A human ipsc-derived model for tauopathies

Abstract

The present invention relates to a human ectoderm derived brain cell characterized by the following features: (1) the intron between exons 10 and 11 of the MAPT gene encoding the Tau protein has been removed by genome-editing from one or both allele(s); and (2) at least one of the alleles of the MAPT gene of (1) carries at least one mutation enhancing Tau aggregation and at least one mutation enhancing nucleation of Tau aggregation; or (3) one allele of the MAPT gene as defined in claim 1(1) carries at least one mutation enhancing Tau aggregation, preferably in exon 10, and the other allele carries at least one mutation enhancing nucleation of Tau aggregation, preferably in exon 11.

Description

[0001] A human iPSC-derived model for tauopathies

[0002] The present invention relates to a human ectoderm derived brain cell characterized by the following features:

[0003] (1) the intron between exons 10 and 11 of the MAPT gene encoding the Tau protein has been removed by genome-editing from one or both allele(s); and

[0004] (2) at least one of the alleles of the MAPT gene of (1) carries at least one mutation enhancing Tau aggregation and at least one mutation enhancing nucleation of Tau aggregation; or

[0005] (3) one allele of the MAPT gene of (1) carries at least one mutation enhancing Tau aggregation, preferably in exon 10, and the other allele carries at least one mutation enhancing nucleation of Tau aggregation, preferably in exon 11.

[0006] In this specification a number of documents are cited. The disclosure of these documents, while not considered relevant for the patentability of this invention is herewith incorporated by reference in its entirety. More specifically, all reference documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

[0007] Tauopathies are a group of neurodegenerative dementias that include Alzheimer’s disease (AD) and Frontotemporal dementia (FTD) and affect over 50 million patients worldwide. Currently there are no causal treatments targeting Tauopathies available. Tauopathies are characterized by deposition of Tau protein in neurofibrillary tangles in brain neurons and massive cortical neurodegeneration.

[0008] Tau is regarded as an ‘executor’ of cell death, making it a central, unifying disease factor and an attractive drug target. Tau also contributes to disease progression by generating small seeds that may spread pathology between neurons in a prion-like manner (Wang et al., 2016). Tau mainly exists in the axons of neurons under physiological conditions and interacts via its microtubule binding domains with tubulin, promoting assembly and stability of microtubules (Zhang et al., 2022).

[0009] Tau is encoded by the microtubule associated protein Tau (MAPT) gene, located on chromosome 17q21 .3, containing 16 exons and exists in different isoforms due to alternative splicing. Mutations in MAPT lead to the formation of insoluble Tau aggregates due to misfolding of Tau. As of 2018, over 50 different pathogenic MAPT missense, silent, and intronic mutations have been reported (Wang et al., 2016). Those mutations vary in their phenotypes and neuropathology and involvements of mechanisms by which they cause disease, such as microtubule binding and assembly (loss of function), changes in alternative spicing, shifts in protein aggregation kinetics, and prion-like “seeding”. Mutations in MAPT cause FTD, corroborating the central role of Tau in neurodegenerative diseases. Understanding the molecular pathways triggering Tau seeding, aggregation, and neuronal death and identifying modulators of disease are central goals of dementia research. Tau seeding has been described as a mechanism to spread tauopathy in the brain, and may be an early, potentially druggable disease factor.

[0010] Pathological Tau seeds are considered to elicit aggregation of formerly unaffected Tau though conformational templating. Tau aggregation is initiated through formation of Tau oligomers (nucleation) followed by an elongation of these oligomers and Tau aggregation into filaments, which accumulate in pathological structures such as neurofibrillary tangles, neuropil threads, Pick bodies and other structures characteristic of neurodegenerative tauopathies (Combs and Gamblin, 2012

[0011] Due to the inaccessibility of the human brain, experimental tauopathy research has mostly been performed in animal and simple cellular models, which have drawbacks limiting research on disease pathways and drug development. All available tauopathy mouse models showing late-stage disease phenotypes, such as tangle formation and neurodegeneration, are based on overexpression of mutant human Tau. This non-physiological modelling leads to phenotypic artefacts (Joel et al., 2019; Gamache et al., 2019) and limits studies on physiological Tau interactors and modulators. Furthermore, placing human Tau in a ‘foreign’ mouse environment hampers identification of central disease-contributing factors, as those may be absent or differentially regulated. Expression of different Tau isoforms is a critical factor in human tauopathies (Wang et al., 2016). Mouse and human Tau expression differs significantly, especially in splicing of the disease-relevant 3R and 4R isoforms. To alter expression of 4R Tau, neurons regulate alternative splicing of exon 10 (Wang et al., 2016), which encodes the fourth microtubule binding repeat. Most regulatory sequences required for splicing lie in the intron after exon 10. While the adult human brain expresses these isoforms at a 1 :1 ratio, adult mice only express 4R. Increased 4R Tau expression in humans results in FTD - indicating fundamental differences in Tau biology between mice and men.

[0012] These limitations contribute to the lack of translatability of findings in tauopathy models into clinical trials, thus hampering successful drug development.

[0013] To overcome limitations of mouse models, researchers have developed human in vitro models based on induced pluripotent stem cells (iPSCs) from tauopathy patients (Kuhn et al. 2021). iPSC technology allows studying human cortical neurons, the cell type most affected in patients, but also other diseaserelevant brain cells, including astrocytes, oligodendrocytes, and microglia. Inducing disease-relevant phenotypes in iPSC models would allow mechanistic studies and translational drug screening in a more physiological system. However, currently available iPSC tauopathy models show only mild phenotypes, such as increased Tau hyperphosphorylation, but no tangle formation or major neurodegeneration (Kuhn et al., 2021), preventing studies on the mechanisms causing late phenotypes, and drug screening approaches targeting the pathways leading to those phenotypes.

[0014] A central reason for the lack of more progressive phenotypes in iPSC-derived models is that iPSC- derived cortical neurons resemble fetal neurons, which recapitulate many adult markers, such as synapse formation, synaptic transmission, or axonal-dendritic separation of Tau and MAP2, but lack other disease-contributing factors, such as adult 3R to 4R Tau isoform expression. Tau isoform expression is developmentally regulated in human brain neurons (Wang et al., 2016).

[0015] It has been found that for the first 6-12 months in culture, iPSC-derived cortical neurons express mostly the 3R Tau isoform typical forthe fetal human brain, but little 4R Tau. 4R slowly increases with extended culture periods, but it takes months or even years to reach the 3R / 4R 1 :1 ratio characteristic for adult neurons (Sposito et al., 2015, Lovejoy et al., 2020). Importantly, expression of 4R Tau is likely required for adequate recapitulation of major tauopathy phenotypes, such as Tau aggregation, as it is more aggregation prone and part of the insoluble core of Tau aggregates in many Tauopathies (Cox et al., 2016; Zhang et al., 2020).

[0016] The lack of adult Tau isoform expression in human iPSC-derived neurons limits their potential to recapitulate late-stage tauopathy phenotypes, and thus reduces the applicability of such tauopathy models for translational research and drug screening. Tauopathy models for translational research and drug screening require to mimic endogenous late-stage tauopathy phenotypes, thus, there is an urgent need in the art to provide methods and means for expression of adult Tau isoforms which may be used to study tauopathy formation and disease modeling.

[0017] To induce a disease-relevant phenotype, Tauopathy models commonly express Tau with a pathogenic mutation. Patient mutations of Tau which are involved in the pathology of tauopathies such as Alzheimer’s disease, Frontotemporal dementia, progressive supranuclear palsy, corticobasal degeneration, chronic traumatic encephalopathy, Pick’s disease, and Parkinsonism are known in the art (Wang et al., 2016). Experiments in animal models of AD demonstrated that the combination of several disease-causing point mutations in the same transgenic animal can induce pathologies not seen with single mutations in the same time frame e.g., plaque formation in triple knock-in AD mice (Saito et al., 2014).

[0018] The combination of methods to provide the 4R Tau isoform expression in human iPSC-derived cells and the introduction of a mutation associated with tauopathies has been recently described by Bravo et al., 2023. Therein, exon 10 splicing has been modulated to achieve 4R Tau production in a generated heterozygous and homozygous cell line, wherein the Tau aggregation prone mutation P301 S has been introduced in the 4R Tau allele by CRISPR / Cas9. CRISPR / Cas9 is a very versatile genome editing tool that allows cleaving genomes specifically and efficiently at most desired loci (Xiong et al., 2015). To achieve Tau pathology in human iPSC-derived neurons, Bravo et al. contacted the mutated 4R(P301 S) Tau producing neurons with exogenous Tau seeds containing aggregation increasing P301 L-mutated tau. A CRISPR screen for modulators of exogenously seeded Tau aggregation has been further performed in said study. However, due to the required exogenous seeding, Bravo et al. do not achieve formation of endogenous pathology like aggregation and only combine Tau aggregation enhancing mutation P301 S with seeding by exogenous recombinant P301 L K18 fibrils. The seeding model thus lacks the necessary endogenous development of Tau aggregates and seeds required for an endogenous tauopathy mimicking model to effectively study tauopathy formation. Also, regarding the genome editing using CRISPR / Cas9 to modulate exon 10 splicing and to introduce a Tau aggregation enhancing mutation, the FRT site and Tau splicing modulating mutations still remain and may be expressed in the generated Tau protein. Reportedly, the editing to induce 4R Tau expression is based on point mutations at the 3’ and 5’ ends of exon 10, which might result in mutations affecting the coding sequence. A detailed description of how the editing is performed to achieve 4R Tau expression, which would allow a skilled person the reproduce the experiment, is not provided.

[0019] In view of the above, there is still a need to provide a method and means to model tauopathies and endogenous tauopathy pathology formation that are relevant in the development of pharmaceutical agents for battling tauopathies. This need is addressed by the present invention.

[0020] Accordingly, the present invention relates in a first aspect to a human ectoderm derived brain cell characterized by the following features:

[0021] (1) the intron between exons 10 and 11 of the MAPT gene encoding the Tau protein has been removed by genome-editing from one or both allele(s); and

[0022] (2) at least one of the alleles of the MAPT gene of (1) carries at least one mutation enhancing Tau aggregation and at least one mutation enhancing nucleation of Tau aggregation; or

[0023] (3) one allele of the MAPT gene of (1) carries at least one mutation enhancing Tau aggregation, preferably in exon 10, and the other allele carries at least one mutation enhancing nucleation of Tau aggregation, preferably in exon 11.

[0024] As used herein, the term “ectoderm derived brain cell” means a cell type that during human development originates from the neural ectoderm, including neural tube and neural crest, and eventually populates the brain. Thus, ectoderm derived brain cells include neuronal and glial subtypes, in particular brainresident neurons, astrocytes, and oligodendrocytes, of all brain regions. An ectoderm derived brain cell of the present invention may be derived from a subject or be generated from iPSCs.

[0025] Generally, the term “iPSC” means induced pluripotent stem cell, wherein the term “pluripotent stem cell” refers to a cell which is capable of differentiation into cells of all three germ layers, i.e., endoderm, mesoderm and ectoderm. The ectoderm is the outermost layer of the three germ layers and differentiates to form epithelial and neural tissue (spinal cord, peripheral nerves and brain) and is thus of particular importance for the present invention.

[0026] The term “MAPT gene”, as used herein refers to all sequences deposited under the NCBI reference sequence NG_007398, such as NG_007398.2, including both the H1 and H2 haplotypes (specifically deposited under (ENSG00000276155 and ENSG00000277956)) corresponding to Gene ID 4137 and described herein by SEQ ID NO: 1 encoding “microtubule associated protein tau”. Additionally, since the invention can be performed with any so far not uncovered allelic forms of the MAPT gene, the actual sequence of the MAPT gene is not decisive for performing the invention and such sequences consequently also fall under the definition of this term.

[0027] As used herein, the term “gene” means the deoxyribonucleotide sequences comprising the coding region of a structural gene. A “gene” may also include non-translated sequences located adjacent to the coding region on both the 5' and 3' ends such that the gene corresponds to the length of the full-length mRNA. The sequences which are located 5' of the coding region and which are present on the mRNA are referred to as 5' non-translated sequences. The sequences which are located 3' or downstream of the coding region and which are present on the mRNA are referred to as 3' non-translated sequences. The term “gene” encompasses both cDNA and genomic forms of a gene. A genomic form or clone of a gene contains the coding region “exons” interrupted with non-coding sequences termed “introns”. Introns are segments of a gene which are transcribed into heterogenous nuclear RNA (hnRNA); introns may contain regulatory elements such as enhancers. Introns are removed from the nuclear or primary transcript during splicing; introns therefore are absent in the messenger RNA (mRNA) transcript, while exons remain. The mRNA functions during translation to specify the sequence or order of amino acids in a nascent polypeptide.

[0028] In addition to containing introns, genomic forms of a gene may also include sequences located on both the 5' and 3' end of the sequences which are present on the RNA transcript. These sequences are referred to as “flanking” sequences or regions (these flanking sequences are located 5' or 3' to the nontranslated sequences present on the mRNA transcript). The 5' flanking region may contain regulatory sequences such as promoters and enhancers which control or influence the transcription of the gene. The 3' flanking region may contain sequences which direct the termination of transcription, post- transcriptional cleavage and polyadenylation.

[0029] The MAPT gene variant, wherein the intron between exons 10 and 1 1 of the MAPT gene has been removed from only one allele of the two alleles leads to the expression of 4R (from the edited allele) and 3R (from the unedited allele) at a 1 :1 ratio, resembling the adult human brain, while the removal of the intron between exons 10 and 11 of the MAPT gene from both alleles results in only 4R Tau expression.

[0030] The skilled person is aware of methods for gene-editing, also referred to as genome editing, for removing an intron between two exons, or introducing mutations into a gene of interest. Generally, the term “genome editing” refers to a technique capable of introducing a targeted alteration into the nucleotide sequence of a gene in animal, bacterial and plant cells, including human cells, and results in the knockout of a specific gene, the knock-in of specific alterations in a gene, or introduction of a mutation into a non-coding DNA sequence that does not produce protein. In addition, genome editing enables deletion, duplication, inversion, replacement or rearrangement of genomic DNA.

[0031] Methods of genome editing, as used herein are based on methods as described in Paquet et al., 2016; Yusa et al., 2013, and are further described in the Examples herein below. The technology to induce 4R expression of Tau by removal of Intron 10-11 applied herein utilizes the CRISPR / Cas9 editing method, originally derived from the bacterial adaptive immune system in Streptococcus pyrogenes, which consists of the nuclease Cas9 and a 20-bp guide RNA, which directs Cas9 to the targeted locus by RNA-DNA base-pairing adjacent to an NGG PAM on the DNA in combination with the PiggyBac system. As used herein, the term PAM refers to a “protospacer-adjacent motif’, which is together with the guide RNA required for the nuclease activity of Cas9 to introduce double-strand breaks, which occurs 3 bp upstream of the PAM. CRISPR / Cas9-based genome editing of point mutations, such as P301 L or S320F as used herein, is achieved by selecting cells in which the induced double strand breaks are repaired by homology directed repair, rather than the non-homologous end joining pathway or microhomology directed repair, such that nonspecific insertions, deletions or other mutations do not occur. The mutation to be edited is provided on a DNA template with homology to the edited region.

[0032] The efficiency of this editing can be increased by using blocking mutations on the template that prevent re-cutting of correctly edited cells. This blocking mutation can either be the mutation of interest or an additional, usually silent, mutation. Alternatively, the two-step gene editing strategy CORRECT can be used, which takes advantage of CRISPR / Cas-blocking mutations in both steps, enabling efficient scarless introduction of a single intended mutations sequence (scarless editing) while minimizing clone picking. The CORRECT method comprises a first step wherein a blocking mutation B is introduced at both alleles of a gene of interest together with the intended mutation M to prevent nonspecific insertions. In a second step the method comprises the use of a modified CORRECT template to remove B but preserve M. By choosing guide RNAs with specific cut-to-mutation distances or using mixed repair templates, each step of CORRECT allows homozygous or heterozygous incorporation of the intended mutation (Paquet et al., 2016).

[0033] Combination of CRISPR / Cas9 based genome editing with the PiggyBac transposon system allows removal of the complete intron between exon 10 and 11 by cleavage and fusion of exons 10 and 11 with single-base precision. PiggyBac is a naturally occurring transposon system i.e., a mobile genetic element that can ‘jump’ from one locus to another in genomes. It can be used for genome engineering, as the transposable element, which is flanked by typical inverse terminal repeats (ITRs), can be removed scarlessly i.e., without any remaining foreign sequence, from a target position in the genome (Yusa et al., 2013). Efficient selection of cells with fused Exon 10 and 11 as described herein is enabled by a PiggyBac transgene incorporating, among others, an antibiotic resistance gene. This transgene can be removed in a scarless manner by integrating the PiggyBac transgene into an existing TTAA sequence in the genome, followed by excision of the PiggyBac transgene by the PiggyBac transposase, which reestablishes the TTAA sequence without any further insertions or duplications (as would be the case for other transposon systems), thus resulting in an unaltered genomic sequence (apart from any intended alterations).

[0034] Thus, in a preferred embodiment of the first aspect of the present invention, the genome editing is a scarless genome editing, wherein the generated protein sequence and / or mRNA sequence are unchanged, except for the intended alterations, providing a genetic disease model with endogenous pathology formation.

[0035] In the present disclosure, mutations introduced by genome editing are applied to mimic tauopathy phenotypes in patients suffering from tauopathies. Thus, mutations as used herein are without limitation based on patient mutations associated with tauopathies. In addition to patient mutations associated with tauopathies, also artificial (non-patient) mutations are investigated in the art and thus, mutations as used herein are also based on alternative artificial (non-patient) mutations in the gene encoding Tau. Mutations associated with abnormal Tau cause shifted Tau isoform ratios, mislocalized or misfolded and / or abnormal Tau protein. The mutations involved in tauopathies associated with Tau aggregation and nucleation of Tau aggregation are of particular focus within this invention.

[0036] As used herein, the term “tauopathy” refers to a neurodegenerative disease in which Tau aggregation is commonly detected. This comprises a heterogenous group of dementias, including at least 25 different disorders, characterized by Tau inclusions or malfunction. These disorders differ in affected cell type, brain region, and thus also the symptoms they entail. Furthermore, depending on the disease, different combinations of Tau isoforms are found in the aggregates, i.e., either 3R isoforms, 4R isoforms, or a combination of both (Wang et al. 2016). Tauopathies can be classified according to these characteristics.

[0037] As used herein, the term “tau aggregation” refers to aggregation of Tau in multimeric fibrils of multiple Tau monomers characterized by the presence of parallel p-sheets. Tau aggregation is associated with other tauopathy phenotypes, such as Tau hyperphosphorylation and mislocalization, pathological Tau seeding and Tau misfolding.

[0038] As used herein, the term “nucleation of Tau aggregation” refers to the initiation of Tau aggregation by formation of oligomers of abnormal tau, typically initiated by conformational changes.

[0039] The term “enhancing” in conjunction with “tau aggregation” and / or “nucleation of Tau aggregation” means the increase of the propensity of disease-related Tau aggregation, such as naturally occurring disease-related Tau aggregation. Assays to detect / measure Tau aggregation include but are not limited to immunostaining with dyes, such as AmyTracker or X34.

[0040] Phosphorylation of Tau (P-Tau) has been described as a major marker of pathologic Tau, especially if present at multiple disease-relevant epitopes. Normal Tau is phosphorylated to a certain degree at specific epitopes to ensure the dynamic character of the microtubule network (see below). Tau in its aggregated form (as Paired Helical Filaments (PHFs) or Straight Filaments (SF)) contains 5-9 moles of phosphate / mole of the protein, defining it as hyperphosphorylated (Lippens et al., 2007.; Kbpke et al., 1993). The term “hyperphosphorylation” may also be referred to as “abnormal phosphorylation”. Assays to detect / measure P-Tau and localization of phosphorylated Tau include but are not limited to immunostaining with an antibody, such as AT8, AT 100, AT 180, PHF-1 , PHF-13, and antibodies specific for pT181 , pS198, pS199, pT205, pS202+pT205, pT212, pS214, pT231 , pS238, pS262, E178, pS396, pS404, pS409, pS422.

[0041] The term “tau seeding” refers to the spreading of transformation of normal Tau into misfolded protein. Small Tau seeds are generated in neurons upon early Tau misfunction and can spread to other neurons by a poorly understood mechanism, where they likely induce further aggregation and disease (Goedert et al., 2017). The spreading of induced Tau pathology along neuroanatomically connected brain areas is referred to as “prion-like”, because the pattern of spreading resembles that of prions, which are characterized by transmission from cell to cell, tissue to tissue and organism to organism (Mudher et al. 2017). Assays to detect / measure the increase of Tau aggregation resulting from seeding include but are not limited to immunostaining with dyes, such as AmyT racker or X34. Alternatively, the capacity for Tau seeding can be measured using fluorescence resonance energy transfer (FRET)-based Tau biosensor cells. These cells typically express Tau repeat domains fused to FRET-fluorophore pairs, such as CFP and YFP, in two separate constructs. Upon treatment with exogenous seed containing material, these repeat domains aggregate and the resulting FRET signal can be used to measure the extent of aggregation (Hitt et al., 2021). Alternatives fordetecting Tau seeds include electron microscopy and RT-QuIC assays.

[0042] The term “misfolded Tau” refers to Tau protein not folded correctly and therefore failing to fulfil its function of stabilizing microtubules. Misfolded Tau can no longer bind to microtubules and is typically more aggregation prone. Thus, misfolding of Tau is considered one of the initial steps leading to Tau aggregation and thus late-stage pathology. Misfolded Tau may be measured by methods known in the art, such as immunostaining with MC-1.

[0043] As used herein, the term “measure”, in accordance with the present invention means to quantitatively and / or qualitatively detect a component, such as a tau protein, a specific modification or conformation thereof, or polynucleotide in a sample of a cell, directly in a cell, or in a group of cells. Quantitative or qualitative detection of a protein, a polynucleotide, or a factor means relative or absolute quantification in a sample of a cell, directly in said cell, or in a group of cells by methods known in the art. Those methods include but are not limited to immunostaining, western blot, qPCR, RNA-Seq, and FRET-based biosensor cell assays.

[0044] In general, relative and absolute quantification means and methods can be used. In absolute quantification no known standards or controls are needed. The presence, localization and / or level of the protein, polynucleotide etc. can be directly quantified. As is well-known in the art, absolute quantification may rely in certain further embodiments on a predetermined standard curve. In relative quantification, the level is quantified relative to a reference (such as known control levels). Also, in the absence of controls, one can relatively quantify the expression level when comparing e.g., fluorescence intensities.

[0045] Detection or measuring increased levels of tauopathy phenotypes also indicates the enhanced propensity of (i) Tau aggregation and / or (ii) nucleation of Tau aggregation.

[0046] The genetic disease model of the present invention facilitates mimicking of the endogenous tauopathy pathology formation and study of tauopathies in humans, which is superiorto other models not achieving endogenous tauopathy associated Tau protein generation.

[0047] Since mutations have been found in 3R / 4R Tau expressing adult human brain of patients suffering from tauopathies, the combination of a 3R Tau expressing and / or 4R Tau expressing allele with mutations enhancing Tau aggregation and / or mutations enhancing nucleation of Tau aggregation, is included in the first aspect of the invention.

[0048] In a preferred embodiment of the first aspect of the present invention the intron between exons 10 and 11 of the MAPT gene encoding the Tau protein has been removed by gene-editing from one allele (edited allele) of an ectoderm derived brain cell, and the edited allele of the ectoderm derived brain cell carries a mutation enhancing Tau aggregation and a mutation enhancing nucleation of Tau aggregation.

[0049] In another preferred embodiment, the intron between exons 10 and 11 of the MAPT gene encoding the Tau protein has been removed by gene-editing from one allele (edited allele) of an ectoderm derived brain cell, and the unedited allele of the ectoderm derived brain cell carries a mutation enhancing Tau aggregation and a mutation enhancing nucleation of Tau aggregation.

[0050] In another preferred embodiment, the intron between exons 10 and 11 of the MAPT gene encoding the Tau protein has been removed by gene-editing from one allele (edited allele) of an ectoderm derived brain cell, and both, the edited and the unedited allele, of the ectoderm derived brain cell carry a mutation enhancing Tau aggregation and a mutation enhancing nucleation of Tau aggregation.

[0051] In another preferred embodiment, the intron between exons 10 and 11 of the MAPT gene encoding the Tau protein has been removed by gene-editing from one allele (edited allele) of an ectoderm derived brain cell, and the edited allele carries the mutation enhancing Tau aggregation in exon 10 and the unedited allele carries the mutation enhancing nucleation of Tau aggregation in exon 11.

[0052] In another preferred embodiment, the intron between exons 10 and 11 of the MAPT gene encoding the Tau protein has been removed by gene-editing from one allele (edited allele) of an ectoderm derived brain cell, and the unedited allele carries the mutation enhancing Tau aggregation in exon 10 and the edited allele carries the mutation enhancing nucleation of Tau aggregation in exon 11.

[0053] In another preferred embodiment, the intron between exons 10 and 11 of the MAPT gene encoding the Tau protein has been removed by gene-editing from both alleles of an ectoderm derived brain cell, and one allele of the ectoderm derived brain cell carries a mutation enhancing Tau aggregation and a mutation enhancing nucleation of Tau aggregation.

[0054] In another preferred embodiment, the intron between exons 10 and 11 of the MAPT gene encoding the Tau protein has been removed by gene-editing from both alleles of an ectoderm derived brain cell, and both alleles of the ectoderm derived brain cell carry a mutation enhancing Tau aggregation and a mutation enhancing nucleation of Tau aggregation.

[0055] In another preferred embodiment, the intron between exons 10 and 11 of the MAPT gene encoding the Tau protein has been removed by gene-editing from both alleles of an ectoderm derived brain cell, and one allele carries the mutation enhancing Tau aggregation in exon 10 and the other allele carries the mutation enhancing nucleation of Tau aggregation in exon 11.

[0056] In accordance with the present invention, the introduction of mutations enhancing (i) Tau aggregation and (ii) nucleation of Tau aggregation in the unedited MAPT allele, is particularly useful for mimicking the tauopathies that are associated with mutated 3R Tau isoform of a 1 :1 3R / 4R Tau ratio expressing brain cell, while the introduction of mutations enhancing (i) Tau aggregation and (ii) nucleation of Tau aggregation in the edited MAPT allele, wherein the intron between exon 10 and 11 has been removed, is particularly useful for mimicking the tauopathies that are associated with mutated 4R Tau isoform or a 1 :1 3R / 4R Tau ratio expressing brain cell.

[0057] Introducing a mutation enhancing Tau aggregation and a mutation enhancing nucleation of Tau aggregation in the same unedited (3R Tau encoding) or edited (4R encoding) gene ensures the expression of 3R and 4R Tau isoforms, respectively, wherein aggregation of the 3R and 4R Tau isoforms, respectively, and nucleation of said aggregation is enhanced.

[0058] The removal of the intron between exons 10 and 11 of the MAPT gene from both alleles, facilitates the expression of only the 4R Tau isoform and therefore provides a basis for observable phenotypes of tauopathy pathogenesis which are associated with the endogenous 4R Tau isoform. Thus, removal of the intron from both alleles is particularly advantageous for studies targeted towards 4R associated tauopathies, such as corticobasal degeneration.

[0059] Introducing a mutation enhancing Tau aggregation and a mutation enhancing nucleation of Tau aggregation in both alleles is particularly useful when tauopathy phenotypes associated with a certain combination of mutations is a focus of the study, since a stronger phenotype is expected if both 3R and / or 4R encoding alleles are mutated. In accordance with the present invention, the combination of different mutations enhancing Tau aggregation and a mutation enhancing nucleation of Tau aggregation on one allele compared to the other allele is included.

[0060] The present invention achieves a modular system for studying tauopathies associated with 3R and / or 4R Tau isoforms and Tau aggregation enhancing and nucleation of Tau aggregation enhancing mutations as further described in the preferred embodiments herein below.

[0061] The nature of mutations in the MAPT gene known to be associated with tauopathies is diverse and includes missense, silent, and intronic mutations, which lead to a diverse range of influences on Tau and its binding to microtubules, such as Tau aggregation, microtubule assembly, microtubule binding, isoform splicing and exon 10 inclusion. Since Tau aggregation has been primarily linked to tauopathies as described herein, the present disclosure focuses in particular on mutations involved in the formation of Tau aggregates. The process of Tau aggregate formation is characterized by a step of oligomer formation (nucleation) and elongation of Tau aggregates into filaments.

[0062] In a preferred embodiment of the first aspect of the invention, at least one mutation that enhances Tau aggregation is selected thus from a group comprising: R5H, R5L, K257T, 1260V, L266V, N279K, A280K, P301 L, P301 S, G335V, Q336H, Q336R, V337M, E342V, S352L, P346S, E372G, and N401 H.

[0063] The above listed mutations are reported to be involved in enhancing the Tau aggregation, especially the elongation of aggregation prone Tau oligomers.

[0064] Prior to formation of elongated aggregated Tau oligomers and filaments that are toxic to neurons, either by causing signaling defects or obstructing the cell interior, the formation of Tau aggregates is initiated by mutations that specifically enhance the nucleation of Tau aggregate formation.

[0065] Thus, in another or more preferred embodiment of the first aspect of the invention, at least one mutation that enhances nucleation of Tau aggregation is selected from a group comprising: G272V, G303V, L315R, S320F, S320I, S320V, I328V, S352L, S356T and G389R.

[0066] The above listed mutations are reported to be involved in enhancing the propensity to form Tau aggregates by changing the structure of Tau so that it dissociates from microtubules and forms oligomers, which then aggregate to filamentous, cytoplasmic inclusions characterized by parallel p- sheet structure of their microtubule binding repeats.

[0067] The selection from a group of mutations that (i) enhance Tau aggregation and (ii) enhance nucleation of Tau aggregation ensures that the endogenous pathology of tauopathies, which involve both the nucleation and the aggregation of pathological Tau protein, is reflected by the present disclosure.

[0068] Combining a specific mutation enhancing Tau aggregation and a specific mutation enhancing nucleation of Tau aggregation is capable of inducing endogenous pathology formation as shown by the combination of P301 L with S320F (Strang et al., 2018). Their combination enhances the propensity of Tau to form oligomers and aggregates, leading to rapid formation of AT8- and Thioflavin S positive Tau aggregates in HEK293 cells, as well as pathology formation in mice (Strang et al., 2018; Koller et al., 2019; Croft et al., 2019), while either mutation alone is not sufficient.

[0069] Accordingly, in a particular preferred embodiment of the above preferred embodiments, the mutation that enhances Tau aggregation is P301 L and the mutation that enhances nucleation of Tau aggregation is S320F.

[0070] In order to develop a human tauopathy model, a protocol to efficiently differentiate iPSCs into pure and electrically active human cortical neurons was applied, which is described in detail in the Examples of the present disclosure. During differentiation from iPSCs to active cortical neurons, neural precursor cells (NPC) were generated, which are capable of differentiating into cortical neurons, astrocytes and oligodendrocytes. Specification of the different cell types i.e., neurons, astrocytes, or oligodendrocytes is achieved by culturing the cells at specific cell densities and selecting a different set of exogenously provided growth and specification factors that mimic neuronal, astroglial, or oligodendroglial differentiation during human development, or by expressing lineage-specifying transcription factors respectively, accordingly, to established protocols.

[0071] Accordingly, in another preferred embodiment of the first aspect of the invention the ectoderm-derived brain cell is a neuron, an astrocyte, or an oligodendrocyte.

[0072] As used herein, the term “neuron” refers to an electrically excitable cell that communicates with other neurons via synapses. A neuron is characterized by the presence of synapses and the capability to release neurotransmitters. Neurons typically consist of a cell body (soma), dendrites, and a single axon. Brain neurons form more complex neuronal networks as compared to peripheral neurons and are thus morphologically more complex. Typically, brain neurons express the markers NeuN, p-Tubulin III, and MAP2.

[0073] As used herein the term “astrocyte” refers to a glial cell, which supports the nervous tissue. An astrocyte is typically characterized by their star shaped morphology with numerous branching processes that extend in multiple directions. Astrocytes are only present in the central nervous system. Typically, astrocytes express proteins such as Sox9, GFAP and / or S100p.

[0074] As used herein, the term “oligodendrocyte” refers to a neuroglial cell of the CNS whose function is to myelinate CNS axons. An oligodendrocyte is characterized by the production of myelin, and myelin proteins such as MBP and PLP. Further markers include expression of Olig2 and 04.

[0075] It is understood that the ectoderm derived brain cell of the present disclosure is preferentially a neuron, as Tau pathology is often found in neurons, but it may also be an astrocyte, or an oligodendrocyte.

[0076] As described above, the human adult brain expresses a ratio of approximately 1 :1 of 3R / 4R tau.

[0077] Thus, in a preferred embodiment of the first aspect, the second allele of the MAPT gene of the ectoderm derived brain cell of the first aspect contains the intron between exon 10 and 11 .

[0078] In other terms, in a preferred embodiment the ectoderm-derived brain cell of the first aspect is characterized by two alleles of the MAPT gene, wherein in one allele the intron between exon 10 and 11 has been removed, thereby producing a ratio of 1 :1 of 3R / 4R tau.

[0079] Apart from modulation of the 3R or 4R Tau isoform ratio, an increase in the presence of 4R Tau (over the healthy 1 :1 ratio) leads to the onset of tauopathies. In accordance with the invention, tauopathies caused by an abnormal ratio of 3R and 4R, represented here by the production of only 4R Tau, can also be studied using the genetic disease model of the present disclosure. In another or even more preferred embodiment, the intron between exon 10 and 11 in both alleles of the MAPT gene of the ectoderm derived brain cell of the first aspect has been removed by gene editing.

[0080] Accordingly, in other terms the ectoderm-derived brain cell of the first aspect is characterized by two alleles of the MAPT gene wherein in both alleles the intron between exon 10 and 11 has been removed, thereby producing only 4R tau.

[0081] An ectoderm-derived brain cell which produces only 4R and wherein mutations to enhance (i) Tau aggregation and (ii) nucleation of Tau aggregation have been introduced is particularly useful to mimic endogenous pathology of tauopathies associated with 4R Tau isoform splicing, such as FTD.

[0082] As mentioned, in accordance with the invention, the present disclosure includes the generation of an ectoderm-derived brain cell as defined in the first aspect as part of the herein described genetic disease model to study endogenous tauopathy pathology formation.

[0083] Accordingly, in a second aspect, the present invention relates to a method for producing the ectoderm derived brain cell of the first aspect, the method comprising:

[0084] (i) inducing differentiation in human induced pluripotent stem cells (hiPSCs), thereby obtaining neural precursor cells;

[0085] (ii) expanding the neural precursor cells of step (i);

[0086] (iii) obtaining neuronal, astroglial, or oligodendroglial cells derived from the cells of step (ii);

[0087] (iv) prior to, simultaneously with or after any one of steps (i), (ii) or (iii), removing the intron between exons 10 and 11 of the MAPT gene encoding the Tau protein by genome-editing from one or both allele(s), thereby inducing endogenous 4R Tau expression;

[0088] (v) prior to, simultaneously with or after step (iv),

[0089] (a) inserting or providing at least one mutation enhancing Tau aggregation in the modified allele(s) of step (iv) lacking the intron between exons 10 and 11 of the MAPT gene; or (b) inserting or providing at least one mutation enhancing nucleation of Tau aggregation in the allele containing the intron between exons 10 and 11 of the MAPT gene; and

[0090] (c) inserting or providing at least one mutation enhancing nucleation of Tau aggregation in the modified allele(s) of step (iv); or

[0091] (d) inserting or providing at least one mutation enhancing nucleation of Tau aggregation in the allele containing the intron between exons 10 and 11 of the MAPT gene; or in the case where at least one allele carries the deletion in the intron between exon 10 and 11 , thereby inducing mutated 4R Tau expression, and at least one or no additional mutations enhancing Tau aggregation or enhancing nucleation of Tau aggregation, exogenously providing a Tau variant carrying at least one mutation enhancing Tau aggregation and / or at least one mutation enhancing nucleation of Tau aggregation so that the cell carries at least one mutation enhancing Tau aggregation and at least one mutation enhancing nucleation of Tau aggregation; and (vi) obtaining the ectoderm derived brain cell.

[0092] The method of the invention consequently ensures that the ectoderm derived brain cell carries at least one mutation enhancing the aggregation and at least one mutation enhancing nucleation of tau aggregation in at least one modified allele and / or the allele containing the intron between exons 10 and 11.

[0093] The term “variant” as used herein, refers to a nucleic acid or an amino acid sequence, a virus comprising a nucleic acid or promoting production of an amino acid sequence, which differs in comparison to the wild-type sequence in at least one base or amino acid residue, respectively. The variant maintains or essentially maintains the function of the form carrying a mutation enhancing Tau aggregation and / or mutation enhancing nucleation of Tau aggregation.

[0094] The method of the second aspect includes methods for differentiation and long-term culture of iPSC- derived cortical neurons and especially CRISPR / PiggyBac based genome engineering. The usage of human iPSCs for this method facilitates the generation of specific neuronal cells which follows the development as usually occurring in the human brain. In accordance with the present invention, the generated neural precursor cells are capable to differentiate into ectoderm derived brain cells expressing adult-like Tau of 1 :1 ratio of 3R / 4R Tau by default. This is advantageous over methods applied in the art, wherein iPSC-derived neurons slowly increase 4R Tau expression, which requires months or years to reach a desired 1 :1 ratio of 3R / 4R tau.

[0095] With the unique combination the method of the invention achieves the distinctive properties of an iPSC- based tauopathy model with adult-like Tau isoform expression that recapitulates late-stage disease phenotypes and allows investigating underlying disease mechanisms and screening for genes / proteins and chemical compounds modulating or abrogating tauopathies. Only the unexpected and unprecedented combination of all these aspects allowed to obtain a novel and the first relevant disease model for tauopathies which may be applied in translational research and drug screening.

[0096] In a preferred embodiment of the second aspect, the method comprises in a step (v) prior to, simultaneously with or after step (iv), inserting or providing a mutation, wherein at least one mutation that enhances Tau aggregation is selected from a group comprising: R5H, R5L, K257T, I260V, L266V, N279K, A280K, P301 L, P301 S, G335V, Q336H, Q336R, V337M, E342V, S352L, P346S, E372G, and N401 H; and at least one mutation that enhances nucleation of Tau aggregation is selected from a group comprising: G272V, G303V, L315R, S320F, S320I, S320V, I328V, S352L, S356T and G389R.

[0097] The term “at least one mutation” as used herein means that combinations of mutations are also possible.

[0098] In a more preferred embodiment of the above preferred embodiment, the mutation that enhances Tau aggregation is P301 L.

[0099] In another more preferred embodiment of the above preferred embodiment, the mutation that enhances nucleation of Tau aggregation is S320F.

[0100] The method of the second aspect facilitates the generation of ectoderm-derived brain cells which specifically produce a certain ratio of 3R / 4R Tau isoforms, wherein those isoforms include in this preferred embodiment specifically selected mutations enhancing (i) Tau aggregation and (ii) nucleation of Tau aggregation.

[0101] In a preferred embodiment of step (i) of the second aspect of the invention, the method comprises inducing neural differentiation in hiPSCs (a) by dual-SMAD inhibition; or (b) by expression of transcription factors selected from the group including Ngn2, Ngn1 , ASCL1 , BRN2, MYT1 L, and NEUROD1.

[0102] The term “dual-SMAD inhibition”, in accordance with the present invention, means the inhibition of the SMAD signaling pathway via two SMAD inhibitors to derive neural progenitor cells from iPSCs. The term “SMAD” is an abbreviation of the genes in Caenorhabditis elegans SMA ("small" worm phenotype) and MAD family ("Mothers Against Decapentaplegic") in Drosophila, wherein SMADs were first discovered. SMAD pathways mediate proliferation and transforming growth factor p (TGF-p) and bone morphogenic protein (BMP) signaling through SMAD members and have distinct effects on development and homeostasis (Derynck et al., 1998). Inhibition of SMAD prevents differentiation of non-neural cells and thus increases the number of neural cells during differentiation from iPSCs. Dual-SMAD inhibition typically inhibits sternness of iPSCs as well as mesodermal development through inhibition of TGFp and Activin / Nodal signaling, for example by using SB431542, respectively; and inhibition of ectoderm and trophectoderm development though inhibition of BMP signaling using Noggin or more commonly LDN-193189. The skilled person is aware of SMAD inhibitors described in the art that can be used in accordance with the present invention, such as described by selleckchem (https: / / www.selleckchem.com / TGF-beta.html).

[0103] Generally, differentiation of hiPSC into the desired cell type may be achieved by methods known in the art. For the present disclosure differentiation methods for differentiation into ectoderm derived brain cells, preferably to neurons as described in Dannert et al., 2023, are applied and described in Example 2 herein below. Other cell types include astrocytes and oligodendrocytes, which can be differentiated as described (Perriot et al., 2021 ; Ehrlich et al., 2017)

[0104] As used herein, the term “transcription factor”, refers to a protein or polypeptide that binds specific DNA sequences associated with a genomic locus or gene of interest to control transcription. Transcription factors may promote (as an activator) or block (as a repressor) the recruitment of RNA polymerase to a gene of interest. Transcription factors may perform their function alone or as a part of a larger protein complex. With regards to information on transcriptional factors, mention is made of Latchman and DS (1997) Int. J. Biochem. Cell Biol. 29 (12): 1305-12; Lee Tl, Young RA (2000) Annu. Rev. Genet. 34: 77- 137and Mitchell PJ, Tjian R (1989) Science 245 (4916): 371-8, herein incorporated by reference in their entirety.

[0105] Transcription factors used for induction of hiPSC may be endogenously or exogenously expressed. For differentiation into neural cell lineages as described in the present disclosure, generally exogenous expression is used in the art, as it is technically much simpler to express from an exogenous source, such as a transgene, than inducing overexpression from the endogenous locus.

[0106] Generally, the term “NGN” means “neurogenin” and refers to the NGN family comprising NGN1 , NGN2 and NGN3, which are important throughout development of neurons. NGN2 is transiently expressed in multiple NPC types and downregulated during final differentiation stages to promote pro-neural pathways and subtype specification. NGN2 is preferably used for its ability to rapidly induced neuronal differentiation (Hulme et al., 2022)

[0107] The terms “Ngn2” (Uniprot ID: Q9H2A3; Gene ID: 63973) encoded by all sequences deposited under the NCBI Reference Sequence NP_076924, such as NP_076924.1) and “Ngn1 ” (Uniprot ID: Q92886; Gene ID: 4762) encoded by all sequences deposited under the NCBI Reference Sequence NP_006152, such as NP_006152.2) also refer to allelic variants thereof that are distinct from the hitherto described sequence by at least one base pair, but maintain with increasing preference at least 50%, 80%, 90% or 100% transcription factor activity of the described transcription factor.

[0108] The forced expression of BRN2, ASCL1 and MYT 1 L are known in the art to reprogram mouse fibroblasts into functional neurons and have been tested in human iPSCs.

[0109] The term “ASCL1 ”, as used herein above means the transcription factor "Achaete-Scute Family BHLH Transcription Factor 1 ” and is encoded by all sequences deposited under the NCBI Reference Sequence NG_008950, such as NG_008950.1 ; and Gene ID: 429, Uniprot ID: P50553. The term “ASCL1 ” also refers to allelic variants thereof that are distinct from the hitherto described sequence by at least one base pair, but maintain with increasing preference at least 50%, 80%, 90% or 100% transcription factor activity of the described transcription factor. ASCL1 is sufficient to drive direct neuronal differentiation of both human and mouse PSCs.

[0110] The term “BRN2”, as used herein above refers to a POU domain transcription factor encoded by the POU3F2 gene and is encoded by all sequences deposited under the NCBI Reference sequence NP_005595, such as NP_005595.2; and Gene ID: 5454; Uniport ID: P20265. The term “BRN2” also refers to allelic variants thereof that are distinct from the hitherto described sequence by at least one base pair, but maintain with increasing preference at least 50%, 80%, 90% or 100% transcription factor activity of the described transcription factor. BRN2 likely contributes to neuronal maturation by increasing morphological complexity. The term “MYT1 L”, as used herein above means the transcription factor “Myelin Transcription Factor 1 Like” and is encoded by all sequences deposited underthe NCBI Reference Sequence: NP_001289981 , such as NP_001289981 .1 ; and Gene ID: 23040, Uniprot ID: Q9UL68. The term “MYT1 L” also refers to allelic variants thereof that are distinct from the hitherto described sequence by at least one base pair, but maintain with increasing preference at least 50%, 80%, 90% or 100% transcription factor activity of the described transcription factor. MYT1 L likely contributes to neuronal maturation by increasing morphological complexity. Forced expression of this gene in combination with the basic helix-loop-helix transcription factor NeuroDI and the transcription factors POU class 3 homeobox 2 and achaete-scute family basic helix-loop-helix transcription factor 1 can convert fetal and postnatal human fibroblasts into induced neuronal cells, which are able to generate action potentials.

[0111] The term “NEUROD1 ”, as used herein above means the transcription factor “Neurogenic Differentiation 1 ”. NEUROD1 is encoded by all sequences deposited under the NCBI Reference Sequence: NP_002491 , such as NP_002491.3; and Gene ID: 4760, Uniprot ID: Q13562. The term “NEUROD1 ” also refesr to allelic variants thereof that are distinct from the hitherto described sequence by at least one base pair, but maintain with increasing preference at least 50%, 80%, 90% or 100% transcription factor activity of the described transcription factor. The forced expression of the NEUROD1 is known in the art to differentiate hiPSCs into excitatory neurons.

[0112] The differentiation using dual-SMAD allows differentiation of human iPSC-derived neurons mimicking human neurodevelopment and are typically used in the art for generating neurons form iPSC. As 4R Tau as well as Tau mutations are suspected to also influence neuronal differentiation, related aspects can better be studied in neurons differentiated using morphogens that imitate human cortical development than using transcription factor overexpression. Thus, dual-SMAD inhibition is preferred over the expression of transcription factors for the induction of neural differentiation in hiPSCs.

[0113] Thus, in a more preferred embodiment of the above-preferred embodiment of the second aspect of the invention, the neural differentiation in hiPSCs is induced by dual-SMAD inhibition.

[0114] The differentiation using expression of transcription factors enables fast differentiation of very high yields of neurons, which can easily be used for large scale drug and genetic screens. Models using transgenic transcription factor-differentiated neurons are also regularly used for iPSC-derived models of dementia. However, the purity and neuronal fate of the resulting cultures is still under debate, making dual-SMAD differentiated neurons more preferrable.

[0115] In a more preferred embodiment of the above preferred embodiment in item (b), neural differentiation is induced by the expression of at least one, at least two, at least three, at least four, and at least five transcription factors selected from the group including Ngn2, Ngn1 , ASCL1 , BRN2, MYT1 L, and NEUROD1. In another more preferred embodiment of the above-preferred embodiment of item (b), neural differentiation is induced by the expression of Ngn2. In another preferred embodiment of the second aspect, the method includes a step (ii), wherein the neural precursor cells of step (i) of the second aspect are expanded in neural rosettes.

[0116] The term “neural rosettes” refers to NPCs which are plated to achieve maximum cell density while allowing sufficient nutrient supply when fed once per day.

[0117] Feeding of the cells includes addition of new culture medium typically including at least one of the following nutrients: nutrient mix Ham’s F12 (ThermoFisher), Neurobasal (ThermoFisher), Penicillin / Streptomycin (ThermoFisher), B27 supplement (503) (with Vit. A) (ThermoFisher), GlutaMax (ThermoFisher), non-essential amino acids (ThermoFisher), N-2 supplement (ThermoFisher), insulin (Sigma-Aldrich), 2-mercaptoethanol (ThermoFisher); with optional addition of bFGF (STEMCELL Technologies), to further promote expansion of neuronal rosettes.

[0118] Culturing conditions are, for example, 37°C, 5% CO2 in neural maturation medium (0,5 x Neurobasal (ThermoFisher), 0,5x DMEM / F-12 (ThermoFisher), 100 U / ml and 0,1 mg / ml Penicillin (10.000 U / mL) / Streptomycin (10 mg / mL) (ThermoFisher), 0,5 x B27 supplement (503) (with Vit. A) (ThermoFisher), 2mM GlutaMax (ThermoFisher), 1x non-essential amino acids (ThermoFisher), 0,5x N- 2 supplement (ThermoFisher), 2,5 pg / ml insulin (SigmaAldrich), 0.05 mM 2-mercaptoethanol (ThermoFisher); with optional addition of 20 ng / pl bFGF (STEMCELL Technologies).

[0119] Neuronal differentiation can be induced using dual-SMAD inhibition, followed by high-density culture in spots starting on day in vitro (DIV)7. Neural rosette selection can be performed between DIV20-23. After one additional passage, pure late neural progenitor cells (NPCs)Zearly neurons can be frozen in liquid nitrogen between DIV37-40 until future use. For long-term culture, NPCs can be thawed in neural maintenance medium (Neurobasal PLUS / B27 PLUS (ThermoFisher, A3582901 and A3582801)) supplemented with Penicillin / Streptomycin (e.g., ThermoFisher 15140-122) and 0,5 mM GlutaMAX (e.g., ThermoFisher, 35050)) and cultured for one week. Subsequently, the cells can be plated onto poly-ornithine (e.g., Sigma-Aldrich, P4957) / Laminin (ThermoFisher, 23017015) coated plates around DIV45 and cultured in Neurobasal PLUS / B27 PLUS medium (e.g., ThermoFisher, A3653401) for up to 1 year. In the first two weeks of long-term culture, media can be supplemented with DAPT / 5-FU as described in Dannert et al., 2023 to remove contaminating glial progenitors from the culture and finalize differentiation of any remaining NPCs. It is of note that these steps can be performed independently from each other.

[0120] In a preferred embodiment of the second aspect, the method includes a step (v), wherein the mutations are inserted by genome-editing.

[0121] Methods for genome-editing are provided elsewhere in the present disclosure.

[0122] In another preferred embodiment of the second aspect, the method includes a step (v), wherein at least one of the mutations is derived from a patient, while at least one is inserted by genome-editing. In accordance with the invention, introduction of mutations by genome-editing facilitates that the produced mutated Tau specifically includes only the mutations desired and makes it better comparable to isogenic non-edited cell lines, which facilitates identification of mutation-specific pathological effects and modulation thereof. The usage of a mutation that is derived from a patient in form of a patient cell line is another alternative. The use of patient mutations has the advantage that also the entire genetic background of the patient is reflected in the generated iPSCs and the neurons derived thereof. In particular, specific aspects of pathology might be enhanced / modulated through the patient’s genetic background. Optionally, this might allow patient-specific drug screening in the light of personalized medicine.

[0123] The term “patient” as used herein, means a subject, preferably mammalian, most preferably human, suffering from at least one of the tauopathies as defined herein, and / or experiencing at least one of the tauopathy phenotypes as defined in the present disclosure, or carrying a mutation that is known to lead to development of tauopathies even if the individual is not yet affected.

[0124] Generally, the term “tauopathy phenotype” as used herein, means but is not limited to abnormal Tau phosphorylation / tau hyperphosphorylation, Tau mislocalization, Tau misfolding, Tau seeding and / orTau aggregation. Further Tau phenotypes associated with tauopathies are included herein, such as nuclear malformations, cellular transport abnormalities, impaired microtubule dynamics, impaired microtubule function, impaired axonal transport, altered neuronal differentiation, impaired DNA maintenance, electrophysiological abnormalities, lysosomal / autophagy / proteasomal abnormalities, and impaired mitochondrial biology including increases in reactive oxygen species.

[0125] The presence of tauopathy phenotypes is necessary to ensure the model of the present invention can be used as a model to study said tauopathy formation.

[0126] Thus, in a preferred embodiment of the second aspect, the method comprises in a further step (vii) characterizing a tauopathy phenotype in the obtained ectoderm derived brain cell.

[0127] The term “characterizing”, refers to observing and classifying the phenotypes as described above, wherein usually biochemical assays are applied, such as immunostaining, microscopy, Western Blot, Sarkosyl extraction and sequential ultracentrifugation to isolate insoluble Tau, FRET-based Tau biosensor cells, RT-QuIC assays, qPCR, RNAseq Mass Spectrometry, electron microscopy, PCRs, electrophysiology, detection of fluorescence using plate readers, flow cytometry or fluorescence activated cell sorting.

[0128] In a third aspect, the present invention relates to a composition comprising the ectoderm derived brain cell according to the invention.

[0129] The term “composition”, as used herein, means a mixture containing the ectoderm-derived brain cell and at least one other component. Such a component as used herein may be without limitation a protein, a reagent, such as a chemical compound, a buffer etc. In accordance with the present invention, a composition may be used to induce tauopathy pathology formation in vitro and / or in vivo for research studies. The combination of the ectoderm-derived brain cell with a second component may, without limitation, prevent further undesired differentiation of the ectoderm-derived brain cell, and / or augment viability of said cell, or production of the desired Tau isoform.

[0130] In a preferred embodiment of the third aspect, the composition is a kit. The various components of the kit may be packaged into one or more containers such as one or more vials. The vials may, in addition to the components, comprise preservatives or buffers for storage. The kit may comprise instructions how to use the kit.

[0131] In accordance with the present invention, the ectoderm-derived brain cell may not only be used to study tauopathy pathology formation, but also to provide information of the effectiveness of drugs against the tauopathy displayed by the ectoderm-derived brain cell.

[0132] Thus, in a fourth aspect, the present invention refers to the use of ectoderm-derived brain cells as defined in any of the first to third aspects for screening drugs useful in preventing ortreating tauopathies.

[0133] As used herein, the term “drug screening” refers to the identification of compounds or drugs that are useful in the treatment or prevention of tauopathies. “Drug screening” also includes toxicity assays.

[0134] The term “toxicity assay” means assays commonly used in the art fortesting the influence of compounds by incubating the tested sample, e.g., a cell, a tissue, an organism, with the compounds and determining the survival and viability of said sample. For example, see the adherent cell differentiation and cytotoxicity assay (ACDC assay) (see Barrier et al., 2010)

[0135] The term “preventing”, as used herein, refers to slowing down of a tauopathy or the onset of a tauopathy, disorder or condition or the symptoms and conditions thereof in a subject.

[0136] As used herein, the term “treating” refers to reversing, alleviating or inhibiting the progress of a tauopathy, or one or more symptoms of such tauopathy, to which such term applies. The term “treating” also means improving, enhancing, ameliorating the conditions of a subject suffering from a disease associated with tauopathies as described herein.

[0137] As will be immediately understood, the use of the present invention in drug screening allows to assess the effectiveness of known and / or unknown drugs or drug candidates in a model that resembles the endogenous tauopathy pathology and therefore provides more reliable information for the production of drugs.

[0138] In a fifth aspect, the present invention thus refers to a method of screening drugs useful in the development of the prevention or treatment of tauopathies, the method comprising:

[0139] (a) contacting the cell of the first or second aspect with potential drugs under conditions that allow the interaction of the drugs with the Tau proteins produced by the cells obtained in the first or second aspect;

[0140] (b) observing at least one of the following phenotypes

[0141] (ba) Tau hyperphosphorylation;

[0142] (bb) Tau mislocalization;

[0143] (be) formation of Tau seeds, optionally, pathological Tau seeds;

[0144] (bd) Tau misfolding; and

[0145] (be) Tau aggregation; wherein modulation of at least one of said phenotypes indicates that the drug is useful in the development of the prevention or treatment of tauopathies.

[0146] Conditions that allow the interactions of the drugs are, for example, addition of the drug to the culture medium of the cells, injection of the drugs into the cells, intracellular application of the drugs using carrier molecules, expression of bioactive compounds either endogenously in the cell or through exogenous addition of expression vectors or mRNA, or addition of LNPs to the culture medium, or delivery of the bioactive compounds using virus like particles.

[0147] In accordance with the present invention, the method of screening drugs is a method to assess the influence of the potential drug on the specific mutated Tau protein and provides qualitative, preferably quantitative information on the effect of the potential drug in view of the specifically mutated Tau protein. The method enables the provision of the direct relationship between mutated Tau mimicking endogenous pathologic conditions of tauopathy formation and the potential drug. In case the drug alleviates any of the above phenotypes, so that a phenotype towards a non-tauopathy phenotype is developed, the drug may be useful as a medicament or may be useful in the development of a medicament. Accordingly, in one preferred embodiment, modulation is alleviation, such as reduction of any of the phenotypes developed for at least 50%, more preferably at least 80%, even more preferred at least 90%, even more preferred at least 95% and most preferred total eradication of at least one, two, three, four and most preferably all phenotypes as defined in items (ba) to (be) of the above aspect.

[0148] In case any of the above phenotypes is aggravated, the drug may provide useful insights into pathways involved in the development of tauopathies, which in turn is also useful in the development of medicaments for treating or preventing tauopathies.

[0149] In an even more preferred embodiment of the fifth aspect, the method comprises a step (b), wherein at least one, preferably at least two, more preferably at least three, more preferably at least four and most preferably five of the phenotypes (ba) to (be) are observed.

[0150] In another, even more preferred embodiment, the method in step (b), wherein observing at least one of the phenotypes includes observation of (be) formation of Tau seeds, optionally, pathological Tau seeds. In another, even more preferred embodiment, the method in step (b), wherein observing at least one of the phenotypes includes observation of (be) Tau aggregation.

[0151] The occurrence of Tau seeds, optionally, pathological Tau seeds, is one of the earliest phenotypes observable and thus most suitable for screening.

[0152] Thus, in a preferred embodiment of the fifth aspect, the method comprises observing at least two, preferably at least three and more preferably at least four phenotypes of (ba) to (be).

[0153] In another, even more preferred embodiment, the method in step (b), requiring observing at least one of the phenotypes, includes observation of (ba) and (bb). In another, even more preferred embodiment, the method in step (b), requiring observing at least one of the phenotypes includes observation of (ba) and (be). In another, even more preferred embodiment, the method in step (b), requiring observing at least one of the phenotypes includes observation of (ba) and (bd). In another, even more preferred embodiment, the method in step (b), requiring observing at least one of the phenotypes includes observation of (ba) and (be).

[0154] In another, even more preferred embodiment, the method in step (b), requiring observing at least one of the phenotypes includes observation of (bb) and (be). In another, even more preferred embodiment, the method in step (b), requiring observing at least one of the phenotypes includes observation of (bb) and (bd). In another, even more preferred embodiment, the method in step (b), requiring observing at least one of the phenotypes includes observation of (bb) and (be).

[0155] In another, even more preferred embodiment, the method in step (b), requiring observing at least one of the phenotypes includes observation of (be) and (bd). In another, even more preferred embodiment, the method in step (b) requiring observing at least one of the phenotypes includes observation of (be) and (be).

[0156] In another, even more preferred embodiment, the method in step (b) requiring observing at least one of the phenotypes includes observation of (bd) and (be).

[0157] In another, even more preferred embodiment, the method in step (b) requiring observing at least one of the phenotypes includes observation of (ba), (bb) and (be). In another, even more preferred embodiment, the method in step (b) requiring observing at least one of the phenotypes includes observation of (ba), (bb) and (bd). In another, even more preferred embodiment, the method in step (b), requiring observing at least one of the phenotypes includes observation of (ba), (bb) and (be).

[0158] In another, even more preferred embodiment, the method in step (b), requiring observing at least one of the phenotypes includes observation of (ba), (be) and (bd). In another, even more preferred embodiment, the method in step (b), requiring observing at least one of the phenotypes includes observation of (ba), (be) and (be).

[0159] In another, even more preferred embodiment, the method in step (b), requiring observing at least one of the phenotypes includes observation of (ba), (bd) and (be).

[0160] In another, even more preferred embodiment, the method in step (b), requiring observing at least one of the phenotypes includes observation of (bb), (be) and (bd). In another, even more preferred embodiment, the method in step (b), requiring observing at least one of the phenotypes includes observation of (bb), (be) and (be). In another, even more preferred embodiment, the method in step (b), requiring observing at least one of the phenotypes includes observation of (bb), (bd) and (be).

[0161] In another, even more preferred embodiment, the method in step (b), requiring observing at least one of the phenotypes includes observation of (ba), (bb), (be) and (bd). In another, even more preferred embodiment, the method in step (b), requiring observing at least one of the phenotypes includes observation of (ba), (bb), (be) and (be). In another, even more preferred embodiment, the method in step (b), requiring observing at least one of the phenotypes includes observation of (ba), (be), (bd) and (be). In another, even more preferred embodiment, the method in step (b), requiring observing at least one of the phenotypes includes observation of (bb), (be), (bd) and (be).

[0162] Apart from the use of the cell of the present invention to find potential drugs, the use to elucidate unknown genes or pathways associated with mutated Tau is another object of the present invention.

[0163] In a sixth aspect, the present invention refers to the use of ectoderm-derived brain cells as defined in any of the first to fifth aspect for genetic screening to identify genes / proteins and pathways involved in tauopathy pathogenesis.

[0164] The term “genetic screening” refers to the process of testing an ectoderm derived brain cell to identify genes of those cells mediating an increased risk of having or developing tau pathology or carrying a genetic variant causing a tauopathy. The term also includes to identify genes of those cells with an involvement of decreased risk of having or developing tau pathology or promoting the presence of non- pathologic effects.

[0165] In a preferred embodiment of the sixth aspect, the invention refers to the use for genetic screening to identify genes / proteins involved in enhancing tauopathy pathogenesis.

[0166] In another preferred embodiment of the sixth aspect, the invention refers to the use for genetic screening to identify genes / proteins involved in decreasing tauopathy pathogenesis.

[0167] The use for genetic screening in accordance with the invention provides the basis for understanding the pathways involved in tauopathy formation in an endogenous tauopathy pathogenesis model.

[0168] In a seventh aspect of the present invention, the invention refers to a method for screening for genes involved in the development or modulation of any of tauopathy phenotypes (ba) to (be) as defined in the fifth aspect, the method comprising:

[0169] (a) contacting the ectoderm derived brain cell of the first or second aspect with a guide RNA (gRNA) or a plurality of gRNAs under conditions that allow the interaction of the gRNA(s) with the DNA in a cell obtained the method of the second aspect;

[0170] (b) expressing a genome-editing system in the cell of (a) that:

[0171] (ba) reduces or prevents expression of a target gene by cleavage of its genomic locus; or (bb) reduces or prevents expression of a target gene by its downregulation; or

[0172] (be) increases expression of a target gene by its upregulation;

[0173] (c) observing in the cell of (b) whether an alteration of at least one of the following phenotypes occurs:

[0174] (ca) Tau hyperphosphorylation;

[0175] (cb) Tau mislocalization;

[0176] (cc) formation of Tau seeds, optionally, pathological Tau seeds;

[0177] (cd) Tau misfolding; and

[0178] (ce) Tau aggregation; wherein alteration of at least one of said phenotypes indicates that the genetic alteration is involved in a pathway modulating the generation of tauopathies; and

[0179] (d) Identifying the gene(s) involved in the development or modulation of any of tauopathy phenotypes (ba) to (be) by identifying the active guide RNA(s) from (a), preferably in cases where a plurality of guide RNAs were used.

[0180] The term “alteration”, as used herein, refers to any change of the phenotypes observed in comparison to an ectoderm derived brain cell of the first or second aspect wherein none of the steps (a) to (d) of the seventh aspect of the invention have been performed. It is intended that an increase, decrease, as well as a loss of previously observed tauopathy phenotype or presence of previously not observed tauopathy phenotype is included in the term “alteration”.

[0181] The “genome-editing system”, as used herein, refers to a system, such as CRISPR / Cas9, CRISPR / Cas12a, CRISPR base editing, CRISPR prime editing, CRISPRi, CRISPRa, Zinc Finger nucleases, or TALENs.

[0182] The method of the seventh aspect of the present invention provides information on the development or modulation of tauopathy phenotypes based on mutated Tau mimicking endogenous pathologic Tau protein. The usage of CRISPR / Cas9 for genetic screens is known in the art. However, the novel combination of the CRISPR / Cas9 based genetic screen with an ectoderm-derived cell producing mutated Tau mimicking endogenous pathologic Tau protein, provides a more reliable basis, compared to methods known in the art not using endogenous pathologic Tau protein with advanced pathology, for identifying genes associated with the alteration of the tauopathy phenotypes described herein.

[0183] Tauopathies as described above may be associated with at least one of the tauopathy phenotypes of item (ca) to (ce) of the seventh aspect of the present disclosure.

[0184] In a preferred embodiment of the fourth or sixth aspect or the method of the fifth or seventh aspect, the tauopathies are selected from the group of Alzheimer’s disease, Frontotemporal dementia, progressive supranuclear palsy, corticobasal degeneration, chronic traumatic encephalopathy, Pick’s disease, and Parkinsonism. In accordance with the present invention, the screening of drugs and / or genetic screening as defined in the fourth or sixth aspect or the method of the fifth or seventh aspect will be particularly important for advancing the understanding of pathways and pathology of the tauopathies as defined herein above.

[0185] In an eighth aspect of the present invention, the invention refers to a method for testing the potential of a Positron-Emission-Tomography (PET)-tracer for detecting tau aggregates comprising:

[0186] (i) contacting and incubating (a) the ectoderm-derived brain cells as defined in the first aspect, the cells produced by the method according to the second aspect, or the composition of the third aspect, wherein the cells produce tau aggregates, and (b) control cells, wherein the control cells do not produce tau aggregates, with a PET-tracer,

[0187] (ii) removing the medium and excess PET-tracer by washing the cells / aggregates,

[0188] (iii) testing for binding of the PET-tracer to tau aggregates by measuring the radioactivity in the cells obtained in step (ii), wherein a radioactive signal shows the binding of the PET-tracer with tau aggregates,

[0189] (iv) normalizing the measured radioactivity obtained in step (iii) to the number of cells and;

[0190] (v) comparing the normalized measurements obtained in step (iv) of cells producing tau aggregates to control cells not producing tau aggregates, wherein increased normalized measurements vs. control measurements positively correlates with the potential of the PET-tracer for detecting tau aggregates.

[0191] The term “Positron-Emission-Tomography (PET)” as used herein is a non-invasive imaging technique and is based on the development and synthesis of molecules labeled with positron-emitting radioisotopes, including18F,64Cu,nC,15O,13N,66Ga,68Ga,76Br,89Zr,94mTc,86Y and1241. The principle of PET is known in the art. In brief, the labeling radioisotopes, referred to as radio ligands or radio tracers, emit positrons. Those ejected positrons collide with electrons resulting in the emission of radioactive y (gamma) rays, which can then be detected by a PET imaging scanner. The detected y rays are converted into flat tomographic images that show the distribution of the used radio ligand over time.

[0192] The term “PET-tracer”, as used herein, refers to a radio tracer molecule comprising a radioactive ligand, i.e., a radioisotope, and a compound / biomolecule which can interact with a compound / biomolecule of interest to enable detection by Positron-Emission-tomography. Preferably, the radioisotope has a halflife long enough to enable synthesis of the PET-tracer, quality control of the resulting PET-tracer, use of the PET tracer in vitro and / or in vivo, removal of non-targeted signal, and detection of the signal by a method capable of measuring radioactive y-rays, i.e. gamma emission measurement, either directly or indirectly, such as PET imaging, but also autoradiography or a gamma counter. The steps for application of a general PET-tracer for detection of a compound / biomolecule of interest in patients is known in the art and has been described for example for the PET-tracer18F-PI-2620 (Mueller et al., 2020). PET- tracers suitable for use in patients may be used similarly for the method of the present invention in vitro (Slemann et al., 2024). The term “increased normalized measurements”, as used herein, refers to a statistically significant increase of the normalized measurements in comparison to the control measurements, wherein the a- error (also called type-1 -error) is defined as p<0,05. It is understood that this increase may also comprises an increase of at least 5%, at least 10 %, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% or at least 50% with increasing preference.

[0193] In a preferred embodiment of the eighth aspect of the present invention, step (i)(a) and (b) are contacted with a PET-tracer for at least 5 minutes, for at least 10 minutes, for at least 15 minutes, for at least 20 minutes, for at least 25 minutes or for at least 30 minutes.

[0194] In a preferred embodiment of the eighth aspect of the present invention, the cells (a) of step (i) produce 3R or 4R tau aggregates.

[0195] In another preferred embodiment of the eighth aspect of the present invention, the radioactivity in step

[0196] (iii) is measured by methods known in the art, such as gamma counter and autoradiography.

[0197] In another preferred embodiment of the eighth aspect of the present invention, the method comprises prior or after step (i), but before step (ii) a step (0) of detaching, disrupting and / or lysing (a) the ectoderm- derived brain cells as defined in the first aspect, the cells produced by the method according to the second aspect, or the composition of the third aspect, wherein the cells produce tau aggregates, and (b) control cells, wherein the control cells do not produce tau aggregates. The results of the method according to this preferred embodiment may be compared to the method of the eighth aspect without step (0). Comparison thereof is proposed to provide further hints on how and whether the binding of the PET-tracer to tau aggregates is improved in disrupted cells vs. whole cells.

[0198] In another preferred embodiment of the eighth aspect of the present invention, wherein the cells in step (i) (a) and (b) are (i) ectoderm-derived brain cells as defined in the first aspect, (ii) the cells produced by the method according to the second aspect, or (iii) cells comprised in the composition of the third aspect, wherein the cells of (a) produce a first type of a tau aggregate exhibiting a certain tau aggregate conformation and wherein the cells of (b) produce a second type of a tau aggregate exhibiting another tau aggregate conformation. These conformations might be determined by e.g., the tau mutations present in cells (a) and (b) and include but are not limited to conformations comprising different isoforms of Tau within the aggregates. The first and the second type of tau aggregates produced by cells of (a) and (b), respectively, are distinct from each other.

[0199] Accordingly, wherein the method of the above preferred embodiment further comprises the steps (ii) to

[0200] (iv) as defined in the eighth aspect and a step (v), wherein the normalized measurements obtained in step (iv) of cells (a) and (b), wherein the cells of (a) produce the first type of a tau aggregate and the cells of (b) produce the second type of a tau aggregate, are compared to each other. An increased normalized measurements of any tau conformation in the aggregates positively correlates with the potential of the PET-tracer for detecting the type of tau aggregate of the respective conformation, i.e., first or second type of tau aggregates. The method according to this preferred embodiment allows the refinement ofthe screening potential of the PET-tracer for discriminating between aggregates consisting of different conformations.

[0201] In a ninth aspect of the present invention, the invention refers to the use of the ectoderm-derived brain cells as defined in the first aspect, the cells produced by the method according to the second aspect or the composition as defined in the third aspect for testing the capacity of PET-tracers to detect tau aggregates.

[0202] As regards the embodiments characterized in this specification, in particular in the claims, it is intended that each embodiment mentioned in a dependent claim is combined with each embodiment of each claim (independent or dependent) said dependent claim depends from. For example, in case of an independent claim 1 reciting 3 alternatives A, B and C, a dependent claim 2 reciting 3 alternatives D, E and F and a claim 3 depending from claims 1 and 2 and reciting 3 alternatives G, H and I, it is to be understood that the specification unambiguously discloses embodiments corresponding to combinations A, D, G; A, D, H; A, D, I; A, E, G; A, E, H; A, E, I; A, F, G; A, F, H; A, F, I; B, D, G; B, D, H; B, D, I; B, E, G; B, E, H; B, E, I; B, F, G; B, F, H; B, F, I; C, D, G; C, D, H; C, D, I; C, E, G; C, E, H; C, E, I; C, F, G; C, F, H; C, F, I, unless specifically mentioned otherwise.

[0203] Similarly, and also in those cases where independent and / or dependent claims do not recite alternatives, it is understood that if dependent claims refer back to a plurality of preceding claims, any combination of subject-matter covered thereby is considered to be explicitly disclosed. For example, in case of an independent claim 1 , a dependent claim 2 referring back to claim 1 , and a dependent claim 3 referring back to both claims 2 and 1 , it follows that the combination of the subject-matter of claims 3 and 1 is clearly and unambiguously disclosed as is the combination of the subject-matter of claims 3, 2 and 1 . In case a further dependent claim 4 is present which refers to any one of claims 1 to 3, it follows that the combination of the subject-matter of claims 4 and 1 , of claims 4, 2 and 1 , of claims 4, 3 and 1 , as well as of claims 4, 3, 2 and 1 is clearly and unambiguously disclosed.

[0204] The Figures show:

[0205] Fig. 1 : iPSC differentiation to cortical neurons. A) Immunofluorescence (IF) stainings of neuronal precursor cells (NPCs) on day-in-vitro (DIV) 10 demonstrating loss of pluripotency marker Oct-4 and increased expression of neural markers Nestin and Pax6. B) RT-q-PCR analysis on iPSCs (DIVO), late NPCs / early neurons (DIV40) and neurons (NE, DIV70) showing downregulation of pluripotency factors Oct4 and Nanog from DIVO to DIV40, upregulation of NPC markers Nestin and Pax6 before DIV40, and their downregulation during neuronal maturation from DIV40 to DIV70. C) IF staining of early neurons on DIV30 confirming expression of NPC markers Pax6 and Nestin as well as deep-layer neuron marker Tbr1 . D) IF staining of cortical neurons on DIV70 confirming expression of neuron markers p3-Tubulin, Tau, and MAP2. E) RT-q-PCR analysis on iPSCs (DIVO), late NPCs / early neurons (DIV40) and cortical neurons (DIV70) showing upregulation of neuronal markers MAP2, Tau, PSD95, Synapsin-1 and NMDAR1 over time. Data are represented as Mean ± SD. F) IF staining showing expression of deep-cortical-layer marker Ctip2 and mid-cortical-layer marker Satb2 on DIV70. G) IF stainings showing long-term survival of neurons and increasing density of axonal networks positive for Tau in cultures on DIV70 (top), DIV 180 (middle) and DIV320 (bottom). All scale bars 50 pm.

[0206] Fig. 2: Data on genome editing and 4R Tau expression. A) Editing strategy to achieve 1 :1 splice ratios between 3R and 4R Tau from the endogenous human Tau locus. Using CRISPR / Cas9, the intron containing regulatory sequences for alternative splicing of Exon 10 is removed from the endogenous human genomic MAPT locus. As a result, the edited locus expresses 4R Tau while the unedited locus retains 3R Tau expression according to the developmental immaturity of iPSC-derived neurons. B) Detailed strategy for scarless removal of the intron between exons 10 and 11. (1) gRNA binding and location of CRISPR-induced DSBs, repair by HDR using PB transposon containing repair plasmid; (2) positive selection of clones using puromycin resistance; (3) scarless removal of transgene via PiggyBac transposase and TK negative selection of correct clones; PB = PiggyBac transposon, TTAA = PB target sequence, puro = puromycin antibiotic resistance, TK = thymidine kinase. For simplification only the edited allele is shown. C) 4R expression can be modulated by editing one or both alleles: lines with editing at one allele express 50% 4R Tau, while those with both alleles edited express 100% 4R Tau. D-E) 50% 4R expression on protein level in iPSC line with one allele edited, as shown by 4R-specific antibody in IF (D, red channel), and presence of 4R band on western blot (E, red arrow). F) Sanger sequencing traces illustrating successful fusion of exons 10 and 11 and insertion of P301 L and S320F FTD mutations.

[0207] Fig. 3: Preliminary data on assay development and tauopathy phenotypes. A) Overview of iPSC lines generated with genotype, line ID and abbreviations used in text and figures. B) Presence of hyperphosphorylated Tau is shown by AT8 staining; presence of oligomeric Tau in the somata of neurons is shown using the oligomeric tau specific T-22 antibody. C) Sarkosyl extracts from WT and mutant lines were transfected into Tau biosensor HEK293 cells (Holmes et al., 2014) and quantified by FACS. Note presence of aggregates in biosensor cells (middle), and presence of FRET+ population in FACS assay after 4 months in culture; presence of FRET-positivity quantified over a time course of 200 days (right) and directly compared to other cell lines after 130 days (graphic created with Biorender.com). D) Mutant neurons display misfolded Tau in MC-1 ‘paperclip’ conformation after 5-6 months. Presence of the pathological paperclip conformation was confirmed with Alz50 staining. E) Presence of AmyTracker- and X34-positive aggregates in mutant iPSC-neurons after 8-10 months.

[0208] Fig. 4: Acceleration of phenotype formation in 100% mutant 4R Tau expressing cells. A) Sarkosyl extracts (isolated after 4 months) from the homozygous P301 L / S320F / 4R line were transfected into Tau biosensor HEK293 cells (Holmes et al., 2014) and quantified by FACS. B) Representation of FACS measurements of Tau biosensor HEK293 cells in different cell lines. Note strong increase in pathology in the homozygous line compared to the other cell lines. C) Presence of FRET-positivity quantified over a time course of 200 days. D) Mutant neurons display misfolded Tau in MC-1 ‘paperclip’ conformation. E) Quantification of the presence of MC-1 misfolded Tau in neurons of different cell lines after 5 months in culture. F) Staining for synapses using PSD-95 (postsynapse) and Synapsin (pre-synapse) in WT and P301 L / S320F / 4R homozygous cells. G) Quantification of the number of synapses in different cell lines, demonstrating loss of synapses in mutant cell lines. H) Partial rescue of FRET positivity in P301 L / S320F / 4R-homozygous neurons treated with the compound Anle-138 for 3 months after the onset of pathology demonstrating modulability of the observed phenotype. I) Scheme representing incubation of in vitro neuron cultures with Tau PET Tracer PI-2620 followed by measurement of radioactivity using a gamma-counter. J) Quantification of TAU PET Tracer PI-2620 binding to WT and P301 L / S320F / 4R homozygous cultures.

[0209] Fig. 5: Overview of plasmid map and schemes for editing of the MAPT gene. A) Plasmid map for CRISPR editing repair template to achieve deletion of the intron. The repair template is used to replace the sequence between the two CRISPR-induced double strand breaks via homology directed repair (HDR). The repair template contains the directly fused Exons 10 and 11 that replace the endogenous sequence (orange). These are flanked by sequences homologous to the endogenous MAPT locus (3’ and 5’ arm, green) that are required to allow HDR. In addition, the repair template contains transgenic sequences flanked by PiggyBac terminal repeats (blue) that are placed at a TTAA sequence in Exon 10. The PiggyBac construct allows later scarless removal of these transgenic sequences (grey and black). The transgenic sequences are an eukaryotic antibiotic resistance gene (here Puromycin as an example) to facilitate selection of edited clones and a Thymidine kinase used for negative selection after scarless removal of the transgene, both in black, including promotor and polyA in grey. The plasmid further contains an origin of replication (black) and a bacterial antibiotic resistance gene (black, Spectinomycin as an example) to allow for bacterial amplification of the plasmid. B) - D) Scheme of the endogenous MAPT locus after each editing step. B) overview of endogenous MAPT locus before editing; the sequence highlighted in purple is replaced through HDR with the sequence in the repair template, thereby removing the intron. The binding sites of the two gRNAs are highlighted. C) Endogenous MAPT locus with integrated repair template after HDR after first editing step, cells that correctly integrated the repair template can be enriched for using the antibiotic resistance gene; D) Fusion of Exon 10 and 11 in the endogenous MAPT locus after removal of the transgene by PiggyBac transposase after the second editing step, cells that successfully removed the PiggyBac with transgenic sequences can be enriched for using FIAU-selection of Thymidine kinase negative cells.

[0210] The invention is illustrated by the examples.

[0211] Example 1. Materials and Methods

[0212] The Materials and Methods of Example 1 are further described in Examples 2 to 5 herein.

[0213] Sequences

[0214] SEQ ID NO:1 is the sequence of human MAPT described by all sequences deposited under the NCBI reference sequence NG_007398, such as NG_007398.2, including both the H1 and H2 haplotypes (specifically deposited under (ENSG00000276155 and ENSG00000277956)) corresponding to Gene ID 4137.

[0215] Table 1 : Guide RNAs: The sequences as shown in the table are defined with SEQ ID numbers. The sequence of gRNA2 is defined herein as SEQ ID NO:2. The sequence of gRNA5 is defined herein as SEQ ID NO:3. The sequence of gRNA84 is defined herein as SEQ ID NO:4. The sequence of gRNA134 is defined herein as SEQ ID NO:5. gRNA2 WT MAPT Exon 11 GTTTGTAGACTATTTGCACC DSB intron-exon 11 border

[0216] 3R / 4R editing gRNAS WT MAPT Exon 10 GATAATATCAAACACGTCCC DSB exon 10-intron border

[0217] Insertion of gRNA84 Fused exon 10-11 AATATCAAACACGTGCTGGG KI of P301L mutation mutations in 4R- edited lines (if not incorporated in gRNA134 Fused exon 10-11 GCCTAATGAGCCACACTTGA KI of S320F mutation repair template)

[0218] Repair Template: the plasmid map is shown in Fig. 4A.

[0219] Plasmid sequence of the repair template (SEQ ID NO: 6): tccacaggtgattctgatgcccggcaggcttgagaacagccgcagggagttctctgggaatgtgccggtgggtctagccaggtgtgagtggagatgccgggga acttcctattactcactcgtcagtgtggccgaacacatttttcacttgacctcaggctggtgaacgctcccctctggggttcaggcctcacgatgccatccttttgtgaa gtgaggacctgcaatcccagcttcgtaaagcccgctggaaatcactcacacttctgggatgccttcagagcagccctctatcccttcagctcccctgggatgtgac tcaacctcccgtcactccccagactgcctctgccaagtccgaaagtggaggcatccttgcgagcaagtaggcgggtccagggtggcgcatgtcactcatcgaa agtggaggcgtccttgcgagcaagcaggcgggtccagggtggcgtgtcactcatccttttttctggctaccaaaggtgcagataattaaccctagaaagatagtct gcgtaaaattgacgcatgcattcttgaaatattgctctctctttctaaatagcgcgaatccgtcgctgtgcatttaggacatctcagtcgccgcttggagctcccgtga ggcgtgcttgtcaatgcggtaagtgtcactgattttgaactataacgaccgcgtgagtcaaaatgacgcatgattatcttttacgtgacttttaagatttaactcatacg ataattatattgttatttcatgttctacttacgtgataacttattatatatatattttcttgttatagatatcaactagaatgctagcatgggcccatctcgaccgccaattcaat atggcgtatatggactcatgccaattcaatatggtggatctggacctgtgccaattcaatatggcgtatatggactcgtgccaattcaatatggtggatctggacccc agccaattcaatatggcggacttggcaccatgccaattcaatatggcggacttggcactgtgccaactggggaggggtctacttggcacggtgccaagtttgagg aggggtcttggccctgtgccaagtccgccatattgaattggcatggtgccaataatggcggccatattggctatatgccaggatcaatatataggcaatatccaat atggccctatgccaatatggctattggccaggttcaatactatgtattggccctatgccatatagtattccatatatgggttttcctattgacgtagatagcccctcccaa tgggcggtcccatataccatatatggggcttcctaataccgcccatagccactcccccattgacgtcaatggtctctatatatggtctttcctattgacgtcatatgggc ggtcctattgacgtatatggcgcctcccccattgacgtcaattacggtaaatggcccgcctggctcaatgcccattgacgtcaataggaccacccaccattgacgt caatgggatggctcattgcccattcatatccgttctcacgccccctattgacgtcaatgacggtaaatggcccacttggcagtacatcaatatctattaatagtaactt ggcaagtacattactattggaaggacgccagggtacattggcagtactcccattgacgtcaatggcggtaaatggcccgcgatggctgccaagtacatccccat tgacgtcaatggggaggggcaatgacgcaaatgggcgttccattgacgtaaatgggcggtaggcgtgcctaatgggaggtctatataagcaatgctcgtttagg gaaccgccattctgcctggggacgtcggagcaagcttgatttaggtgacactatagaatacaagctacttgttctttttgcaggatcccatcgattcgaattccatgg ggaccgagtacaagcccacggtgcgcctcgccacccgcgacgacgtcccccgggccgtacgcaccctcgccgccgcgttcgccgactaccccgccacgcg ccacaccgtcgacccggaccgccacatcgagcgggtcaccgagctgcaagaactcttcctcacgcgcgtcgggctcgacatcggcaaggtgtgggtcgcgg acgacggcgccgcggtggcggtctggaccacgccggagagcgtcgaagcgggggcggtgttcgccgagatcggcccgcgcatggccgagttgagcggttc ccggctggccgcgcagcaacagatggaaggcctcctggcgccgcaccggcccaaggagcccgcgtggttcctggccaccgtcggcgtctcgcccgaccac cagggcaagggtctgggcagcgccgtcgtgctccccggagtggaggcggccgagcgcgccggggtgcccgccttcctggagacctccgcgccccgcaacc tccccttctacgagcggctcggcttcaccgtcaccgccgacgtcgaggtgcccgaaggaccgcgcacctggtgcatgacccgcaagcccggtgccggatcca tgcccacgctactgcgggttatatagacggtcctcacgggatggggaaaaccaccaccacgcaactgctggtggccctgggtcgcgcgacgatatcgtctac gtacccgagccgatgactactggcaggtgctgggggctccgagacaatcgcgaacatctacaccacacaacaccgcctcgaccagggtgagatatcggc cggggacgcggcggtggtaatgacaagcgcccagataacaatgggcatgcctatgccgtgaccgacgccgtctggctcctcatatcgggggggaggctgg gagctcacatgccccgcccccggccctcaccctcatctcgaccgccatcccatcgccgccctcctgtgctacccggccgcgcgatacctatgggcagcatga ccccccaggccgtgctggcgtcgtggccctcatcccgccgacctgcccggcacaaacatcgtgtgggggccctccggaggacagacacatcgaccgcct ggccaaacgccagcgccccggcgagcggctgacctggctatgctggccgcgatcgccgcgttacgggctgcttgccaatacggtgcggtatctgcagggc ggcgggtcgtggcgggaggatggggacagctttcggggacggccgtgccgccccagggtgccgagccccagagcaacgcgggcccacgaccccatatc ggggacacgtattaccctgttcgggcccccgagtgctggcccccaacggcgacctgtacaacgtgtttgcctgggcctggacgtcttggccaaacgcctcc gtcccatgcacgtcttatcctggatacgaccaatcgcccgccggctgccgggacgccctgctgcaactacctccgggatggtccagacccacgtcaccacc cccggctccataccgacgatctgcgacctggcgcgcacgttgcccgggagatgggggaggctaactgagctctagagctcgctgatcagcctcgactgtgcct tctagtgccagccatctgtgttgcccctcccccgtgcctcctgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaatgcatcgcatgt ctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggatgggaagacaatagcaggcatgctggggatgcggtgggctct atggcttctgaggcgggaagtcctatactttctagagaataggaactcactagtaaaagttttgtactttatagaagaaattgagtttgtttttttaataaataa ataaacataaataaatgttgtgaatttatatagtatgtaagtgtaaatataataaaactaatatctatcaaataataaataaacctcgatatacagaccgata aaacacatgcgtcaattttacgcatgatatcttaacgtacgtcacaatatgatatctttctagggtaataagaagctggatctagcaacgtccagtccaagtgtg gctcaaaggataatatcaaacacgtgctgggaggcggcagtgtgcaaatagtctacaaaccagtgacctgagcaaggtgacctcaagtgtggctcatagg caacatccatcataaaccaggtagccctgtggaaggtgagggtgggacgggagggtgcagggggtggaggagtcctggtgaggctggaactgctccagac ttcagaaggggctggaaaggatattttaggtagacctacatcaaggaaagtgtgagtgtgaaactgcgggagcccaggaggcgtggtggctccagctcgctc ctgcccaggccatgctgcccaagacaaggtgaggcgggagtgaagtgaaataaggcaggcacagaaagaaagcacatattctcggccgggcgctgtggct cacgcctgtaatccagcacttgggaggccaaggtgggtggatcatgaggtcaggagatgagaccatcctggctaacacagtgaaaccccgtctctactaaa aatacaaaaaatagccgggcgtggtggtgggcgcctgtagtcccagctactccggaggctgaggcaggaaaatggcgtgaacccggaaggcggagctg cagtgagcggagtgagcagagggagacgaagggcgaatcgacccagctttctgtacaaagtggcatataaaaaataatgctcatcaattgtgcaacg aacaggtcactatcagtcaaaataaaatcatattgccatccagctgatatcccctatagtgagtcgtatacatggtcatagctgtttcctggcagctctggcccgt gtctcaaaatctctgatgtacatgcacaagataaaaatatatcatcatgcctcctctagaccagccaggacagaaatgcctcgactcgctgctgcccaaggtg ccgggtgacgcacaccgtggaaacggatgaaggcacgaacccagtggacataagcctgtcggtcgtaagctgtaatgcaagtagcgtatgcgctcacgca actggtccagaacctgaccgaacgcagcggtggtaacggcgcagtggcggtttcatggctgtatgactgttttttggggtacagtctatgcctcgggcatcca agcagcaagcgcgtacgccgtgggtcgatgttgatgtatggagcagcaacgatgttacgcagcagggcagtcgccctaaaacaaagtaaacatcatgag ggaagcggtgatcgccgaagtatcgactcaactatcagaggtagtggcgtcatcgagcgccatctcgaaccgacgtgctggccgtacatttgtacggctccgc agtggatggcggcctgaagccacacagtgatatgattgctggtacggtgaccgtaaggctgatgaaacaacgcggcgagcttgatcaacgaccttgga aacttcggctcccctggagagagcgagatctccgcgctgtagaagtcaccatgtgtgcacgacgacatcatccgtggcgtatccagctaagcgcgaactg caattggagaatggcagcgcaatgacatctgcaggtatcttcgagccagccacgatcgacatgatctggctatctgctgacaaaagcaagagaacatagc gttgcctggtaggtccagcggcggaggaactctttgatccggtcctgaacaggatctattgaggcgctaaatgaaacctaacgctatggaactcgccgcccg actgggctggcgatgagcgaaatgtagtgctacgtgtcccgcattggtacagcgcagtaaccggcaaaatcgcgccgaaggatgtcgctgccgactgggc aatggagcgcctgccggcccagtatcagcccgtcatactgaagctagacaggctatcttggacaagaagaagatcgctggcctcgcgcgcagatcagtg gaagaatttgtccactacgtgaaaggcgagatcaccaaggtagtcggcaaataaccctcgagccacccatgaccaaaatccctaacgtgagtacgcgtcgt tccactgagcgtcagaccccgtagaaaagatcaaaggatctctgagatccttttctgcgcgtaatctgctgctgcaaacaaaaaaaccaccgctaccagc ggtggttgttgccggatcaagagctaccaactctttttccgaaggtaactggctcagcagagcgcagataccaaatactgtcctctagtgtagccgtagtagg ccaccactcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtctaccgggtggactc aagacgatagtaccggataaggcgcagcggtcgggctgaacggggggtcgtgcacacagcccagctggagcgaacgacctacaccgaactgagatac ctacagcgtgagcatgagaaagcgccacgctcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacga gggagcttccagggggaaacgcctggtatctttatagtcctgtcgggttcgccacctctgactgagcgtcgattttgtgatgctcgtcaggggggcggagcctat ggaaaaacgccagcaacgcggccttttacggtcctggccttttgctggcctttgctcacatgtcttcctgcgtatcccctgatctgtggataaccgtataccgc cttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctct ccccgcgcgtggccgatcataatgcagctggcacgacaggttcccgactggaaagcgggcagtgagcgcaacgcaataatacgcgtaccgctagcca ggaagagttgtagaaacgcaaaaaggccatccgtcaggatggcctctgctagtttgatgcctggcagtttatggcgggcgtcctgcccgccaccctccgggc cgtgctcacaacgtcaaatccgctcccggcggattgtcctactcaggagagcgtcaccgacaaacaacagataaaacgaaaggcccagtcttccgactg agcctttcgttattgatgcctggcagtccctactctcgcgtaacgctagcatggatgttcccagtcacgacgtgtaaaacgacggccagtctaagctcggg ccccaaataatgattttattgactgatagtgacctgtcgtgcaacaaatgatgagcaatgctttttataatgccaactttgtacaaaaaagcaggctccgaatc gccctt

[0220] As an example, puromycin is depicted as an antibiotic resistance gene. Any other eukaryotic antibiotic resistance gene can be used for selection of correctly edited clones together with the corresponding antibiotic.

[0221] Sequence modifications at human MAPT locus: a) Before editing: endogenous MAPT locus (Fig. 4B)

[0222] DNA Sequence of genomic region before editing (SEQ ID NO: 7): agactcctagattctaattctaggtcagggctaggggctgagattgtaaaaatccacaggtgattctgatgcccggcaggcttgagaacagccgcagggagttct ctgggaatgtgccggtgggtctagccaggtgtgagtggagatgccggggaacttcctattactcactcgtcagtgtggccgaacacatttttcacttgacctcaggc tggtgaacgctcccctctggggttcaggcctcacgatgccatccttttgtgaagtgaggacctgcaatcccagcttcgtaaagcccgctggaaatcactcacacttc tgggatgccttcagagcagccctctatcccttcagctcccctgggatgtgactcaacctcccgtcactccccagactgcctctgccaagtccgaaagtggaggcat ccttgcgagcaagtaggcgggtccagggtggcgcatgtcactcatcgaaagtggaggcgtccttgcgagcaagcaggcgggtccagggtggcgtgtcactca tccttttttctggctaccaaaggtgcagataattaataagaagctggatcttagcaacgtccagtccaagtgtggctcaaaggataatatcaaacacgtcccggga ggcggcagtgtgagtaccttcacacgtcccatgcgccgtgctgtggcttgaattattaggaagtggtgtgagtgcgtacacttgcgagacactgcatagaataaat ccttcttgggctctcaggatctggctgcgacctctgggtgaatgtagcccggctccccacattcccccacacggtccactgttcccagaagccccttcctcatattct aggagggggtgtcccagcatttctgggtcccccagcctgcgcaggctgtgtggacagaatagggcagatgacggaccctctctccggaccctgcctgggaag ctgagaatacccatcaaagtctccttccactcatgcccagccctgtccccaggagccccatagcccattggaagttgggctgaaggtggtggcacctgagactg ggctgccgcctcctcccccgacacctgggcaggttgacgttgagtggctccactgtggacaggtgacccgtttgttctgatgagcggacaccaaggtcttactgtc ctgctcagctgctgctcctacacgttcaaggcaggagccgattcctaagcctccagcttatgcttagcctgcgccaccctctggcagagactccagatgcaaaga gccaaaccaaagtgcgacaggtccctctgcccagcgttgaggtgtggcagagaaatgctgcttttggcccttttagatttggctgcctcttgccaggagtggtggct cgtgcctgtaattccagcactttgggagactaaggcgggaggttcgcttgagcccaggagttcaagaccagcctgggcaacaatgagacccctgtgtctacaa aaagaattaaaattagccaggtgtggtggcacgcacctgtagtcccagctacttgggaggctgaggtgggaggattgcctgagtccgggaggcggaagttgca aggagccatgatcgcgccactgcacttcaacctaggcaacagagtgagactttgtctcaaaaaacaatcatataataattttaaaataaatagatttggcttcctct aaatgtccccggggactccgtgcatcttctgtggagtgtctccgtgagattcgggactcagatcctcaagtgcaactgacccacccgataagctgaggcttcatca tcccctggccggtctatgtcgactgggcacccgaggctcctctcccaccagctctcttggtcagctgaaagcaaactgttaacaccctggggagctggacgtatg agacccttggggtgggaggcgttgatttttgagagcaatcacctggccctggctggcagtaccgggacactgctgtggctccggggtgggctgtctccagaaaat gcctggcctgaggcagccacccgcatccagcccagagggtttattcttgcaatgtgctgctgcttcctgccctgagcacctggatcccggcttctgccctgaggcc ccttgagtcccacaggtagcaagcgcttgccctgcggctgctgcatggggctaactaacgcttcctcaccagtgtctgctaagtgtctcctctgtctcccacgccctg ctctcctgtccccccagtttgtctgctgtgaggggacagaagaggtgtgtgccgcccccacccctgcccgggcccttgttcctgggattgctgttttcagctgtttgag ctttgatcctggttctctggcttcctcaaagtgagctcggccagaggaggaaggccatgtgctttctggttgaagtcaagtctggtgccctggtggaggctgtgctgct gaggcggagctggggagagagtgcacacgggctgcgtggccaacccctctgggtagctgatgcccaaagacgctgcagtgcccaggacatctgggacctc cctggggcccgcccgtgtgtcccgcgctgtgttcatctgcgggctagcctgtgacccgcgctgtgctcgtctgcgggctagcctgtgtcccgcgctctgcttgtctgc ggtctagcctgtgacctggcagagagccaccagatgtcccgggctgagcactgccctctgagcaccttcacaggaagcccttctcctggtgagaagagatgcc agcccctggcatctgggggcactggatccctggcctgagccctagcctctccccagcctgggggccccttcccagcaggctggccctgctccttctctacctggg acccttctgcctcctggctggaccctggaagctctgcagggcctgctgtccccctccctgccctccaggtatcctgaccaccggccctggctcccactgccatcca ctcctctcctttctggccgttccctggtccctgtcccagcccccctccccctctcacgagttacctcacccaggccagagggaagagggaaggaggccctggtcat accagcacgtcctcccacctccctcggccctggtccaccccctcagtgctggcctcagagcacagctctctccaagccaggccgcgcgccatccatcctccctg tcccccaacgtcctgccacagatcatgtccgccctgacacacatgggtctcagccatctctgccccagtaactccccatccataaagagcacatgccagctga caccaaaataatcgggatggtccagttagacctaagtggaaggagaaaccaccacctgccctgcacctgtttttggtgacctgataaaccatctcagcca tgaagccagctgtctcccaggaagctccagggcggtgctcctcgggagctgactgataggtgggaggtggctgccccctgcaccctcaggtgaccccacac aaggccactgctggaggccctggggactccaggaatgtcaatcagtgacctgccccccaggccccacacagccatggctgcatagaggcctgcctccaagg gacctgtctgtctgccactgtggagtccctacagcgtgccccccacaggggagctggtcttgactgagatcagctggcagctcagggtcatcatcccagagg gagcggtgccctggaggccacaggcctcctcatgtgtgtctgcgtccgctcgagctactgagacactaaatctgtggttctgctgtgccacctacccaccctgtt ggtgttgcttgtcctatgctaaagacaggaatgtccaggacactgagtgtgcaggtgcctgctggtctcacgtccgagctgctgaactccgctgggtcctgcta ctgatggtctttgctctagtgcttccagggtccgtggaagcttcctggaataaagcccacgcatcgaccctcacagcgcctcccctcttgaggcccagcagata ccccactcctgccttccagcaagatttcagatgctgtgcatactcatcatatgatcactttttctcatgcctgatgtgatctgtcaatttcatgtcaggaaaggga gtgacatttacacttaagcgttgctgagcaaatgtctgggtctgcacaatgacaatgggtccctgttttcccagaggctcttgtctgcagggatgaagacac tccagtcccacagtccccagctcccctggggcagggtggcagaattcgacaacacatttccaccctgactaggatgtgctcctcatggcagctgggaacca ctgtccaataagggcctgggctacacagctgctctcatgagtacaccctaataaaataatcccattttatcctttttgtctctctgtctcctctctctctgccttcctct tctctctcctcctctctcatctccaggtgcaaatagtctacaaaccagtgacctgagcaaggtgacctccaagtgtggctcataggcaacatccatcataaacca ggtagccctgtggaaggtgagggtgggacgggagggtgcagggggtggaggagtcctggtgaggctggaactgctccagactcagaaggggctggaaa ggatattttaggtagacctacatcaaggaaagtgtgagtgtgaaacttgcgggagcccaggaggcgtggtggctccagctcgctcctgcccaggccatgctgc ccaagacaaggtgaggcgggagtgaagtgaaataaggcaggcacagaaagaaagcacatattctcggccgggcgctgtggctcacgcctgtaatccagc acttgggaggccaaggtgggtggatcatgaggtcaggagatgagaccatcctggctaacacagtgaaaccccgtctctactaaaaatacaaaaaatagcc gggcgtggtggtgggcgcctgtagtcccagctactccggaggctgaggcaggaaaatggcgtgaacccggaaggcggagctgcagtgagcggagtgagc agagatcgcgccactgcactccagcctgggcgacagagcgagactccgtctcaaaaaaaaaaagcacatgtt b) After first editing step: endogenous MAPT locus with integrated repair plasmid (Fig. 4C)

[0223] DNA Sequence of genomic region after first editing step (SEQ ID NO: 8):

[0224] Agggctctcaaacctggctgtgtgtcagaatcaccaggggaacttttcaaaactagagagactgaagccagactcctagatctaatctaggtcagggctagg ggctgagatgtaaaaatccacaggtgatctgatgcccggcaggctgagaacagccgcagggagtctctgggaatgtgccggtgggtctagccaggtgtga gtggagatgccggggaactcctatactcactcgtcagtgtggccgaacacatttcactgacctcaggctggtgaacgctcccctctggggtcaggcctcac gatgccatccttttgtgaagtgaggacctgcaatcccagctcgtaaagcccgctggaaatcactcacactctgggatgcctcagagcagccctctatccctca gctcccctgggatgtgactcaacctcccgtcactccccagactgcctctgccaagtccgaaagtggaggcatcctgcgagcaagtaggcgggtccagggtgg cgcatgtcactcatcgaaagtggaggcgtcctgcgagcaagcaggcgggtccagggtggcgtgtcactcatccttttttctggctaccaaaggtgcagataata accctagaaagatagtctgcgtaaaatgacgcatgcatctgaaatatgctctctcttctaaatagcgcgaatccgtcgctgtgcatttaggacatctcagtcgc cgctggagctcccgtgaggcgtgctgtcaatgcggtaagtgtcactgattgaactataacgaccgcgtgagtcaaaatgacgcatgatatcttttacgtgact ttaagatttaactcatacgataatatatgtatttcatgtctactacgtgataactatatatatatattctgtatagatatcaactagaatgctagcatgggcccat ctcgaccgccaatcaatatggcgtatatggactcatgccaatcaatatggtggatctggacctgtgccaatcaatatggcgtatatggactcgtgccaatcaat atggtggatctggaccccagccaatcaatatggcggactggcaccatgccaatcaatatggcggactggcactgtgccaactggggaggggtctactggc acggtgccaagttgaggaggggtcttggccctgtgccaagtccgccatatgaatggcatggtgccaataatggcggccatattggctatatgccaggatcaat atataggcaatatccaatatggccctatgccaatatggctatggccaggtcaatactatgtatggccctatgccatatagtattccatatatgggttcctattgac gtagatagcccctcccaatgggcggtcccatataccatatatggggctcctaataccgcccatagccactcccccatgacgtcaatggtctctatatatggtcttc ctattgacgtcatatgggcggtcctatgacgtatatggcgcctcccccatgacgtcaattacggtaaatggcccgcctggctcaatgcccatgacgtcaatagg accacccaccatgacgtcaatgggatggctcatgcccatcatatccgtctcacgccccctatgacgtcaatgacggtaaatggcccactggcagtacatca atatctataatagtaactggcaagtacatactatggaaggacgccagggtacatggcagtactcccatgacgtcaatggcggtaaatggcccgcgatggc tgccaagtacatccccatgacgtcaatggggaggggcaatgacgcaaatgggcgttccatgacgtaaatgggcggtaggcgtgcctaatgggaggtctatat aagcaatgctcgttagggaaccgccatctgcctggggacgtcggagcaagctgattaggtgacactatagaatacaagctactgtcttttgcaggatccca tcgatcgaatccatggggaccgagtacaagcccacggtgcgcctcgccacccgcgacgacgtcccccgggccgtacgcaccctcgccgccgcgtcgccg actaccccgccacgcgccacaccgtcgacccggaccgccacatcgagcgggtcaccgagctgcaagaactcttcctcacgcgcgtcgggctcgacatcggc aaggtgtgggtcgcggacgacggcgccgcggtggcggtctggaccacgccggagagcgtcgaagcgggggcggtgtcgccgagatcggcccgcgcatg gccgagtgagcggtcccggctggccgcgcagcaacagatggaaggcctcctggcgccgcaccggcccaaggagcccgcgtggtcctggccaccgtcg gcgtctcgcccgaccaccagggcaagggtctgggcagcgccgtcgtgctccccggagtggaggcggccgagcgcgccggggtgcccgcctcctggagac ctccgcgccccgcaacctcccctctacgagcggctcggctcaccgtcaccgccgacgtcgaggtgcccgaaggaccgcgcacctggtgcatgacccgcaa gcccggtgccggatccatgcccacgctactgcgggtttatatagacggtcctcacgggatggggaaaaccaccaccacgcaactgctggtggccctgggtcg cgcgacgatatcgtctacgtacccgagccgatgactactggcaggtgctgggggctccgagacaatcgcgaacatctacaccacacaacaccgcctcgac cagggtgagatatcggccggggacgcggcggtggtaatgacaagcgcccagataacaatgggcatgcctatgccgtgaccgacgccgtctggctcctcat atcgggggggaggctgggagctcacatgccccgcccccggccctcaccctcatctcgaccgccatcccatcgccgccctcctgtgctacccggccgcgcgat acctatgggcagcatgaccccccaggccgtgctggcgtcgtggccctcatcccgccgacctgcccggcacaaacatcgtgttgggggccctccggaggac agacacatcgaccgcctggccaaacgccagcgccccggcgagcggctgacctggctatgctggccgcgatcgccgcgttacgggctgctgccaatacg gtgcggtatctgcagggcggcgggtcgtggcgggaggatggggacagcttcggggacggccgtgccgccccagggtgccgagccccagagcaacgcg ggcccacgaccccatatcggggacacgtattaccctgttcgggcccccgagtgctggcccccaacggcgacctgtacaacgtgtttgcctgggcctggac gtcttggccaaacgcctccgtcccatgcacgtctttatcctggatacgaccaatcgcccgccggctgccgggacgccctgctgcaactacctccgggatggtcc agacccacgtcaccacccccggctccataccgacgatctgcgacctggcgcgcacgttgcccgggagatgggggaggctaactgagctctagagctcgctg atcagcctcgactgtgcctctagtgccagccatctgtgttgcccctcccccgtgcctcctgaccctggaaggtgccactcccactgtccttcctaataaaatga ggaaatgcatcgcatgtctgagtaggtgtcatctatctggggggtggggtggggcaggacagcaagggggaggatgggaagacaatagcaggcatgct ggggatgcggtgggctctatggctctgaggcgggaagtcctatacttctagagaataggaactcactagtaaaagttgttacttatagaagaaattttgagt ttgttttttttaataaataaataaacataaataaatgttgtgaatttatatagtatgtaagtgtaaatataataaaactaatatctattcaaataataaataaacct cgatatacagaccgataaaacacatgcgtcaattttacgcatgatatcttaacgtacgtcacaatatgatatctttctagggtaataagaagctggatcttagca acgtccagtccaagtgtggctcaaaggataatatcaaacacgtcccaggaggcggcagtgtgcaaatagtctacaaaccagtgacctgagcaaggtgacct ccaagtgtggctcataggcaacatccatcataaaccaggtagccctgtggaaggtgagggtgggacgggagggtgcagggggtggaggagtcctggtga ggctggaactgctccagactcagaaggggctggaaaggatatttaggtagacctacatcaaggaaagtgtgagtgtgaaactgcgggagcccaggagg cgtggtggctccagctcgctcctgcccaggccatgctgcccaagacaaggtgaggcgggagtgaagtgaaataaggcaggcacagaaagaaagcacatat tctcggccgggcgctgtggctcacgcctgtaatccagcacttgggaggccaaggtgggtggatcatgaggtcaggagatgagaccatcctggctaacacag tgaaaccccgtctctactaaaaatacaaaaaatagccgggcgtggtggtgggcgcctgtagtcccagctactccggaggctgaggcaggaaaatggcgtga acccggaaggcggagctgcagtgagcggagtgagcagagatcgcgccactgcactccagcctgggcgacagagcgagactccgtctcaa c) After second editing step: fused Exon 10 and 11 in the endogenous MAPT locus (Fig. 4D) DNA Sequence of genomic region after second editing step (SEQ ID NO: 9): aactagagagactgaagccagactcctagattctaattctaggtcagggctaggggctgagattgtaaaaatccacaggtgattctgatgcccggcaggcttga gaacagccgcagggagttctctgggaatgtgccggtgggtctagccaggtgtgagtggagatgccggggaacttcctattactcactcgtcagtgtggccgaac acatttttcacttgacctcaggctggtgaacgctcccctctggggttcaggcctcacgatgccatccttttgtgaagtgaggacctgcaatcccagcttcgtaaagcc cgctggaaatcactcacacttctgggatgccttcagagcagccctctatcccttcagctcccctgggatgtgactcaacctcccgtcactccccagactgcctctgc caagtccgaaagtggaggcatccttgcgagcaagtaggcgggtccagggtggcgcatgtcactcatcgaaagtggaggcgtccttgcgagcaagcaggcgg gtccagggtggcgtgtcactcatccttttttctggctaccaaaggtgcagataattaataagaagctggatcttagcaacgtccagtccaagtgtggctcaaaggat aatatcaaacacgtgcTgggaggcggcagtgtgcaaatagtctacaaaccagttgacctgagcaaggtgaccttcaagtgtggctcattaggcaacatccatc ataaaccaggtagccctgtggaaggtgagggttgggacgggagggtgcagggggtggaggagtcctggtgaggctggaactgctccagacttcagaaggg gctggaaaggatattttaggtagacctacatcaaggaaagtgttgagtgtgaaacttgcgggagcccaggaggcgtggtggctccagctcgctcctgcccagg ccatgctgcccaagacaaggtgaggcgggagtgaagtgaaataaggcaggcacagaaagaaagcacatattctcggccgggcgctgtggctcacgcctgt aattccagcactttgggaggccaaggtgggtggatcatgaggtcaggagattgagaccatcctggctaacacagtgaaaccccgtctctactaaaaatacaaa aaattagccgggcgtggtggtgggcgcctgtagtcccagctactccggaggctgaggcaggaaaatggcgtgaacccggaaggcggagcttgcagtgagc ggagtgagcagagatcgcgccactgcactccagcctgggcgacagagcgagactccgtctcaaaaaaaaaaag CRISPR design and efficiency testing gRNAs for CRIPSR-editing were designed using CRISPOR (http: / / crispor.tefor.net)and selected based on their predicted efficiency and specificity. The five most probable off-targets predicted by both the MIT and CFD scoring system embedded into CRISPOR were selected for later off-target analysis (see below), respectively. 3-5 gRNAs for each edit were tested using a plasmid-based transfection approach to perform CRISPR / Cas9 editing in HEK293 cells and selected the most efficient candidate for editing in iPSCs. This was determined by seguencing the pool of edited HEK293 cells and assessing the editing efficiency using the ICE CRISPR Analysis Tool from Synthego (https: / / ice.synthego.com). To achieve a knock in of the different mutations, simple symmetric repair oligonucleotides were designed that contained 50 base pair homology arms flanking the desired point mutation. Forthe large-scale 4R-splice editing, the large repair template for HDR was designed as a plasmid. To generate this plasmid, 500 base pair long MAPT homology arms, the terminal repeats reguired for recognition by PiggyBac transposase, a puromycin resistance, a thymidine kinase, and the fused Tau Exons 10 and 11 were cloned into the pCR™8 / GW / TOPO™ TA cloning vector (ThermoFisher, K252020)). The details of genome editing to induce 4R Tau expression are described in Example 3.

[0225] CRISPR editing of iPSC to generate mutant cell lines

[0226] CRISPR-editing was performed as described previously (Paguet et al., 2016;). Briefly, iPSCs were cultured as single cells for 48h prior to electroporation with either (1) a plasmid-based approach for 4R splice editing (or (2) a ribonucleotide particle (RNP) approach for knock in of the different mutations.

[0227] For the (1) plasmid-based approach, Cas9-puromycin plasmid (pSpCas9(BB)-2A-Puro (PX459) V2.0, a gift from F. Zhang, Addgene 62988) the gRNA cloned into the BsmBI restriction site of plasmid MLM3636 (a gift from K. Joung, Addgene 43860) and a large repair template provided as plasmid were electroporated into the cells. Briefly, two million cells were resuspended in 100 pL cold BTXpress electroporation solution (VWR 732-1285) with 20 pg Cas9, 5 pg gRNA plasmid for each of the two gRNAs, and 20 pg repair plasmid. Cells were electroporated with 2 pulses at 65 mV for 20 ms in a 1 mm cuvette (Fisher Scientific, 15437270). Following electroporation, cells were cultured at very low density as single cells on Geltrex-coated (ThermoFisher, A1413302) 10 cm dishes in StemFlex Medium (ThermoFisher, A3349401) supplemented with RevitaCell (ThermoFisher, ThermoFisher). Cells that integrated the PiggyBac repair plasmid were selected using 350 ng / ml Puromycin dihydrochloride (VWR J593) starting two or three days after electroporation.

[0228] For the (2) RNP approach, 30 pmol Cas9 protein (Alt-R® S.p. HiFi Cas9 Nuclease V3, IDT, 1081060) was pre-complexed with 60 pmol gRNA (ordered from Synthego) for 20 mins. 250k iPSCs were resuspended in 20 pl P3 buffer with supplement (P3 Primary Cell 4D X Kit, Lonza, V4XP-3032) and added to the RNP complex together with 120 pmol repair oligonucleotide (100 base pair oligonucleotides ordered as Ultramer from IDT). The suspension was transferred to a cuvette (P3 Primary Cell 4D X Kit, Lonza, V4XP-3032) and electroporated with the CA137 program of the Lonza 4D-Nucleofactor. Following electroporation, iPSCs were plated in 1 well of a Geltrex-coated 12-well plate in StemFlex Medium supplemented with RevitaCell. Cells were split at low density 3 days after electroporation onto Geltrex-coated 10 cm dishes. When iPSC-colonies had formed after about 7-10 days in culture, single colonies were manually selected and genotyped as described below.

[0229] For the 4R splice edited iPSC, the PiggyBac selection cassette was removed in a second step by electroporating the cells with integration deficient PiggyBac transposase which allows scarless editing and thus scarless fusion of Exons 10 — 11. The cells were selected for loss of the selection cassette using 1-(2-deoxy-2-fluoro-p-darabinofuranosyl)-5-iodouracil (FIAU, 0,5 pM) for 48h starting 72h after editing. FIAU kills all cells still containing the transgenic Thymidine kinase. Single cell clones were selected as described above and subjected to genotyping. The details of the 4R splice editing steps are described in example 3.

[0230] Genotyping of edited cell lines

[0231] Selected colonies were analyzed in 96-well format for the desired edit via PCR and restriction fragment length polymorphism (RFLP). To genotype the edited cell lines and confirm correct editing, a set of different PCRs was used, in particular to confirm large edits including exon 10-11 fusion and intron deletion. Successful knock in of the different mutations was additionally confirmed using RFLP analysis after restriction enzyme digests according to manufacturer’s instructions (NEB). The edits that induced 4R expression from one or both alleles were further confirmed using qPCRs to determine the number of WT alleles and alleles with exon 10-11 fusion remaining after editing. Successful removal of the PB transposon was further confirmed with absence of amplification in a PB specific PCR and qPCR. The different sets of primers and restriction enzymes are summarized in the table below. All relevant PCRs were Sanger sequenced to confirm scarless editing and correct integration of mutations in the absence of any other undesired edits. Positive clones were expanded and subjected to extensive quality controls, including repeated genotyping, pluripotency testing, testing for iPSC-typical triplication of the Bcl2l1 locus, testing for clonality of the established lines, line identity, on-target effects, off-target effects, and molecular karyotyping as described in (Weisheit et al., 2021).

[0232] Example 2. Differentiation of iPSC-derived brain cells for long-term culture:

[0233] The published protocol (Dannert et al., 2023) has been optimized to efficiently differentiate iPSCs into pure and electrically active human cortical neurons, which display typical mature morphologies, and activities (Fig. 1A-F). Protocols for long-term (1-12 months) culture were established, which is critical to allow enough time for development of late-stage phenotypes (Fig. 1G). Protocols for astrocyte differentiation are published by Perriot et al., 2021 , methods for oligodendrocyte differentiation are described by Ehrlich et al, 2017. Culturing conditions of iPSCs are described in Dannert et al., 2023.

[0234] Example 3. Genome-editing to induce 4R Tau expression:

[0235] To alter expression of 4R Tau, the intron between exons 10 and 11 was removed and said exons fused with single-base precision by genome engineering, thus forcing expression of 4R Tau from the physiological locus without any non-physiological overexpression or alterations of the protein sequence (Fig. 2A).

[0236] To achieve a 1 :1 splice ratio between 3R and 4R Tau in human iPSC-derived neurons early after differentiation, CRISPR / Cas9 editing was used to force expression of 4R Tau from one allele, while the second unedited allele continues to express 3R Tau (Fig. 2C). Alternatively, to generate a cell line that expresses 100% 4R Tau, both alleles are edited (Fig.2C), resulting in both alleles expressing 4R Tau by default. In order to force 4R Tau expression in human iPSC-derived neurons, iPSCs are edited using a three-step approach combining CRISPR / Cas9 genome editing (Paquet et al., 2016) and the scarless PiggyBac transposon system (Figure 2). This allows for seamless editing of a desired locus when inserted into a site containing the sequence TTAA (Wang et al., 2017, Yusa et al., 2013).

[0237] (1) In a first step, to mediate removal of intron 10-11 and precise fusion of the adjacent exons, CRISPR / Cas9 is targeted to the endogenous human MAPT locus using two guide RNAs (gRNA, Table 1) to remove the entire intron between exons 10 and 11 along with the 3’ and 5’ end of exons 10 and 11 , respectively (Figure 2B). This intron contains many regulatory elements for alternative splicing of Exon 10 including the splice acceptor. The region between the resulting double strand breaks (DSB) is replaced with a PiggyBac transposon via homology directed repair (HDR) using a repair-template encoding plasmid (see SEQ ID NO: 5). For precise insertion, the template also contains arms with homology to the Tau locus, and the ITR-flanked resistance gene is inserted between the TT and AA sequences at a TTAA PiggyBac target site present on the endogenous sequence. The transposon harbors a drug selection cassette which allows for both positive antibiotic selection and negative selection via Thymidin kinase. It was confirmed for all clones by PCR analysis that the transgene was correctly inserted. Moreover, it contains the scarless fused 3’ and 5’ ends of exons 10 and 11 to replace the previously removed exonal parts (Figure 2B).

[0238] (2) In a second step, antibiotic selection is used to enrich for single cell clones that successfully integrated the PiggyBac transposon to counteract the low efficiency of this large-scale edit.

[0239] (3) In the third editing step, integration deficient PiggyBac transposase is used to excise the entire transgenic selection cassette, leaving the scarlessly fused exons 10 and 11 in the endogenous human MAPT locus. PiggyBac transposase was expressed in the cells to remove the ITR-flanked resistance transgene between the TT and AA of the endogenous TTAA site, effectively fusing the original sequence upstream of TT and downstream of AA. As a result, Exons 10 and 11 are fused with single-base-pair precision, which were confirmed by PCR and sequencing analysis (Fig. 2B). The HDR repair template is designed in a way that the PiggyBac transposon is integrated at a naturally existing TTAA site within Exon 10, thus removal of the transposon is scarless and does not leave any alterations other than the intended ones in the genomic MAPT locus. Negative selection for removal of the transgene is performed using FIAU which enriches for clones without the Thymidin-kinase containing PiggyBac transposon.

[0240] (4) The aggregation-inducing mutation (e.g., P301 L) and the nucleation promoting mutation (e.g., S320F) are either directly incorporated into the HDR repair template or alternatively added in subsequent editing steps using CRISPR / Cas9 or other editing tools on the same or the other allele. Using patient lines that already contain one of the mutations is another alternative.

[0241] If only one allele is edited this way, it is critical to confirm that the unedited allele has not been modified by undesired genome editing events, which we confirmed by PCR, qPCR, and sequencing analysis. Importantly, this strategy maintains physiological expression of Tau with an unchanged primary amino acid sequence.

[0242] Editing only one of the two alleles in a cell leads to expression of 4R (from edited allele) and 3R (from unedited allele) at the desired 1 :1 ratio; editing both alleles, for example by double-selection with two different antibiotic resistance genes on the two alleles, or by sequential editing of one allele followed by the other allele, we can obtain 100% 4R expression (Fig. 2C). The inventors have confirmed 3R / 4R expression by qPCR, Western blotting, and immunofluorescence (IF) stainings (Fig. 2D-E).

[0243] Example 4. Genome-editing to insert synergistic P301 L and S320F mutations:

[0244] Two pathogenic Tau mutations P301 L and S320F that synergistically promote Tau aggregate nucleation and elongation (Combs et al., 2012) either alone or in combination into iPSC-lines expressing different 3R / 4R Tau isoform ratios were inserted (Fig. 2F). Insertion can either be done alongside editing of 4R expression as described above by adding the respective mutations to homology arms, or subsequently by knock-in into pre-edited lines using our CRISPR / Cas9 genome editing pipeline. Successful integration of mutations was confirmed by PCR and sequencing analysis.

[0245] Overall, a panel of 8 isogenic iPSC lines with combinations of splicing and pathogenic mutations, as well as controls (Table 1 and Fig. 3A) has been established. All lines have been validated by standard iPSC quality controls, as laid out in Weisheit et al., 2021 .

[0246] Example 5. Characterization of tauopathy phenotypes:

[0247] In orderto characterize formation of disease-relevant pathology in the present iPSC model, the inventors set up assays to confirm that the model recapitulates late-stage tauopathy phenotypes: a. Tau hyperphosphorylation and mislocalization

[0248] There is a large panel of phospho-specific antibodies used by the field to detect these modifications (Wang et al., 2016). The inventors assayed the model with various antibodies for P-Tau, such as AT8, and found accumulation of phosphorylated Tau in the somata of affected neurons. From the P301 L / S320F / 4R cell line after 4-5 months in culture (Fig. 3A). Furthermore, the inventors confirmed presence of oligomeric Tau in the somata of mutant neurons using the T-22 antibody (Fig. 3A) b. Formation of pathologic Tau seeds

[0249] To detect seeding-competent material in our cultures, protein from the cells was extracted, subjected to Sarkosyl extraction and analyzed its soluble and insoluble fractions for seeding competence in a published, highly sensitive Tau FRET Biosensor cell line (Holmes et al., 2014), which is broadly used in the field to investigate formation of seeding-competent material. The inventors established microscopy and FACS-based readouts to detect the FRET signal produced by the biosensor cells and found that a significant number of seeds is produced endogenously in the P301 L / S320F / 4R cell line after 3-4 months in culture (Fig. 3B) and in the P301 L / S320F / 4R-hom cell line (Fig. 4A-C). c. Tau misfolding

[0250] It is believed that Tau becomes not only phosphorylated, but also misfolded before it aggregates in tangles. Misfolding of Tau can be detected with specific antibodies, such as MC-1 or Alz50 (Jicha et al., 1999). The inventors found that Tau becomes misfolded in the P301 L / S320F / 4R cell line in a stereotyped manner after a culture period of about 5-7 months, with a gradual increase in MC-1 positive cells (Fig. 3D) and in the P301 L / S320F / 4R-hom line (Fig. 4 D-E). d. Tau aggregation

[0251] Tau phosphorylation and misfolding promote aggregation of Tau in tangles containing parallel betasheets, which can be stained with dyes, such as AmyTracker, X34, or pFTAA. The data in Fig. 3E suggests that in the P301 L / S320F / 4R cell line Tau starts to aggregate after about 8-9 months, forming Amytracker, X34, and pFTAA-positive cell bodies.

[0252] The present disclosure is the first iPSC-based model that reproducibly recapitulates multiple late-stage phenotypes of tauopathies, such as Tau misfolding and aggregation solely based expression of Tau from the endogenous locus. Furthermore, it is also the first model displaying endogenous formation of a significant amount of Tau seeds. This aspect of the present disclosure is particularly interesting, as this assay in principle allows screening for disease modulators. This way, the present disclosure is the first model in which identification of human disease pathways and compounds modulating them as a treatment for patients may become reality. e. Acceleration and intensification of phenotype formation

[0253] Investigating the cell line in which not one but both alleles are edited to express P301 L / S320F / 4R Tau (referred to as the P310L / S320F / 4R-hom cell line, see Fig. 3A) showed that while the same phenotypes are observed as in neurons expressing 3R and 4R Tau in a 1 :1 ratio, the extent of affected neurons was larger, and the timeline of phenotype formation was accelerated. After 4 months in culture, lysates from P301 L / S320F / 4R-hom neurons resulted in 40% conversion of HEK293 Tau biosensor cells compared to 5% conversion achieved with neurons with one edited allele (Fig. 4A-B). Formation of seeding competent material is accelerated by about 2-3 months (Fig. 4C). In addition, misfolding of Tau as detected with the MC-1 antibody is strongly increased, resulting in a 4-5-fold increase in detected MC- 1 positive structures (Fig. 4D-E). f. Detection of neurodeqeneration

[0254] One of the most advanced hallmarks of Tauopathy is neurodegeneration. First signs of neurodegeneration are detectable in heterozygous and homozygous P301 L / 320F / 4R neurons after 5 months in culture, as indicated by a decrease in detectable synapses (Fig. 4F-G). The extent of pathology is larger in 100% mutant 4R expressing cells compared to heterozygous neurons. g. Application of the model for testing modulators of Tau pathology

[0255] The ability to modulate the observed phenotype is confirmed by treatment with the disaggregating compound Anle-138 (Wagner et al. 2015) as shown in Fig. 4H, thus confirming usability of this phenotype for drug screening in principle. h. Application of model to test PET-tracers

[0256] The inventors determined that the aggregates that are formed in affected neurons are detectable by the Tau PET-Tracer PI-2620 (Mueller et al. 2020) that is validated for detection of aggregates in patient brain (Fig. 41-J). In vitro testing of this tracer in Tau mutant neurons demonstrates a specific increase of radioactive signal in P301 L / S320F / 4R-hom neurons compared to WT, thus underlining the usability of the presented model to screen potential Tau PET-T racers.

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Claims

CLAIMS1 . A human ectoderm derived brain cell characterized by the following features:(1) the intron between exons 10 and 11 of the MAPT gene encoding the Tau protein has been removed by genome-editing from one or both allele(s); and(2) at least one of the alleles of the MAPT gene of (1) carries at least one mutation enhancing Tau aggregation and at least one mutation enhancing nucleation of Tau aggregation; or(3) one allele of the MAPT gene of (1) carries at least one mutation enhancing Tau aggregation, preferably in exon 10, and the other allele carries at least one mutation enhancing nucleation of Tau aggregation, preferably in exon 11.

2. The ectoderm derived brain cell of claim 1 , wherein at least one mutation that enhances Tau aggregation is selected from a group comprising: R5H, R5L, K257T, I260V, L266V, N279K, A280K, P301 L, P301 S, G335V, Q336H, Q336R, V337M, E342V, S352L, P346S, E372G, and N401 H.

3. The ectoderm derived brain cell of claim 1 or 2, wherein at least one mutation that enhances nucleation of Tau aggregation is selected from a group comprising: G272V, G303V, L315R, S320F, S320I, S320V, I328V, S352L, S356T and G389R.

4. The ectoderm derived brain cell of claims 2 or 3, wherein the mutation that enhances Tau aggregation is P301 L and the mutation that enhances nucleation of Tau aggregation is S320F.

5. The ectoderm derived brain cell of any one of claims 1 to 4, wherein said cell is a neuron, an astrocyte, or an oligodendrocyte.

6. The ectoderm derived brain cell of any one of claims 1 to 5, wherein the second allele of the MAPT gene contains the intron between exon 10 and 11 .

7. The ectoderm derived brain cell of any one of claims 1 to 5, wherein the intron between exon 10 and 11 in both alleles of the MAPT gene has been removed by gene editing.

8. A method for producing the ectoderm derived brain cell of any one of claims 1 to 7, the method comprising:(i) inducing differentiation in human induced pluripotent stem cells (hiPSCs), thereby obtaining neural precursor cells;(ii) expanding the neural precursor cells of step (i);(iii) obtaining neuronal, astroglial, or oligodendroglial cells derived from the cells of step (ii);(iv) prior to, simultaneously with or after any one of steps (i), (ii) or (iii), removing the intron between exons 10 and 11 of the MAPT gene encoding the Tau protein by genome-editingfrom one or both allele(s), thereby inducing endogenous 4R Tau expression;(v) prior to, simultaneously with or after step (iv),(a) inserting or providing at least one mutation enhancing Tau aggregation in the modified allele(s) of step (iv) lacking the intron between exons 10 and 11 of the MAPT gene; or(b) inserting or providing at least one mutation enhancing Tau aggregation in the allele containing the intron between exons 10 and 11 of the MAPT gene, and(c) inserting or providing at least one mutation enhancing nucleation of Tau aggregation in the modified allele(s) of step (iv) or(d) inserting or providing at least one mutation enhancing nucleation of Tau aggregation in the allele containing the intron between exons 10 and 11 of the MAPT gene; or in the case where at least one allele carries the deletion in the intron between exon 10 and 11 , thereby inducing mutated 4R Tau expression, and at least one or no additional mutations enhancing Tau aggregation or enhancing nucleation of Tau aggregation, exogenously providing a Tau variant carrying at least one mutation enhancing Tau aggregation and / or at least one mutation enhancing nucleation of Tau aggregation so that the cell carries at least one mutation enhancing Tau aggregation and at least one mutation enhancing nucleation of Tau aggregation; and(vi) obtaining the ectoderm derived brain cell.

9. The method of claim 8(v), wherein: at least one mutation that enhances Tau aggregation is selected from a group comprising: R5H, R5L, K257T, I260V, L266V, N279K, A280K, P301 L, P301 S, G335V, Q336H, Q336R, V337M, E342V, S352L, P346S, E372G, and N401 H, more preferably P301 L; and at least one mutation that enhances nucleation of Tau aggregation is selected from a group comprising: G272V, G303V, L315R, S320F, S320I, S320V, I328V, S352L, S356T and G389R, more preferably S320F.

10. The method of claim 8(i), wherein the method comprises inducing neural differentiation in hiPSCs (a) by dual-SMAD inhibition; or (b) by expression of transcription factors selected from the group including Ngn2, Ngn1 , ASCL1 , BRN2, MYT 1 L, and NEUROD1 .

11. The method of claim 8(i) and (ii), wherein the neural precursor cells are expanded in neural rosettes.

12. The method of claim 8(v), wherein the mutations are inserted by genome-editing.

13. The method of claim 8(v), wherein one of the mutations is derived from a patient, while the other is inserted by genome-editing.

14. The method of claim 8, the method comprising in a further step (vii) characterizing a tauopathy phenotype in the obtained ectoderm derived brain cell.

15. A composition comprising the ectoderm derived brain cell according to any one of claims 1 to 7 or the cells produced by the method according to any one of claims 8 to 14.

16. The composition of claim 15, which is a kit.

17. Use of ectoderm-derived brain cells as defined in any of the preceding claims for screening drugs useful in preventing or treating tauopathies.

18. A method of screening drugs useful in the development of the prevention or treatment of tauopathies, the method comprising:(a) contacting the cell of any one of claims 1 to 8 with potential drugs under conditions that allow the interaction of the drugs with the Tau proteins produced by the cells obtained in any one of claims 8 to 13;(b) observing at least one of the following phenotypes(ba) Tau hyperphosphorylation;(bb) Tau mislocalization;(be) formation of Tau seeds, optionally, pathological Tau seeds;(bd) Tau misfolding; and(be) Tau aggregation; wherein modulation of at least one of said phenotypes indicates that the drug is useful in the development of the prevention or treatment of tauopathies.

19. The method of claim 18, comprising observing at least two, preferably at least three and more preferably at least four phenotypes of (ba) to (be).

20. Use of ectoderm-derived brain cells as defined in any of the preceding claims for genetic screening to identify genes / proteins and pathways involved in tauopathy pathogenesis.21 . A method for screening for genes involved in the development or modulation of any of the tauopathy phenotypes as defined in claim 18 (ba) to (be), the method comprising:(a) contacting the ectoderm derived brain cell of any one of claims 1 to 8 with a guide RNA (gRNA) or a plurality of gRNAs under conditions that allow the interaction of the gRNA(s) with the DNA in a cell obtained in any one of claims 8 to 13;(b) expressing a genome-editing system in the cell of (a) that:(ba) reduces or prevents expression of a target gene by cleavage of its genomic locus; or(bb) reduces or prevents expression of a target gene by its downregulation; or(be) increases expression of a target gene by its upregulation;(c) observing in the cell of (b) whether an alteration of at least one of the following phenotypesoccurs:(ca) Tau hyperphosphorylation;(cb) Tau mislocalization;(cc) formation of Tau seeds, optionally, pathological Tau seeds;(cd) Tau misfolding; and(ce) Tau aggregation; wherein alteration of at least one of said phenotypes indicates that the genetic alteration is involved in a pathway modulating the generation of tauopathies; and(d) Identifying the gene(s) involved in the development or modulation of any of tauopathy phenotypes (ba) to (be) by identifying the active guide RNA(s) from (a).

22. The use of claims 17 or 20 or the method of any one of claims 18, 19, and 21 , wherein the tauopathies are selected from the group of Alzheimer’s disease, Frontotemporal dementia, progressive supranuclear palsy, corticobasal degeneration, chronic traumatic encephalopathy, Pick’s disease, and Parkinsonism.

23. A method fortesting the potential of a Positron-Emission-Tomography (PET) tracer for detecting tau aggregates comprising:(vi) contacting and incubating(a) the ectoderm-derived brain cells as defined in any one of claims 1 to 7, the cells produced by the method according to any one of claims 8 to 14, or the composition of claim 15, wherein the cells produce tau aggregates, and(b) control cells, wherein the control cells do not produce tau aggregates, with a PET-tracer,(vii) removing the medium and excess PET tracer by washing the cells / aggregates,(viii) testing for binding of the PET-tracer to tau aggregates by measuring the radioactivity in the cells obtained in step (ii), wherein a radioactive signal shows the binding of the PET-tracer with tau aggregates,(ix) normalizing the measured radioactivity obtained in step (iii) to the number of cells and;(x) comparing the normalized measurements obtained in step (iv) of cells producing tau aggregates to control cells not producing tau aggregates, wherein increased normalized measurements vs. control measurements positively correlates with the potential of the PET-tracer for detecting tau aggregates.

24. Use of the ectoderm-derived brain cells as defined in any one of claims 1 to 7, the cells produced by the method according to any one of claims 8 to 14 or the composition as defined in claim 15 for testing the capacity of PET-tracers to detect tau aggregates.

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