR4A2, TLE4, PAX6, FOXG1, FEZF2, EMX1 and variants thereof in a cell population and culturing the cell population to obtain glutamatergic neurons.

2. The method of claim 1, wherein the transcription factors comprise the combination of NGN2 and two or more additional transcription factors selected from the list consisting of: TBR1, NFIA, BCL11B, MEF2C, ZBTB18, POU4F2, POU4F1, FOXP1, NR4A2, TLE4, PAX6, FOXG1, FEZF2 and EMX1.

3. The method of claim 1 or claim 2, wherein the transcription factors comprise the combination of NGN2 and one or more additional transcription factors selected from the list consisting of: TBR1, NFIA, BCL11B, MEF2C, ZBTB18, POU4F2, POU4F1, FOXP1, NR4A2, TLE4, PAX6 and FOXG1.

4. The method of claim 1 or claim 2, wherein the transcription factors comprise the combination of NGN2 and one or more additional transcription factors selected from the list consisting of: TBR1, NFIA, BCL11B, ZBTB18, FOXP1, NR4A2, TLE4, FOXG1, FEZF2 and EMX1.

5. The method of claim 1 or claim 2, wherein the transcription factors comprise the combination of NGN2 and one or more additional transcription factors selected from the list consisting of: TBR1, NFIA, BCL11B, MEF2C, ZBTB18, POU4F2, POU4F1, FOXP1, NR4A2, TLE4, PAX6 and FEZF2.

6. The method of claim 5, wherein the transcription factors comprise the combination of NGN2 and one or more additional transcription factors selected from the list consisting of: TBR1, NFIA, BCL11B, ZBTB18, FOXP1, NR4A2, TLE4 and FEZF2.BIT-C-P3888PCT547. The method of claim 5, wherein the transcription factors comprise the combination of NGN2 and one or more additional transcription factors selected from the list consisting of: TBR1, NFIA, POU4F2 and POU4F1.

8. The method of claim 7, wherein the transcription factors comprise the combination of NGN2 and one or more additional transcription factors selected from the list consisting of: NFIA, POU4F2 and POU4F1.

9. The method of claim 7, wherein the transcription factors comprise the combination of NGN2 and one or more additional transcription factors selected from the list consisting of: TBR1 and NFIA.

10. The method of claim 4, wherein the transcription factors comprise one of the following combinations:(i) NGN2, TBR1 and EMX1;(ii) NGN2, TBR1 and FOXG1;(iii) NGN2, BCL11 B and EMX1 ;(iv) NGN2, BCL11 B and FOXG1 ;(v) NGN2, FEZF2 and EMX1; or(vi) NGN2, FEZF2 and FOXG1.

11. The method of any one of claims 1 to 10, wherein the glutamatergic neurons are deep layer glutamatergic neurons or upper layer glutamatergic neurons.

12. The method of claim 11, wherein, where the glutamatergic neurons are deep layer glutamatergic neurons, the transcription factors comprise the combination of NGN2 and one or more additional transcription factors selected from the list consisting of: NFIA, BCL11B, FOXP1, FOXG1, POU4F2, ZBTB18 and EMX1.

13. The method of claim 11, wherein, where the glutamatergic neurons are upper layer glutamatergic neurons, the transcription factors comprise the combination of NGN2 and one or more additional transcription factors selected from the list consisting of: NR4A2, TLE4, PAX6, MEF2C, POU4F1 and TBR1.

14. The method of any one of claims 1 to 13, wherein the cell population comprises pluripotent stem cells, in particular induced pluripotent stem cells.BIT-C-P3888PCT5515. The method of any one of claims 1 to 14, wherein the method comprises generating glutamatergic neurons by forward programming of pluripotent stem cells or induced pluripotent stem cells, preferably human induced pluripotent stem cells.

16. The method of any one of claims 1 to 15, wherein the glutamatergic neurons are human glutamatergic neurons.

17. The method of any one of claims 1 to 16, which additionally comprises monitoring the cell population for at least one characteristic of a glutamatergic neuron.

18. The method of claim 17, wherein the characteristic is selected from one or more of:(i) endogenous expression of one or more cell markers, such as a glutamate transporter protein, VGLUT1, VGLUT2, POU3F2, SATB2, BCL11B, PCP4, FOXP2, FEZF2, TSHZ3, TLE4, TBR1, and synaptic vesicle proteins, SV2A and SV2B or a combination thereof;(ii) glutamatergic neuron morphological features; and(iii) show spontaneous electrophysiological properties that can be inhibited pharmacologically by known inhibitors of glutamatergic neurotransmission.

19. The method of any one of claims 1 to 18, wherein the expression of the transcription factors is increased by contacting the cell population with one or more exogenous expression cassettes encoding one or more of the genes, or one or more agents that activate or increase the expression or amount of the transcription factors.

20. The method of claim 17, wherein the expression of the transcription factors is increased by contacting the cell population with a single exogenous expression cassette encoding genes for all of the transcription factors.

21. The method of claim 19 or claim 20, wherein expression of the genes is under controlled transcription.

22. The method of any one of claims 1 to 21, wherein a sequence encoding one or more of the transcription factors is introduced into the cell population using a method comprising:BIT-C-P3888PCT56- insertion, preferably targeted insertion, of a coding sequence for a transcriptional regulator protein into a first genomic safe harbour site of a source cell present in the cell population; and- insertion, preferably targeted insertion, of an inducible cassette into a second genomic safe harbour site of the source cell, wherein said inducible cassette comprises said sequence encoding one or more transcription factors operably linked to an inducible promoter, and said promoter is regulated by the transcriptional regulator protein.

23. The method of any one of claims 1 to 22, which comprises culturing under suitable conditions for at least 14 days, such as at least 17 days, in particular about 21 days.

24. A method for the production of glutamatergic neurons from a source cell, preferably a human cell, comprising the steps of:a) insertion, preferably targeted insertion, of a gene encoding a transcriptional regulator protein into a first genomic safe harbour site of the source cell; andb) insertion, preferably targeted insertion, of at least one nucleotide sequence encoding one or more polypeptides having the activity of two or more transcription factors and / or encoding two or more transcription factors, the transcription factors comprising NGN2 and one or more transcription factors selected from the group consisting of: TBR1, NFIA, BCL11B, MEF2C, ZBTB18, POU4F1, POU4F2, FOXP1, NR4A2, TLE4, PAX6, FOXG1, FEZF2, EMX1 and variants thereof, operably linked to an inducible promoter into a second genomic safe harbour site of the source cell, wherein said inducible promoter is regulated by the transcriptional regulator protein; andc) culturing the source cell(s) comprising the insertions to obtain glutamatergic neurons.

25. A composition comprising one or more vectors, preferably one vector, encoding polypeptides having the activity of two or more transcription factors, wherein the transcription factors comprise a combination of NGN2 and one or more transcription factors selected from the group consisting of: TBR1, NFIA, BCL11B, MEF2C, ZBTB18, POU4F1, POU4F2, FOXP1, NR4A2, TLE4, PAX6, FOXG1, FEZF2 and EMX1.

26. The composition of claim 25, comprising a vector encoding NGN2, and a vector encoding one or more transcription factors selected from the group consisting of: TBR1, NFIA,BIT-C-P3888PCT57BCL11B, MEF2C, ZBTB18, P0U4F1, POU4F2, F0XP1, NR4A2, TLE4, PAX6, F0XG1, FEZF2 and EMX1.

27. Use of one or more polypeptides having the activity of two or more transcription factors and / or two or more transcription factors, wherein the transcription factors comprise the combination of NGN2 and one or more transcription factors selected from the group consisting of: TBR1, NFIA, BCL11B, MEF2C, ZBTB18, POU4F1, POU4F2, FOXP1, NR4A2, TLE4, PAX6, FOXG1, FEZF2, EMX1 and variants thereof, to generate glutamatergic neurons, preferably human glutamatergic neurons.

28. A cell obtainable by any one of the methods defined in claims 1 to 24.

29. A cell, preferably a human cell, comprising one or more, preferably one, exogenous expression cassettes comprising nucleotide sequences encoding one or more polypeptides having the activity of two or more transcription factors and / or encoding two or more transcription factors, wherein the transcription factors comprise the combination of NGN2 and one or more transcription factors selected from the group consisting of: TBR1, NFIA, BCL11B, MEF2C, ZBTB18, POU4F1, POU4F2, FOXP1, NR4A2, TLE4, PAX6, FOXG1, FEZF2, EMX1 and variants thereof.

30. The cell of claim 28 or claim 29, wherein the nucleotide sequences encoding the one or more transcription factors are integrated into the genome of the cell.

31. The cell of claim 30, wherein the nucleotide sequences encoding the one or more transcription factors are integrated into a target site of the cell.

32. A cell of any one of claims 28 to 31 , for use in therapy.

33. A cell of any one of claims 28 to 31 , for use in in vitro diagnostics or drug screening.

34. A kit for differentiating a cell, preferably a human cell, into a glutamatergic neuron comprising:(i) a source cell and an agent that activates or increases the expression or amount of at least one or more transcription factors; and / orBIT-C-P3888PCT58(ii) one or more expression cassette(s) comprising nucleotide sequences encoding one or more polypeptides having the activity of two or more transcription factors and / or encoding two or more transcription factors,wherein the transcription factors comprise the combination of NGN2 and one or more transcription factors selected from the group consisting of: TBR1, NFIA, BCL11B, MEF2C, ZBTB18, POU4F1, POU4F2, FOXP1, NR4A2, TLE4, PAX6, FOXG1, FEZF2, EMX1 and variants thereof.

35. Use of a kit as defined in claim 34, for differentiating a cell, preferably a pluripotent stem cell, more preferably an induced pluripotent stem cell, into a glutamatergic neuron.

36. A method of drug screening comprising contacting a glutamatergic neuron generated using the method as defined in any one of claims 1 to 24, or a cell as defined in any one of claims 28 to 31, with a drug and observing a change in the glutamatergic neuron induced by the drug.

37. A method of treating a subject having or at risk of a disease or disorder comprising administering to the subject a therapeutically effective amount of glutamatergic neurons generated using the method as defined in any one of claims 1 to 24, or cells as defined in any one of claims 28 to 31.

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