Cell model of pathogenic short tandem repeat diseases and associated cell-based assay

Abstract

The present invention relates to an expression construct and a vector comprising said construct for generating a cellular model of unstable short tandem repeats which are pathogenic to humans. The invention further relates to a cell-based phenotypic assay using said cells, wherein the assay can be used to determine whether a candidate therapeutic agent modulates the somatic instability of said short tandem repeats, and thereby identify those candidates which have therapeutic potential to treat diseases caused by said short tandem repeats.

Description

CELL MODEL OF PATHOGENIC SHORT TANDEM REPEAT DISEASES AND ASSOCIATED CELL-BASED ASSAY FIELD OF THE INVENTION

[0001] The present invention relates to components such as an expression construct and a vector comprising said construct for generating a cellular model of unstable short tandem repeats which are pathogenic to humans. The invention further relates to a cell-based phenotypic assay using said cells, wherein the assay can be used to determine whether a candidate therapeutic agent modulates the somatic instability of said short tandem repeats, and thereby identify those candidates which have therapeutic potential to treat diseases caused by said short tandem repeats. In particular, the invention is directed towards a cell model of Huntington's disease and its use thereof in such an assay method. BACKGROUND

[0002] Short tandem repeats (STR) represent one of the most abundant class of variations in human genomes, are polymorphic by nature and become highly unstable in a length dependent fashion. The expansion of repeat length above a threshold is a well-established feature of more than 50 hereditary human disorders mainly affecting the nervous system, with many likely remaining to be identified due to improvements in detection by sequencing-based methods.

[0003] Coding repeat expansion diseases include but are not limited to brachydactyly and cleidocranial dysplasia (BCCD); blepharophimosis, ptosis and epicanthus inversus (BPES); cleidocranial dysplasia (CCD); congenital central hypoventilation syndrome (CCHS); dentatorubropallidoluysian atrophy (DRPLA); early infantile epileptic encephalopathy type 1 (EIEE1); Huntington’s disease (HD); huntington disease-like 2 (HDL2); hand-foot-genital syndrome (HFGS); holoprosencephaly type 5 (HPE5); mental retardation with isolated growth hormone deficiency (MRGH); oculopharyngeal muscular dystrophy (OPMD); spinobulbar muscular atrophy (SBMA); spinocerebellar ataxias type 1, 2, 3, 6, 7, 17 (SCA1, SCA2, SCA3, SCA6, SCA7, SCA17); synpolydactyly (SPD); x-linked mental retardation and abnormal genitalia (XLAG); X-linked mental retardation with or without growth hormone deficiency (XLMR, XLMRGHD). Non-coding repeat expansion diseases include but are not limited to benign adult familial myoclonic epilepsy (BAFME); Baratela-Scott syndrome (BSS); cerebellar ataxia, neuropathy and vestibular areflexia syndrome (CANVAS); myotonic dystrophy type 1(DM1); myotonic dystrophy type 2 (DM2); progressive myoclonus epilepsy type 1 or Unverricht-Lundborg diseae (EPM1); familial adult myoclonic epilepsy (FAME); Fuch’s endothelial corneal dystrophy type 3 (FECD3); fragile XE syndrome (FRAXE);; Friedreich ataxia (FRDA) frontotemporal dementia / amyotrophic lateral sclerosis (FTD / ALS); fragile X- associated premature ovarian infertility (FXPOI); fragile X syndrome (FXS); fragile X- associated tremor ataxia syndrome (FXTAS); global development delay, progressive ataxia and elevated glutamine (GDPAG); neuronal intranuclear inclusion disease (NIID); oculopharyngodistal myopathy type 1 (OPDM1); oculopharyngodistal myopathy type 2 (OPDM2); oculopharyngeal myopathy with leukoencephalopathy type 1 (OPML1), spinocerebellar ataxias types 8, 10, 12, 31, 36, 37 (SCA8, SCA10, SCA12, SCA31, SCA36, SCA37); X-linked dystonia parkinsonism (XDP).

[0004] Repeat expansion diseases can be dominant (e.g. Huntington’s disease) or recessive (e.g. Friedreich’s ataxia) and have the common feature of inheritance of an expansion of a normally polymorphic repeat in the disease-associated gene above a defined threshold. The number of repeats required to pass this threshold varies for each disease, although exonic repeats generally have lower thresholds than intronic repeats. Coding or exonic repeat diseases generally lead to a toxic gain of function of the resulting protein gene product, whereas non- coding intronic repeats generally lead to reduced expression of the protein gene product. In both cases this leads to dysfunction and death in vulnerable cell types, eventually resulting in clinical signs and symptoms. In addition to inheritance of an expanded allele there is now strong evidence that these repeat expansions are unstable and expand further during the lifespan of the individual. This expansion happens at the individual cell level (i.e. somatic expansion) and the extent of expansion in a given cell type / tissue typically correlates tightly with how vulnerable it is to degeneration during the course of the disease. The faster the expansion in mutation carriers, the greater the impact on disease measures such as age-of-onset, progression and severity.

[0005] Huntington’s disease (HD) is employed as a prototypic repeat expansion disease. It is a heritable neurodegenerative disorder, characterised by motor dysfunctions, psychiatric changes, and cognitive decline. The disease is caused by an expanded CAG repeat in exon 1 of the huntingtin gene (HTT), which encodes an expanded polyglutamine (polyQ) tract in the huntingtin protein (HTT). Individuals with 40 or more CAG repeats will develop Huntington’s disease in a normal life span, while 65 or more CAG repeats will lead to disease onset inchildhood or adolescence. The CAG repeat is unstable in both somatic and germ cells, and large expansions up to approximately 1000 CAG repeats have been observed in patient brains. The HTT protein is aggregation-prone in a repeat length and protein-length dependent manner (i.e. more truncated forms of repeat-containing HTT proteins are more toxic). HD pathology is characterised by the deposition of nuclear and cytoplasmic inclusions, as well as synaptic and neuronal loss in the striatum and other regions of the brain. Nuclear huntingtin aggregation is linked to transcriptional dysregulation, a molecular phenotype observed in mouse models and post-mortem brains.

[0006] HD clinical onset is inversely correlated with CAG repeat length. Genetic and environmental modifiers also contribute to the variation in age of onset, disease progression and somatic expansion of the CAG repeat. Genome-wide association studies (GWAS) have identified potential genetic modifiers including MSH3, MLH1, MLH3, PMS1, PMS2 FAN1 and LIG1, which are all associated with the DNA mismatch repair (MMR) pathway. Ablation of specific mismatch repair genes (Msh2, Msh3, Mlh1 and Mlh3) reduced somatic CAG repeat instability in mouse models of Huntington’s disease. Further, variants of mouse Msh3 and levels of MSH3 expression in Huntington’s disease patients modify the rate of somatic expansion. Reduction in MutSβ function (such as point mutations in the ATPase domain) also block somatic expansion. Biochemically, MutSβ (but not MutSα) has been shown to be sufficient for driving CAG repeat expansion in an acellular assay with purified recombinant proteins from the mismatch repair pathway.

[0007] It is now widely accepted that the somatic expansion (also referred to as somatic instability) of a mutant CAG repeat in brain cells is the first step in the molecular pathogenesis of Huntington’s disease, followed by a second process that leads to cell dysfunction once a CAG repeat threshold has been achieved. The pathogenic repeat threshold in brain is longer than 40 CAG, as measured in blood, and is currently unknown. Therefore, it is the rate of somatic CAG repeat expansion that drives the age of onset and rate of progression of the disease. Somatic CAG repeat expansion is a repeat length-, time- and cell-type dependent.

[0008] Thus, methods of arresting, or reversing, somatic repeat expansions is hypothesised to be an effective therapeutic approach to slow or stop repeat disease progression. Research in this field have collectively highlighted MSH3, part of the MutSβ heterodimer, as being of particular interest as a therapeutic target.

[0009] Mismatch repair (MMR) is a highly conserved DNA repair pathway that promotes genome stability in all organisms which recognises and repairs erroneous insertion, deletion or mis-incorporation of nucleotides that can arise during DNA replication, repair or recombination. MMR is thus one of the key pathways that participates in the cellular response to certain types of DNA damage. In addition to DNA damage response roles, MMR is employed during meiosis in eukaryotes and immunoglobulin maturation / diversification in mammals.

[0010] MMR predominantly corrects DNA base mismatches and insertion / deletion (indel) loops that can occasionally arise during normal DNA replication processes. Base pair mismatches occur when incorrect nucleotides (for example, in response to oxidative damage) are inserted into the newly synthesised DNA strand and escape the proofreading function of DNA polymerases. Indel loops commonly arise in the context of microsatellites which are highly polymorphic, short repetitive DNA sequences (microsatellites) distributed throughout genomes. Typically, at microsatellites, especially in the context of longer repetitive sequences, the template and primer strands are prone to slippage (dissociation and reannealing) during replication; this can result in loop structures and ultimately result in a discordant number of repeat units between the template and newly synthesised strand.

[0011] Mammalian MMR can be separated into four steps: (1) mismatch recognition mediated by MutS heterodimers; (2) recruitment of MutL heterodimers which connect the mismatch recognition signal to where the DNA strand scission begins in a strand-specific fashion; (3) excision of the errant, error-containing DNA strand and (4) re-synthesis of the excision gap using the remaining DNA strand as a template.

[0012] In humans, mismatch recognition by MutSα (comprised of MSH2 and MSH6 gene products) or MutSβ (comprised of MSH2 and MSH3 gene products) heterodimers initiate the MMR pathway. These obligate heterodimers differ in their substrate preferences but there are considerable overlaps between the substrates recognised and processed. MutSα recognizes base-base mismatches and some insertion-deletion mismatches with a preference for shorter indels (1-3 nucleotides), whereas MutSβ predominantly processes larger indels of 3+ nucleotides. MutSα also contributes to the recognition of certain types of DNA damage such as oxidised nucleotides and can participate in the checkpoint response to such lesions. Similarly, MutSβ may cooperate with the nucleotide excision repair machinery in the repair of interstrand cross-links (6). Furthermore, both heterodimers have been implicated in the production ofcertain genetic variants. MutSα participates in the somatic hypermutation phase of immunoglobulin gene affinity maturation, and MutSβ is required for the triplet repeat expansions that are responsible for several neurodegenerative diseases. Importantly, for therapeutic potential, unlike other DNA mis-match repair genes, loss of function of MSH3 does not cause Lynch syndrome, which is associated with a high risk of colon cancer and a spectrum of other malignancies.

[0013] Binding of MutSα or MutSβ heterodimers to the mismatch results in the ATP- dependent recruitment of a MutL heterodimer (either MutLα comprising MLH1 and PMS2 gene products, MutLβ comprising MLH1 and PMS1 or MutLγ comprising MLH1 or MLH3 gene products) to form a ternary complex. This ternary complex forms interactions with additional proteins including proliferating cell nuclear antigen (PCNA) and exonuclease 1 (EXO1) which enable successful completion of MMR sequelae.

[0014] The mismatch repair gene MSH3 has become a major focus for therapeutic development, as unlike other mismatch repair genes, nullizygosity for MSH3 does not cause malignancies associated with mismatch repair deficiency. Potential treatments targeting MSH3 currently under development include gene therapy, biologics and small molecules.

[0015] The preclinical testing of novel MSH3 targeting therapeutics is not straightforward. Ideally, the chosen model will develop robust phenotypes within a timescale amenable to the administration and durability of the potential therapeutic agents.

[0016] Somatic CAG repeat expansion is a conserved and well-documented biological process in HD mouse models. In contrast, somatic CAG repeat expansion is challenging to assess in cell models of HD, and no robust cellular model yet exists for diseases caused by STRs. Previous attempts to produce a cell model have been made either utilising plasmid-based shuttle vectors or chromosomally integrated reporters. In shuttle vector systems, repeat- containing plasmids are introduced into mammalian cells which are then subjected to perturbation prior to using the plasmids to transform into E.coli or S.cerevisiae for instability readouts (either repeat-length dependent resistance to an agent or digestion with restriction enzymes and measuring product length). These shuttle systems are relatively rapid but labour intensive and low throughput. Integrated reporters in cells have been based on CAG-length dependent HPRT or APRT activity which can be selected for; or on GFP fluorescence. While higher throughput, these constructs have low sensitivity. Other disadvantages of both systems include a bias toward rare event, and in some cases these models are only sensitive to specifictypes of repeat length changes. Other mammalian cell models allow for direct detection of repeat length changes by PCR which include cell lines stably transfected with plasmids harbouring repeats at ectopic loci, or patient-derived cells including lymphoblastoid and fibroblasts and stem cells (including either embryonic or induced pluripotent stem cells). However, lymphoblastoid and fibroblast cells do not show high levels of repeat instability and therefore do not provide a realistic pathogenic model. Stem cells and in vitro differentiated counterparts (i.e. specific cell types showing high instability such as medium spiny neuron like cells) do have increased rates of repeat instability but require long-term culturing of weeks to months and are furthermore labour intensive and low-throughput.

[0017] The present invention was devised with the foregoing in mind, and aims to solve one or more of the above-mentioned problems in the art. In particular, the invention aims to provide a realistic cellular model of pathogenic short tandem repeat somatic instability for use in assays to screen potential therapies for the treatment of diseases resulting from said short tandem repeats. STATEMENTS OF INVENTION

[0018] According to a first aspect of the present invention there is provided an expression construct comprising: an inducible promoter operably linked to a nucleic acid sequence comprising a pathogenic short tandem repeat region, fused via a transcriptional or translational split mechanism, to a nucleic acid sequence encoding a reporter protein.

[0019] According to a second aspect of the present invention there is provided a vector comprising the expression construct of the first aspect.

[0020] According to a third aspect of the present invention there is provided a cell comprising the expression construct of the first aspect or the vector of the second aspect.

[0021] In one embodiment of the third aspect, the cell is a U2OS cell.

[0022] According to a fourth aspect of the present invention there is provided a U2OS cell comprising an expression construct or a vector comprising an expression construct, wherein the expression construct comprises: an inducible promoter operably linked to a nucleic acid sequence encoding a pathogenic short tandem repeat region, fused via a transcriptional or translational split mechanism, to a nucleic acid sequence encoding a reporter protein.

[0023] In one embodiment, the inducible promoter is a Tet-on promoter.

[0024] In one embodiment, the nucleic acid sequence comprising a pathogenic short tandem repeat region comprises exon 1 of the HTT gene.

[0025] In one embodiment, the transcriptional or translational split mechanism comprises a nucleic acid encoding a P2A self-cleaving peptide.

[0026] In one embodiment, the reporter protein is a luciferase reporter protein.

[0027] According to a fifth aspect of the present invention there is provided a population of the cells of the third or fourth aspects.

[0028] According to a sixth aspect of the present invention there is provided a method of screening one or more candidate therapeutic agents for the ability to inhibit MutSβ activity, the method comprising: a. Providing a cell according to the invention, suitably according to the third or fourth aspect; b. Culturing the cell under suitable conditions to induce expression of the expression construct therein, and to expand the number of short tandem repeats in the short tandem repeat region to a desired level; c. Exposing the cell to one or more candidate therapeutic agents under suitable conditions to inhibit MutSβ activity; d. Determining the effect of each candidate therapeutic agent on the short tandem repeat region of the expression construct; and e. Selecting those candidate therapeutic agents which cause the number of short tandem repeats in the short tandem repeat region to arrest or contract, indicating inhibition of MutSβ activity.

[0029] In one embodiment, the cell comprises an expression construct of the first aspect which comprises a pathogenic short tandem repeat region.

[0030] In one embodiment, step (b) of culturing the cell to induce expression of the expression construct therein causes expression of the reporter protein, and optionally expression of any proteins encoded by the short tandem repeat region, suitably which may be pathogenic proteins as discussed hereinbelow.

[0031] In one embodiment, successful expression of the construct in the cell is determined by expression of the reporter protein. In one embodiment, the level of expression of the reporter protein further indicates the expansion of the short tandem repeats. Therefore, suitably step (b) may comprise culturing the cell under suitable conditions to induce expression of the construct therein to expand the number of short tandem repeats in the short tandem repeat region to a desired level as indicated by the level of expression of the reporter protein.

[0032] In one embodiment, the method may comprise a step of determining the number of short tandem repeats in the repeat region, suitably before step (c), and suitably after step (c).

[0033] In one embodiment, determining the effect of each candidate therapeutic agent comprises determining whether the number of short tandem repeats in the repeat region expands, arrests or contracts in response to the therapeutic agent.

[0034] In one embodiment the method further comprises a step of determining whether each candidate therapeutic agent is toxic to the cell, and optionally selecting those candidate therapeutic agents which are not toxic to the cell.

[0035] In one embodiment the method further comprises a step of identifying one or more selected candidate therapeutic agents.

[0036] In one embodiment the method further comprises a step of formulating the one or more selected candidate therapeutic agents, suitably into a pharmaceutical composition, suitably for use in therapy.

[0037] According to a seventh aspect of the present invention there is provided a primer pair comprising a first primer capable of annealing to a site within exon 1 of the HTT gene, and a second primer capable of annealing to a site within a transcriptional or translational split mechanism or within a nucleic acid sequence encoding a reporter protein.

[0038] In one embodiment, the second primer is capable of annealing to a site within a nucleic acid sequence encoding a P2A self-cleaving peptide.

[0039] In one embodiment, the first primer has a sequence according to SEQ ID NO:9 and the second primer has a sequence according to SEQ ID NO:10.

[0040] According to an eighth aspect of the present invention there is provided use of the primer pair of the fourth aspect for determining the number of short tandem repeats in a nucleic acid sequence comprising exon 1 of the HTT gene.

[0041] In one embodiment the primer pair is for use in determining the number of short tandem repeats in an expression construct according to the first aspect in which the nucleic acid sequence comprising a pathogenic short tandem repeat region comprises exon 1 of the HTT gene.

[0042] The present invention solves the problem of providing a reliable in vitro cell model of short tandem repeat disorders which cause diseases in humans, where the model accurately reflects the in vivo somatic instability of such tandem repeats whilst being non-toxic to the hostcell. Advantageously such a cell-based model can be used to screen potential therapies for treating diseases caused by short tandem repeat regions, prior to expensive and risky in vivo testing, thereby increasing the efficiency of finding treatments for such diseases. Prior to the present invention, attempts to make cellular models of short tandem repeat instability for diseases such as Huntington's have failed for a variety of reasons. One key challenge is the timeframe in which repeat instability occurs which makes it impractical for therapeutic screening (exemplified by iPSC and iPSC-MSN models which only exhibit 1-2 and 2-3 CAG repeat increases respectively over a long period of 8-10 weeks). A second challenge is that published methods are highly labour intensive and low throughput. A third challenge is repeat distribution and reproducibility / variability: other assay systems can be challenged with bimodal or multimodal distributions and variable expansion trajectories within cell populations leading to heterogenous results when these populations are used in research. A fourth challenge is that if reporter signals are used, they are small and decrease further with repeat expansion which challenges measurement and interpretation. Typically, the short tandem repeats in the context of expression products are not well tolerated by most cells and are therefore toxic. Furthermore, the short tandem repeat regions in some prior constructs are not unstable, so they do not expand when expressed in the cell and do not reflect the somatic instability of the in vivo disease conditions. Finally, in some constructs inducing the expression thereof has not resulted in reliable and reproducible repeat expansion or in expansion which can be controlled by an inducer. DEFINITIONS

[0043] While the making and using of various embodiments of the present invention are discussed in detail below, it should be appreciated that the present invention provides many applicable inventive concepts that can be embodied in a wide variety of specific contexts. The specific embodiments discussed herein are merely illustrative of specific ways to make and use the invention and do not delimit the scope of the invention.

[0044] The discussion of the background to the invention herein is included to explain the context of the invention. This is not to be taken as an admission that any of the material referred to was published, known, or part of the common general knowledge in any country as of the priority date of any of the claims.

[0045] Throughout this disclosure, various publications, patents and published patent specifications are referenced by an identifying citation. All documents cited in the presentspecification are hereby incorporated by reference in their entirety. In particular, the teachings or sections of such documents herein specifically referred to are incorporated by reference.

[0046] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Current Protocols in Molecular Biology (Ausubel, 2000, Wiley and son Inc, Library of Congress, USA); Molecular Cloning: A Laboratory Manual, Third Edition, (Sambrook et al, 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press); Oligonucleotide Synthesis (M. J. Gait ed., 1984); U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (Harries and Higgins eds. 1984); Transcription and Translation (Hames and Higgins eds. 1984); Culture of Animal Cells (Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells and Enzymes (IRL Press, 1986); Perbal, A Practical Guide to Molecular Cloning (1984); the series, Methods in Enzymology (Abelson and Simon, eds. -in-chief, Academic Press, Inc., New York), specifically, Vols.154 and 155 (Wu et al. eds.) and Vol. 185, “Gene Expression Technology” (Goeddel, ed.); Gene Transfer Vectors For Mammalian Cells (Miller and Calos eds., 1987, Cold Spring Harbor Laboratory); Immunochemical Methods in Cell and Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook of Experimental Immunology, Vols. I-IV (Weir and Blackwell, eds., 1986); and Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

[0047] To facilitate the understanding of this invention, a number of terms are defined or explained below. Terms used herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present invention. Terms such as “a”, “an” and “the” are not intended to refer to only a singular entity, but include the general class of which a specific example may be used for illustration. The terminology herein is used to describe specific embodiments of the invention, but their usage does not delimit the invention, except as outlined in the claims.

[0048] As used herein, the phrase “promoter” refers to a region of DNA that generally is located upstream of a nucleic acid sequence to be transcribed that is needed for transcription to occur, i.e. which initiates transcription. Promoters permit the proper activation or repression of transcription of a coding sequence under their control. A promoter typically contains specific sequences that are recognized and bound by plurality of transcription factors. These bind to thepromoter sequences and result in the recruitment of RNA polymerase, an enzyme that synthesizes RNA from the coding region of the gene. Many diverse promoters are known in the art.

[0049] As used herein, “minimal promoter” (also known as the “core promoter”) refers to a typically short DNA segment which is inactive or largely inactive by itself but can mediate transcription when combined with other transcription regulatory elements. Minimal promoter sequences can be derived from various different sources, including prokaryotic and eukaryotic genes.

[0050] The term “nucleic acid sequence” as used herein typically refers to an oligomer or polymer (preferably a linear polymer) of any length composed essentially of nucleotides. A nucleotide unit commonly includes a heterocyclic base, a sugar group, and at least one, e.g. one, two, or three, phosphate groups, including modified or substituted phosphate groups. Heterocyclic bases may include inter alia purine and pyrimidine bases such as adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U) which are widespread in naturally- occurring nucleic acids, other naturally-occurring bases (e.g., xanthine, inosine, hypoxanthine) as well as chemically or biochemically modified (e.g., methylated), non-natural or derivatised bases. Sugar groups may include inter alia pentose (pentofuranose) groups such as preferably ribose and / or 2-deoxyribose common in naturally-occurring nucleic acids, or arabinose, 2- deoxyarabinose, threose or hexose sugar groups, as well as modified or substituted sugar groups. Nucleic acids as intended herein may include naturally occurring nucleotides, modified nucleotides or mixtures thereof. A modified nucleotide may include a modified heterocyclic base, a modified sugar moiety, a modified phosphate group or a combination thereof. Modifications of phosphate groups or sugars may be introduced to improve stability, resistance to enzymatic degradation, or some other useful property. The term “nucleic acid” further preferably encompasses DNA, RNA and DNA RNA hybrid molecules, specifically including hnRNA, pre-mRNA, mRNA, cDNA, genomic DNA, amplification products, oligonucleotides, and synthetic (e.g., chemically synthesised) DNA, RNA or DNA RNA hybrids. A nucleic acid can be naturally occurring, e.g., present in or isolated from nature; or can be non-naturally occurring, e.g., recombinant, i.e., produced by recombinant DNA technology, and / or partly or entirely, chemically or biochemically synthesised. A “nucleic acid” can be double-stranded, partly double stranded, or single-stranded. Where single-stranded, the nucleic acid can be the sense strand or the antisense strand. In addition, nucleic acid can be circular or linear.

[0051] The terms “identity” and “identical” and the like refer to the sequence similarity between two polymeric molecules, e.g., between two nucleic acid molecules, such as between two DNA molecules. Sequence alignments and determination of sequence identity can be done, e.g., using the Basic Local Alignment Search Tool (BLAST) originally described by Altschul et al. 1990 (J Mol Biol 215: 403-10), such as the “Blast 2 sequences” algorithm described by Tatusova and Madden 1999 (FEMS Microbiol Lett 174: 247-250).

[0052] Methods for aligning sequences for comparison are well-known in the art. Various programs and alignment algorithms are described in, for example: Smith and Waterman (1981) Adv. Appl. Math. 2:482; Needleman and Wunsch (1970) J. Mol. Biol. 48:443; Pearson and Lipman (1988) Proc. Natl. Acad. Sci. U.S.A. 85:2444; Higgins and Sharp (1988) Gene 73:237- 44; Higgins and Sharp (1989) CABIOS 5:151-3; Corpet et al. (1988) Nucleic Acids Res. 16:10881-90; Huang et al. (1992) Comp. Appl. Biosci. 8:155-65; Pearson et al. (1994) Methods Mol. Biol. 24:307-31; Tatiana et al. (1999) FEMS Microbiol. Lett. 174:247-50. A detailed consideration of sequence alignment methods and homology calculations can be found in, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-10.

[0053] The National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST™; Altschul et al. (1990)) is available from several sources, including the National Center for Biotechnology Information (Bethesda, MD), and on the internet, for use in connection with several sequence analysis programs. A description of how to determine sequence identity using this program is available on the internet under the “help” section for BLAST™. For comparisons of nucleic acid sequences, the “Blast 2 sequences” function of the BLAST™ (Blastn) program may be employed using the default parameters. Nucleic acid sequences with even greater similarity to the reference sequences will show increasing percentage identity when assessed by this method. Typically, the percentage sequence identity is calculated over the entire length of the sequence.

[0054] For example, a global optimal alignment is suitably found by the Needleman-Wunsch algorithm with the following scoring parameters: Match score: +2, Mismatch score: -3; Gap penalties: gap open 5, gap extension 2. The percentage identity of the resulting optimal global alignment is suitably calculated by the ratio of the number of aligned bases to the total length of the alignment, where the alignment length includes both matches and mismatches, multiplied by 100.

[0055] “Transfection” in the present application refers broadly to any process of deliberately introducing nucleic acids into cells, and covers introduction of viral and non-viral vectors, and includes or is equivalent to transformation, transduction and like terms and processes. Examples include, but are not limited to: transfection with viral vectors; transformation with plasmid vectors; electroporation (Fromm et al. (1986) Nature 319 :791-3) ; lipofection (Feigner et al. (1987) Proc. Natl. Acad. Sci. USA 84 :7413-7) ; microinjection (Mueller et al. (1978) Cell 15:579-85); Agrobacterium-mediated transfer (Fraley et al. (1983) Proc. Natl. Acad. Sci. USA 80:4803-7); direct DNA uptake; whiskers-mediated transformation; and microprojectile bombardment (Klein et al. (1987) Nature 327:70).

[0056] The term “vector” is well known in the art, and as used herein refers to a nucleic acid molecule, e.g. double-stranded DNA, which may have inserted into it a nucleic acid sequence according to the present invention. A vector is suitably used to transport an inserted nucleic acid molecule into a suitable host cell. A vector typically contains all of the necessary elements that permit transcribing the insert nucleic acid molecule, and, preferably, translating the transcript into a polypeptide. A vector typically contains all of the necessary elements such that, once the vector is in a host cell, the vector can replicate independently of, or coincidental with, the host chromosomal DNA; several copies of the vector and its inserted nucleic acid molecule may be generated.

[0057] The term “operably linked” as used herein refer to the arrangement of various nucleic acid elements relative to each other such that the elements are functionally connected and are able to interact with each other in the manner intended. Such elements may include, without limitation, a promoter, a short tandem repeat region, a transcriptional or translational split mechanism, and a nucleic acid encoding a reporter protein. The nucleic acid sequence elements, when properly oriented or operably linked, act together to modulate the activity of one another, and ultimately may affect the level of expression. By modulate is meant increasing, decreasing, or maintaining the level of activity of a particular element. The position of each element relative to other elements may be expressed in terms of the 5’ terminus and the 3’ terminus of each element or their position upstream or downstream of another element or position (such as a TSS or promoter), and the distance between any particular elements may be referenced by the number of intervening nucleotides, or base pairs, between the elements. As understood by the skilled person, operably linked implies functional activity, and is not necessarily related to a natural positional link.

[0058] The term “pharmaceutically acceptable” as used herein is consistent with the art and means compatible with the other ingredients of the pharmaceutical composition and not deleterious to the recipient thereof.

[0059] The terms “treatment” or “treating” refer to reducing, ameliorating or eliminating one or more signs, symptoms, or effects of a disease or condition. “Treatment,” as used herein thus includes any treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease from occurring in a subject predisposed to the disease or at risk of acquiring the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease.

[0060] "Expression construct" as used herein means a nucleic acid sequence capable of directing expression of a particular nucleic acid sequence in an appropriate host cell, comprising a promoter operably linked to the nucleic acid molecule of interest, which is optionally further operably linked to termination signal sequences. It also typically comprises sequences required for proper translation of the nucleic acid molecule sequence. The expression construct comprising the nucleic acid molecule of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components. The expression construct may also be one that is naturally occurring but has been obtained in a recombinant form useful for heterologous expression. Typically, however, the expression construct is heterologous with respect to the host cell, i.e., the particular nucleic acid molecule of the expression construct does not occur naturally in the host cell and must have been introduced into the host cell or an ancestor of the host cell by a transformation event. The expression of the nucleic acid molecule sequence in the expression construct may be under the control of, for example, an inducible promoter that initiates transcription only when the host cell is exposed to some particular external stimulus. DETAILED DESCRIPTION

[0061] Expression construct

[0062] Inducible Promoter

[0063] Suitably the expression construct comprises an inducible promoter, suitably to control expression of the nucleic acid sequences comprising the short tandem repeat region and encoding the reporter protein. Suitably the inducible promoter is capable of driving expression of the entire construct i.e. the nucleic acid sequence comprising the pathogenic short tandemrepeat region, the transcriptional or translational split mechanism, and the nucleic acid sequence encoding the reporter protein.

[0064] Inducible promoters allow production of the expression product to be induced at a desired point, which is useful in many ways. An inducible promoter can be used to produce expression products which are toxic to cells or which would inhibit the growth of the cells, as it allows the cells to be grown to a specific density or number before inducing production of the expression product and harvesting.

[0065] Suitably the inducible promoter can be any inducible promoter. For example, the inducible promoter may be a chemically inducible promoter; a temperature inducible promoter; a light inducible promoter; a hypoxia inducible promoter. Other inducible promoters include a promoter capable of inducing expression of the downstream nucleic acid of interest by binding to a complex including a tetracycline antibiotic (tetracycline, doxycycline, or the like) and a tetracycline transactivator in a case where the external stimulus is the presence of the tetracycline antibiotic; a promoter capable of inducing expression of the downstream nucleic acid of interest by release of a tetracycline repressor in a case where the external stimulus is the absence of a tetracycline antibiotic; a promoter capable of inducing expression of the downstream nucleic acid of interest by binding of an ecdysteroid (ecdysone, muristerone A, ponasterone A, or the like) to an ecdysone receptor-retinoid receptor complex in a case where the external stimulus is the presence of the ecdysteroid; and a promoter capable of inducing expression of the downstream nucleic acid of interest by binding of FKCsA to a complex including a Gal4 DNA binding domain fused to FKBP12 and a VP16 activator domain fused to cyclophilin in a case where the external stimulus is the presence of FKCsA.

[0066] Suitably the inducible promoter is a chemically inducible promoter selected from a tetracycline, doxycycline, alcohol, steroid, or other hormone inducible promoter.

[0067] In one embodiment the inducible promoter is a tetracycline / doxycycline inducible promoter.

[0068] Suitably the tetracycline / doxycycline inducible promoter may be any such known promoter, suitably the inducible promoter is a Tet-on promoter. Suitably therefore the inducible promoter comprises a Tet responsive element (TRE), suitably which comprises a plurality of Tet operator (tetO) sequences fused to a minimal promoter. Suitably at least two Tet operator sequences are comprised in the Tet-On promoter. In one embodiment, the Tet operator comprises a sequence according to SEQ ID NO:2, or a sequence having at least 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity thereto, or a functional fragment thereof. The minimal promoter can be any suitable minimal promoter. A wide range of minimal promoters are known in the art. Without limitation, suitable minimal promoters include CMV minimal promoter (CMV-MP), YB-TATA minimal promoter (YB- TABA), HSV thymidine kinase minimal promoter (MinTK), SV40 minimal promoter (SV40- MP), or G6PC-MP (which is a liver-derived non-TATA box MP). The minimal promoter can be a synthetic minimal promoter. Suitably the minimal promoter sequence may be derived from the human cytomegalovirus (hCMV-MP). Suitably the inducible promoter may comprise or consist of a sequence according to SEQ ID NO: 1 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity thereto, or a functional fragment thereof.

[0069] In such embodiments, suitably there is further provided a second expression construct comprising a nucleic acid sequence encoding a TetR repressor protein, suitably operably linked to a constitutive promoter. In some embodiments, the TetR repressor protein may be TetR(B). Alternatively in some embodiments, the second expression construct may encode a reverse tetracycline-controlled transactivator, rtTA. Suitably rtTA is a fusion protein comprised of a modified TetR repressor and a transactivation domain, suitably the VP16 transactivation domain. Suitably the nucleic acid sequence encoding a Tet repressor or rtTA is under the control of a constitutive promoter. Suitable such promoters may be CMV, for example. In one embodiment, said second expression construct is comprised on a vector, and is suitably any available vector comprising a nucleic acid sequence encoding the TetR repressor. In one embodiment said vector is pcDNA6 / TR available from ThermoFisher Scientific (https: / / www.thermofisher.com / uk / en / home / references / protocols / proteins-expression-isolation- and-analysis / protein-expression-protocol / inducible-protein-expression-using-the-trex- system.html#pre). In a further aspect of the invention there may be provided a system comprising the first expression construct of the first aspect, and said second expression construct, optionally which may be provided on the same or different vectors, optionally which may be provided as a first vector of the second aspect, and a second vector comprising the second expression construct. Suitably such system may form a ‘Tet-on’ system of controlling expression of the short tandem repeat region and the reporter protein.

[0070] Suitably, the TetR repressor protein is constitutively expressed from the second expression construct and binds to the Tet operator sequences of the TRE, thereby inhibitingtranscription of the operably linked nucleic acid sequences of the first expression construct. Alternatively, when using a rtTA protein then the rtTA protein is constitutively expressed from the second construct but in the absence of tetracycline / doxycycline it cannot bind to the TRE, therefore transcription of the first expression construct is inhibited thereby. In the presence of tetracycline / doxycycline, suitably the TetR repressor binds to tetracycline / doxycycline and undergoes a conformational change such that is no longer able to bind to the Tet operator sequences of the TRE, thereby allowing transcription of the first expression construct to occur. Alternately, the rtTA protein binds to the tetracycline / doxycycline and recognises and binds to the TRE, allowing transcription of the first expression construct to occur. Suitably the TetR repressor can only be removed from the TRE in the presence of tetracycline / doxycycline. Suitably the rtTA protein can only recognise the Tet operator sequences in the TRE in the presence of the Tetracycline or doxycyline effector.

[0071] Short Tandem Repeat Region

[0072] Suitably the expression construct of the invention is intended to inducibly drive expression across a sequence comprising a region of pathogenic short tandem repeats, to expand the number of repeats in the region, and thereby provide a model of somatic instability present in many human diseases.

[0073] Suitably a short tandem repeat is a back-to-back repeat of 1-6 nucleotides (mono-, di-, tri-, tetra-, penta-, and hexanucleotide repeats). Suitably the short tandem repeat is a repeat of 1-3 nucleotides. In one embodiment the short tandem repeat is a repeat of 3 nucleotides, otherwise known as a trinucleotide repeat.

[0074] Suitably the short tandem repeat may be a repeat of a sequence selected from: CAG, CTG, GAA, CCTG, CGG, GCN, ATTCT, GGCCTG, GCG, and GGGGCC. Suitably the short tandem repeat is a trinucleotide repeat of a sequence selected from: CAG, CTG, GAA, CGG, GCN, and GCG. In one embodiment the short tandem repeat is a CAG repeat.

[0075] Suitably the short tandem repeat region comprises between 15 and 800 repeats, between 20 and 500 repeats, between 25 and 300 repeats, between 30 and 200 repeats, between 35 and 150 repeats. Suitably the short tandem repeat region comprises between 36 and 120 repeats, preferably between 40 and 100 repeats. In one embodiment, the short tandem repeat region comprises about 100 repeats.

[0076] In one embodiment, the short tandem repeat region comprises or consists of about 100 CAG repeats.

[0077] Suitably the short tandem repeat region is unstable. Suitably this means that the number of repeats in the region can change over time or in response to external stimuli. Suitably the number of repeats in the region increases over time. Suitably therefore the short tandem repeat region of the expression construct expands over time. Suitably therefore the short tandem repeat region of the expression construct may be deemed expandable. Suitably this is desirable to reflect the behavior of somatic instability demonstrated by such pathogenic short tandem repeat regions in vivo, which causes disease as discussed further herein below.

[0078] Suitably the number of repeats in the region also increases in response to the inducer of the expression construct. Suitably therefore the short tandem repeat region of the expression construct expands in response to the inducer. Suitably by expanding the short tandem repeat region it is meant increasing the number of repeats in the short tandem repeat region. Equally therefore by contracting the short tandem repeat region it is meant reducing the number of repeats in the short tandem repeat region. Suitably by arresting or stabilizing the short tandem repeat region it is meant that there is no change in the number of repeats in the short tandem repeat region. Suitably the short tandem repeat region in the expression construct is unstable and expandable, such that the number of repeats in the region increases in response to the inducer which is capable of inducing expression i.e. transcription from the inducible promoter of the expression construct. Suitably this is desirable to drive somatic instability within an in vitro cellular environment to the desired level required in a reasonable length of time to provide a suitable model of the in vivo disease.

[0079] Suitably the short tandem repeat region further comprises flanking sequences, preferably flanking genomic DNA sequences, suitably upstream and downstream of the short tandem repeat region, suitably therefore at the 5’ and 3’ ends of the short tandem repeat region. Suitably the flanking regions comprise between 30-200, suitably between 40 -180, suitably between 50-150 nucleotides of genomic DNA. In one embodiment the flanking region comprises about 50 nucleotides of genomic DNA, suitably at the5’ end. In one embodiment the flanking region comprises about 150 nucleotides of genomic DNA, suitably at the 3’ end. Suitably the flanking regions may comprise one or more sequence elements which may modulate or stabilise the short tandem repeat region. Suitably such sequence elements may comprise one or more of: a GC rich region, one or more CAA repeats, a sequence encoding a proline-rich region.

[0080] Suitably the short tandem repeat region may be a coding short tandem repeat or a non- coding short tandem repeat. Suitably both types of repeat cause diseases in humans. In one embodiment, the pathogenic short tandem repeat region is a coding region. Suitably therefore the encoded pathogenic protein may comprise a repeating amino acid sequence, such as a Poly- Q sequence.

[0081] Suitably the short tandem repeat region is pathogenic, by which it is meant that the short tandem repeat region causes or is involved in the physiology of a disease. In some embodiments, the short tandem repeat region may encode a pathogenic protein. Suitably the pathogenic protein causes or is involved in the physiology of a disease. Suitably the pathogenic short tandem repeat region, and / or the resulting pathogenic protein, causes a disease in humans. Suitably therefore the pathogenic short tandem repeat region, and / or the pathogenic protein, is pathogenic to humans. Suitably the pathogenic short tandem repeat region and / or the pathogenic protein causes a disease selected from those listed below. In one embodiment, the pathogenic short tandem repeat region encodes a pathogenic protein which causes Huntington’s disease. In one embodiment, the pathogenic short tandem repeat region encodes the HTT protein or a fragment thereof, suitably exon 1 thereof.

[0082] Suitably the nucleic acid sequence comprising the pathogenic short tandem repeat region may comprise one of the following genes or a fragment thereof: DMPK, ZNF9, ATN1, FMR1, FMR2, FXN, HTT, JPH3, CSTB, PABPN1, AR, ATXN1, ATXN2, CACNA1A, ATXN7, ATXN8OS, ATXN10, PPP2R2B, TBP, NOP56, ARX, FOXL2, RUNX2, PHOX2B, HOXA13, ZIC2, HOXD13, SOX3, and C9orf72. Suitably wherein the fragment thereof comprises the pathogenic short tandem repeat region.

[0083] In one embodiment, the pathogenic short tandem repeat region comprises or consists of exon 1 of the HTT gene. In one embodiment, exon 1 of the HTT gene comprises a sequence according to SEQ ID NO:3, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity thereto, or a functional fragment thereof.

[0084] Suitably the pathogenic short tandem repeat region, and / or the pathogenic protein, causes a short tandem repeat disease, suitably in humans. Suitably the pathogenic short tandem repeat region and / or the pathogenic protein, causes a trinucleotide repeat disorder, suitably in humans. Suitably the pathogenic short tandem repeat region and / or the pathogenic protein may cause a disease selected from: benign adult familial myoclonic epilepsy (BAFME); brachydactyly and cleidocranial dysplasia (BCCD); blepharophimosis, ptosis and epicanthusinversus (BPES); Baratela-Scott syndrome (BSS); cerebellar ataxia, neuropathy and vestibular areflexia syndrome (CANVAS); congenital central hypoventilation syndrome (CCHS); dentatorubropallidoluysian atrophy (DRPLA); early infantile epileptic encephalopathy type 1 (EIEE1); familial adult myoclonic epilepsy (FAME); Fuch’s endothelial corneal dystrophy type 3 (FECD3); Dentatorubral pallidoluysian atrophy; fragile XE syndrome (FRAXE);; Friedreich ataxia (FRDA) frontotemporal dementia / amyotrophic lateral sclerosis (FTD / ALS); fragile X- associated premature ovarian infertility (FXPOI); fragile X syndrome (FXS); fragile X- associated tremor ataxia syndrome (FXTAS); global development delay, progressive ataxia and elevated glutamine (GDPAG); Huntington’s disease (HD); Huntington disease-like 2 (HDL2); hand-foot-genital syndrome (HFGS); holoprosencephaly type 5 (HPE5); mental retardation with isolated growth hormone deficiency (MRGH); myotonic dystrophy type 1 (DM1); myotonic dystrophy type 2 (DM2); progressive myoclonus epilepsy type 1 or Unverricht- Lundborg diseae (EPM1); neuronal intranuclear inclusion disease (NIID); oculopharyngodistal myopathy type 1 (OPDM1); oculopharyngodista myopathy type 2 (OPDM2); oculopharyngeal myopathy with leukoencephalopathy type 1 (OPML1); spinobulbar muscular atrophy (SBMA); spinocerebellar ataxias type 1, 2, 3, 6, 7, 8, 10, 12, 17, 31, 36, 37 (SCA1, SCA2, SCA3, SCA6, SCA7, SCA8, SCA10, SCA12, SCA17, SCA31, SCA36, SCA37); synpolydactyly (SPD); X- linked dystonia parkinsonism (XDP); X-linked mental retardation and abnormal genitalia (XLAG); and X-linked mental retardation with or without growth hormone deficiency (XLMR, XLMRGHD.

[0085] Suitably the pathogenic short tandem repeat region and / or the pathogenic protein may cause a trinucleotide repeat disease selected from: dentatorubropallidoluysian atrophy (DRPLA); Fuch’s endothelial corneal dystrophy type 3 (FECD3); fragile XE syndrome (FRAXE); Friedreich ataxia (FRDA); fragile X-associated premature ovarian infertility (FXPOI); fragile X syndrome (FXS); fragile X-associated tremor ataxia syndrome (FXTAS); Huntington’s disease (HD); Huntington disease-like 2 (HDL2); myotonic dystrophy type 1 (DM1); neuronal intranuclear inclusion disease (NIID)spinobulbar muscular atrophy (SBMA); spinocerebellar ataxias type 1, 2, 3, 6, 7, 8, 17, (SCA1, SCA2, SCA3, SCA6, SCA7, SCA8, SCA17); or X-linked dystonia parkinsonism (XDP). Suitably, the triplet repeat disease or disorder is Huntington’s disease (HD).

[0086] In one embodiment, the pathogenic short tandem repeat region causes Huntington’s disease. In one embodiment, the pathogenic short tandem repeat region encodes a pathogenic protein, suitably exon 1 of HTT, which causes Huntington’s disease.

[0087] Reporter Protein

[0088] Suitably the expression construct of the invention further comprises a nucleic acid sequence encoding a reporter protein, which when expressed, indicates successful integration of the expression construct into a host cell.

[0089] Suitably any reporter protein may be used. Suitably any fluorescent or luminescent protein may be used such as: YFP, CFP, GFP, RFP, EBFP, ECFP, EGFP, YFP, mHoneydew, mBanana, mOrange, tdTomato,mTangerine, mStrawberry, mCherry,mGrape1, mRaspberry, mGrape2 and mPlum. etc. A fluorescent protein allows selection based on the presence or absence of a fluorescent protein (e.g. by FACS). A luminescent protein allows selection based on the presence or absence of a luminescent protein (e.g. by an enzymatic reaction of the luminescent protein with a substrate such as luciferin). In one embodiment, the reporter protein is luciferase. Suitably the luciferase reporter protein is used to give a qualitative indication of the number of repeats in the short tandem repeat region, suitably the luciferase expression can broadly be correlated to the number of repeats in the region unlike other reporter proteins. Suitably any luciferase protein available in the art may be used. In one embodiment, the reporter protein is the Nano-Luc luciferase available from Promega, which advantageously is small.

[0090] Transcriptional or Translational Split Mechanism

[0091] Suitably the nucleic acid sequence comprising the pathogenic short tandem repeat region is fused via a transcriptional or translation split mechanism to the nucleic acid sequence encoding the reporter protein.

[0092] Suitably fusion of the nucleic acid sequences is achieved via a transcriptional or translation split mechanism. Suitably the transcriptional or translation split mechanism is located between the nucleic acid sequence comprising the pathogenic short tandem repeat sequence and the nucleic acid sequence encoding the luciferase reporter.

[0093] Suitably, the transcriptional or translational split mechanism may allow two separate proteins to be created from a single nucleic acid. Suitably, the transcriptional or translational split mechanism may allow two separate proteins to be created from a single nucleic acid via a transcriptional mechanism. For example, the nucleic acids encoding each of the proteins may beoperably linked to separate promoters. Alternatively, the transcriptional or translational split mechanism may allow two separate proteins to be created from a single nucleic acid via a translational mechanism. Suitably the translational mechanism may be during translation or post- translational. Suitably the transcriptional or translational split mechanism may allow two separate proteins to be created from a single nucleic acid by allowing translation initiation in a cap- independent manner. Suitably the transcriptional or translational split mechanism may allow two separate proteins to be created from a single nucleic acid by inducing ribosomal skipping during translation. Suitably the transcriptional or translational split mechanism may be a DNA sequence, an RNA sequence or a protein sequence.

[0094] Suitably the split mechanism is a translational split mechanism, suitably which allows the nucleic acid sequences to be expressed as a polycistronic single transcript and then subsequently translated separately. Suitably, transcription of the nucleic acid sequence comprising the pathogenic short tandem repeat region as a fusion with the nucleic acid sequence encoding the reporter protein reduces the toxicity of the pathogenic short tandem repeat region to the host cell, and more accurately reflects behaviour of any pathogenic proteins in the cell as they would behave in vivo.

[0095] Suitably the transcriptional or translation split mechanism may be an IRES, a furin cleavage site, or a cleavable linker. Internal ribosome entry site is an RNA element that allows for translation initiation in cap-independent manner. 2A peptides are approximately 20 amino acids long and self-cleavage occurs between the last 2 amino acids, glycine and proline. Suitably the transcriptional or translation split mechanism is a self-cleaving peptide linker. Suitably the self-cleaving peptide linker is selected from: F2A, P2A, E2A, T2A, GF2A, GP2A, GE2A and GT2A. In one embodiment, the self-cleaving peptide linker is P2A. Suitably, the 2A linked nucleic acids are translated in one open reading frame and self-cleavage occurs post- translationally to give equal amounts of co-expressed protein. This achieves a 1:1 level of expression of the reporter protein and any pathogenic protein encoded by the short tandem repeat region. In one embodiment, the transcriptional or translation split mechanism encodes a P2A peptide comprising an amino acid sequence according to SEQ ID NO:4, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity thereto, or a functional fragment thereof.

[0096] Other Features of the Expression Construct

[0097] Suitably the elements of the expression construct may be present in any workable order which allows the nucleic acid sequences therein to be expressed. In some embodiments, the elements are present in the recited order (i.e. in an upstream to downstream order, with reference to their position with respect to an operably linked promoter). Suitably, some or all of the recited elements may be positioned adjacent to one other (i.e. without any intervening regulatory elements). Suitably the elements may be contiguous or non-contiguous (i.e. they can be positioned immediately adjacent to one another, or they can be separated by a spacer or other sequence). In some embodiments, some or all of the elements are contiguous. In some embodiments, the elements are provided in the recited order and are adjacent to one another.

[0098] Suitably therefore the nucleic acid sequence comprising the short tandem repeat region and the nucleic acid sequence encoding the reporter protein may be reversed. Suitably the expression construct may comprise in a 5' to 3' order an inducible promoter operably linked to the nucleic acid comprising the pathogenic unstable short tandem repeat region, the transcriptional or translation split mechanism, and the nucleic acid encoding the reporter protein. Alternatively, the expression construct may comprise in a 5' to 3' order an inducible promoter operably linked to the nucleic acid encoding the reporter protein, the transcriptional or translation split mechanism, and the nucleic acid comprising the pathogenic unstable short tandem repeat region.

[0099] Suitably the expression construct may comprise one or more further regulatory components to aid expression of the nucleic acid sequences encoded therein. For example, suitable regulatory elements may include a transcription initiation start site, a ribosome binding site, an RNA processing signal, a transcription termination site, and / or a polyadenylation signal. An expression construct may include transcriptional and translational regulatory sequences. These control regions may be native to a wild-type nucleic acid encoding the short tandem repeat region or may be derived from exogenous sources. In general, the transcriptional and translational regulatory sequences may include, but are not limited to, promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences.

[0100] The expression construct may comprise, as necessary, an enhancer, a silencer, a selection marker gene (for example, a drug resistance gene such as a neomycin resistance gene), an SV40 replication origin, and the like. Expression constructs generally have convenient restriction sites located near the promoter sequence to provide for the insertion ofnucleic acid sequences (such as, in the present invention, a nucleic acid sequence comprising a short tandem repeat region as described herein). A selectable marker operative in the host cell may be present to facilitate selection of cells containing the vector. In addition, the expression construct may include additional elements. For example, the expression vector may have one or two replication systems; thus allowing it to be maintained in different organisms, for example in mammalian cells for expression and in a prokaryotic host for cloning and amplification. In addition the expression construct may contain a selectable marker gene to allow the selection of transformed host cells. Selection genes are well known in the art and will vary with the host cell used.

[0101] The expression constructs may also comprise transcription termination regions. Where transcription terminations regions are used, any termination region may be used in the preparation of the expression constructs. For example, the termination region may be native to the transcriptional initiation region, may be native to the operably linked nucleic acid molecule of interest, may be native to the host cell, or may be derived from another source. In one embodiment, the expression construct comprises a polyadenylation signal. Suitably located at the 3' end of the expression construct. In one embodiment the polyadenylation signal is an HSV TK PolyAdenylation sequence, suitably according to SEQ ID NO:6, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity thereto, or a functional fragment thereof.

[0102] Suitably the expression construct comprises Kozak sequences. Suitably the expression construct is codon optimised, suitably codon optimised for mammalian cells.

[0103] In one embodiment, the expression construct comprises a sequence according to SEQ ID NO 18, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity thereto, or a functional fragment thereof. In one embodiment, the expression construct consists of a sequence according to SEQ ID NO 18.

[0104] Vector

[0105] Suitably the expression construct may be comprised upon a vector, any suitable vector for expression in a host cell may be used.

[0106] Suitably the vector can be any naturally occurring or synthetically generated constructs suitable for uptake, proliferation, expression or transmission of nucleic acids in a cell, e.g. plasmids, minicircles, phagemids, cosmids, artificial chromosomes / mini-chromosomes,bacteriophages, viruses such as baculovirus, retrovirus, adenovirus, adeno-associated virus (AAV), herpes simplex virus, or bacteriophages.

[0107] Methods for the construction of vectors are well known to the person skilled in the art, and they are described in various publications and reference texts. In particular, techniques for constructing suitable vectors, including a description of the functional and regulatory components such as promoters, enhancers, termination and polyadenylation signals, selection markers, origins of replication, and splicing signals, are known to the person skilled in the art.

[0108] Suitably the vector is a eukaryotic expression vector. Eukaryotic expression vectors will typically contain also prokaryotic sequences that facilitate the propagation of the vector in bacteria such as an origin of replication and antibiotic resistance genes for selection in bacteria. A variety of eukaryotic expression vectors, containing a cloning site into which a polynucleotide can be operably linked, are well known in the art and several are commercially available from companies such as Stratagene, La JoIIa, CA; Invitrogen, Carlsbad, CA; and Promega, Madison, Wl. Such a plasmid may include a variety of other functional nucleic acid sequences, such as one or more selectable markers, one or more origins of replication, multiple cloning sites and the like.

[0109] Suitably, the vector is an expression vector for expression in eukaryotic cells. Examples of eukaryotic expression vectors include, but are not limited to, pW-LNEO, pSV2CAT, pOG44, pXTl and pSG available from Stratagene; pSVK3, pBPV, pMSG and pSVL available from Amersham Pharmacia Biotech; and pCMVDsRed2-express, pIRES2-DsRed2, pDsRed2-Mito, pCMV-EGFP available from Clontech. Many other vectors are well-known and commercially available. For mammalian cells adenoviral vectors, the pSV and the pCMV series of vectors are particularly well-known non-limiting examples. There are many well-known yeast expression vectors including, without limitation, yeast integrative plasmids (YIp) and yeast replicative plasmids (YRp). For plants the Ti plasmid of agrobacterium is an exemplary expression vector, and plant viruses also provide suitable expression vectors, e.g. tobacco mosaic virus (TMV), potato virus X, and cowpea mosaic virus.

[0110] Suitably, the vector is a plasmid. Suitably therefore the vector is a eukaryotic plasmid. Such a plasmid may include a variety of other regulatory or functional sequences, such as one or more selectable markers, one or more origins of replication, polycloning sites and the like.

[0111] Suitably the vector comprises one or more nucleic acids encoding screenable markers. Suitable screenable markers include marker genes such as those encoding fluorescent proteins.Suitable fluorescent proteins include YFP, CFP, GFP, RFP, EBFP, ECFP, EGFP, YFP, mHoneydew, mBanana, mOrange, tdTomato,mTangerine, mStrawberry, mCherry,mGrape1, mRaspberry, mGrape2 and mPlum. etc. Expression of the marker by host cells having successfully integrated the expression construct allows the isolation of these cells using methods such as, for example, FACS (fluorescent activated cell sorting).

[0112] Suitably the vector comprises one or more nucleic acids encoding selectable markers. The phrase “selectable marker” refers to a protein that enables the separation of host cells expressing the marker from those that lack or do not express it. Suitably to determine when a cell has successfully been transfected with the expression construct or vector. Suitable selectable markers include antibiotic resistance genes such as: a kanamycin resistance gene, spectinomycin resistance gene, streptomycin resistance gene, ampicillin resistance gene, carbenicillin resistance gene, bleomycin resistance gene, erythromycin resistance gene, polymyxin B resistance gene, tetracycline resistance gene, chloramphenicol resistance gene, hygromycin resistance gene, puromycin resistance gene, neomycin resistance gene, zeomycin resistance gene, or a blasticidin resistance gene. Suitably expression of a selectable marker may confer an advantageous property to the host cell that allows survival of only those cells carrying the gene. For example, the marker protein may allow for the selection of the host cell by conferring an antibiotic resistance to the host cell. Consequently, when host cells are cultured in medium containing said antibiotic, only cell clones expressing the marker protein that mediates antibiotic resistance are capable of propagating, indicating the successful integration of the intended expression construct, or vector.

[0113] In some embodiments of the invention the vector is episomal or it may be integrated into the genome of a cell. Suitably the vector stably integrates into the genome of the cell.

[0114] In one embodiment the vector is a mammalian vector suitable for expression in mammalian cell lines, suitably a mammalian plasmid vector. Suitable mammalian vectors include: pcDNA3.1, pCI or pSI, pCMV, pLenti, pRSC, for example.

[0115] In one embodiment the vector is a viral vector, suitably a lentiviral vector suitable for lentivirus transduction of a mammalian cell line as explained below. In one embodiment the vector is pcDNA4 or pcDNA6 available from ThermoFisher Scientific, suitably pcDNA4 / TO .

[0116] In one embodiment the vector comprises or consist of a sequence according to SEQ ID NO:7, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity thereto, or a functional fragment thereof.

[0117] Cell and Population Thereof

[0118] Suitably the expression construct or a vector comprising the expression construct may be provided within a cell, which may be regarded as a host cell. The cell may be present, for example, in vitro in cell culture.

[0119] Suitably the cell may be any type of cell. Suitably the cell may be prokaryotic or eukaryotic. Suitably the cell is eukaryotic. Suitable cells include, but are not limited to, eukaryotic cells, such as yeast, plant, insect or mammalian cells. For example, the cells may be any type of differentiated cells or may, be oocytes, embryonic stem cells, hematopoietic stem cells or other form. In some preferred embodiments the cell is an animal (metazoan) cell (e.g. a mammalian cell). In one embodiment the cell is a mammalian cell. In some embodiments the mammalian cell is a human, simian, murine, rat, rabbit, hamster, goat, bovine, sheep or pig cell. Particularly preferred cells or "host cells" of interest are human, mice, rat, monkey, or rodent cell lines. Suitable mammalian cell lines include: BHK21, BHK TK-, CHO, CHO-K1, CHO-DUKX, CHO-DUKX B1, PC12, CHO-S and CHO-DG44 cells, or derivatives / progenies of any of such cell lines; human embryonic kidney (HEK) cell, preferably a HEK 293F cell; a retinal cell, e.g. a retinal pigmented epithelium (RPE) cell, for example ARPE-19 (ATCC CRL- 2302); murine myeloma cells, preferably NS0 and Sp2 / 0 cells. Suitable host cells are commercially available, for example, from culture collections such as the DSMZ (Deutsche Sammlung von Mikroorganismen and Zeilkuituren GmbH, Braunschweig, Germany) or the American Type Culture Collection (ATCC).

[0120] In one embodiment the cell is a human cell. Suitable human cell lines include: iPSCs, HEK, HEK293F, RPE, Huh7, C2C12, HEPG2, SVG-A, SAOS2, HeLa, SH-SY-5Y, HT1080, and U2OS.

[0121] Suitably the cell is a human cell which can tolerate unstable or expandable tandem repeat regions. Suitably the cell is a human cell which expresses key pathway proteins of interest to the study of the relevant short tandem repeat region within the expression construct. In one embodiment, when the short tandem repeat region comprises exon 1 of the HTT gene, then suitably the human cell expresses proteins involved in the mismatch repair MMR pathway. Suitably the human cell expresses one or more of: MSH3, MSH2, MLH1, MLH3, PMS1, PMS2 and FAN1. In one embodiment the human cell expresses MSH3.

[0122] In one embodiment the cell is a U2OS cell as described in Goold et al. https: / / pubmed.ncbi.nlm.nih.gov / 30358836 / , which has a high tolerance of tandem repeat regions and their expansion.

[0123] Suitably the expression construct or vector is provided to the cell by any known means, suitably by transfection of the cell. Numerous methods of transfection are known to the ordinarily skilled artisan, for example, by Agrobacterium-mediated transformation, protoplast transformation (including polyethylene glycol (PEG)-mediated transformation, electroporation, protoplast fusion, and microcell fusion), lipid-mediated delivery, liposomes, electroporation, sonoporation, microinjection, particle bombardment and silicon carbide whisker-mediated transformation and combinations thereof (see, e.g., Paszkowski, et al., EMBO J., 3:2717-2722 (1984); Potrykus, et al., Mol. Gen. Genet., 199:169-177 (1985); Reich, et al., Biotechnology, 4:1001-1004 (1986); Klein, et al., Nature, 327:70-73 (1987); U.S. Pat. No. 6,143,949; Paszkowski, et al., in Cell Culture and Somatic Cell Genetics of Plants, Vol. 6, Molecular Biology of Plant Nuclear Genes, (Schell and Vasil, eds., Academic Publishers 1989); and Frame, et al., Plant J., 6:941-948 (1994)); direct uptake using calcium phosphate (Wigler, et al., PNAS U.S.A., 76:1373-1376 (1979)); polyethylene glycol (PEG)-mediated DNA uptake; lipofection (see, e.g., Strauss, Meth. Mol. Biol., 54:307-327 (1996)); microcell fusion (Lambert, PNAS U.S.A., 88:5907-5911 (1991); U.S. Pat. No. 5,396,767; Sawford, et al., Somatic Cell Mol. Genet., 13:279-284 (1987); Dhar, et al., Somatic Cell Mol. Genet., 10:547- 559 (1984); and McNeill-Killary, et al., Meth. Enzymol., 254:133-152 (1995)); lipid-mediated carrier systems (see, e.g., Teifel, et al., Biotechniques, 19:79-80 (1995); Albrecht, et al., Ann. Hematol., 72:73-79 (1996); Holmen, et al., In Vitro Cell Dev. Biol. Anim., 31:347-351 (1995); Remy, et al., Bioconjug. Chem., 5:647-654 (1994); Le Bolch, et al., Tetrahedron Lett., 36:6681-6684 (1995); and Loeffler, et al., Meth. Enzymol., 217:599-618 (1993)); or other suitable methods. Methods for production of synthetic chromosomes are described in U.S. application Ser. No. 09 / 815,979.

[0124] Suitably the cell may be transiently or stably transfected with the expression construct. Suitably the cell is stably transfected with the expression construct. In some embodiments, the expression construct integrates into the genome of the cell, in other embodiments, the expression construct remains cytoplasmic.

[0125] In one embodiment the cell is provided with the expression construct or vector by lentiviral transduction. Suitably lentiviral transduction comprises forming viral particlescomprising the expression construct or vector for delivery into cells, and suitably contacting the cell with the lentiviral particles. Suitably the viral particles are formed by co-transfecting packaging cells such as HEK293 cells with a lentiviral transfer vector comprising the expression construct, and two or more viral helper / packaging vectors, and culturing the packaging cells to produce said viral particles, then subsequently collecting the viral particles therefrom.

[0126] Suitably once the cell has been transformed or transduced with the expression construct or a vector it may go through a selection process, suitably to select those cells that successfully contain and express the expression construct. Suitably the selection process may comprise culturing the cell on a selection media, suitably containing a selective agent such as an antibiotic, to determine whether the cell has successfully taken up and can express the expression construct. Suitably only those cells that have successfully taken up the expression construct or vector will comprise the selectable marker gene, suitably which may be an antibiotic resistance gene, which will allow them to survive the selection media.

[0127] Suitably once the cell has been transformed or transduced with the expression construct or a vector it may be cultured, passaged or expanded, into a population of said cells, suitably into a population of cells each comprising the expression construct or vector of the invention. Suitable culture conditions are identified below. A further aspect of the invention defines a population of cells of the invention, and a yet further aspect of the invention defines a cell culture comprising a population of said cells and a medium. Suitably said medium is sufficient to support growth of the cells, suitable media are defined below.

[0128] Method of Screening

[0129] The cells comprising the expression construct or vector of the invention are intended to provide a realistic in vitro model of unstable / expandable short tandem repeat diseases that can be used to study such diseases, including in assays for screening potential therapeutic agents. The method of screening of the invention provides such an assay.

[0130] Suitably the method of screening is a method of screening candidate therapeutic agents. Suitably the candidate therapeutic agents may be any type of therapeutic agent such as: a chemical compound, or a biological molecule. Suitably the candidate therapeutic agents are chemical compounds such as drugs.

[0131] Suitably the candidate therapeutic agents may be derived or obtained from a drug discovery process. Suitably any drug discovery process which may include in silico or in vitroanalysis. Suitably the candidate therapeutic agents are any therapeutic agents which are suspected to inhibit the activity of the MutSβ complex. Suitably the candidate therapeutic agents are any therapeutic agents which are suspected to inhibit the activity of MSH3 or MSH2 proteins, or the interaction with associated proteins such as MLH1, MLH3, PMS1, PMS2 or FAN1, suitably the interaction with MLH1 and / or PMS2. Suitably the candidate therapeutic agents are any therapeutic agents which are suspected to inhibit the ATP hydrolysis activity of said proteins. In one embodiment, the candidate therapeutic agents are any therapeutic agents which are suspected to inhibit the ATP hydrolysis activity of MSH3 protein. In another embodiment, the candidate therapeutic agents are any therapeutic agents which are suspected to inhibit the mismatch recognition activity of MSH3 protein. In another embodiment, the candidate therapeutic agent are any therapeutic agents which are suspected to alter the expression level of MSH3 or other proteins including MSH2, MLH1, MLH3, PMS1, PMS2 and / or FAN1. In another embodiment, the candidate therapeutic agents are any therapeutic agents which are suspected to inhibit the interaction between mismatch repair proteins selected from any of MSH2, MSH3, MLH1, MLH3, PMS1, PMS2 and / or FAN1).

[0132] Suitably the first step of the method comprises culturing the cell under suitable conditions to induce expression of the construct of the invention therein. Suitable conditions are as defined herein but maybe varied by the skilled person depending on the cell type and specific vector used for example. Suitably culturing the cell under suitable conditions to induce expression of the construct therein causes any proteins encoded by the expression construct to be expressed, suitably at least the reporter protein is expressed, optionally any pathogenic protein encoded by the short tandem repeat region is also expressed. Suitably step (a) may comprise culturing the cell under suitable conditions to (i) induce expression of the reporter protein and optionally any pathogenic proteins encoded by the short tandem repeat region, and to (ii) expand the number of short tandem repeats in the short tandem repeat region to a desired level.

[0133] Suitably the cell is cultured for a total period of up to 6 weeks, suitably for 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or 6 weeks. Suitably the cell cultured for a total period of at least 2 to 3 weeks.

[0134] Suitably the cell is cultured in suitable culture media, suitable media may be any commercially available media such as Ham's F12 (Sigma, Deisenhofen, Germany), RPMI-1640 (Sigma), Dulbecco's Modified Eagle's Medium (DMEM; Sigma), Minimal Essential Medium(MEM; Sigma), Iscove's Modified Dulbecco's Medium (IMDM; Sigma), CD-CHO (Invitrogen, Carlsbad, CA), CHO-S-SFMII (Invtirogen), serum-free CHO Medium (Sigma), protein-free CHO Medium (Sigma), EX-CELL Media (SAFC), CDM4CHO and SFM4CHO (HyClone), which may be supplemented. In one embodiment the media used is McCoy5A, suitably with GlutaMAX supplement. Any of the media may be supplemented as necessary with a variety of compounds examples of which are hormones and / or other growth factors (such as insulin, transferrin, epidermal growth factor, insulin like growth factor), salts (such as sodium chloride, calcium, magnesium, phosphate), buffers (such as HEPES), nucleosides (such as adenosine, thymidine), glutamine, glucose or other equivalent energy sources, antibiotics, trace elements. In one embodiment, zeocin and / or blasticidin are added to the media. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art. In the present invention the use of serum-free medium is preferred, but media supplemented with a suitable amount of serum can also be used for the cultivation of cells. For the growth and selection of cells expressing a selectable marker gene a suitable selection agent is added to the culture medium, such as an antibiotic.

[0135] Also contemplated is supplementation of cell culture medium with mammalian sera. Sera often contain cellular factors and components for viability and expansion. Examples of sera include fetal bovine serum (FBS), bovine serum (BS), calf serum (CS), foetal calf serum (FCS), newborn calf serum (NCS), goat serum (GS), horse serum (HS), human serum, chicken serum, porcine serum, sheep serum, rabbit serum, serum replacements and bovine embryonic fluid. In one embodiment the serum is tetracycline free. In one embodiment the serum is fetal bovine serum (FBS), suitably tetracycline free FBS. It is understood that sera can be heat- inactivated at 55-65°C if deemed necessary to inactivate components of the complement cascade. Additional supplements also can be used advantageously to supply the cells with the trace elements for optimal growth and expansion. Such supplements include insulin, transferrin, sodium selenium and combinations thereof. These components can be included in a salt solution such as, but not limited to, Hanks' Balanced Salt Solution® (HBSS), Earle's Salt Solution®, antioxidant supplements, MCDB-201® supplements, phosphate buffered saline (PBS), ascorbic acid and ascorbic acid-2-phosphate, as well as additional amino acids. Many cell culture media already contain amino acids, however, some require supplementation prior to culturing cells. Such amino acids include, but are not limited to, L-alanine, L-arginine, L- aspartic acid, L-asparagine, L-cysteine, L-cystine, L- glutamic acid, L-glutamine, L-glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L- serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine. It is well within the skill of one in the art to determine the proper concentrations of these supplements.

[0136] Suitably the cell is cultured on a surface such as a well or plate, suitably in a 96 well plate. Suitably the cell is culture at a seeding density of between 2-6K cells / well. Suitably the cell is cultured at a temperature of about 15°C to 40°C. Suitably, at a temperature of about 20°C to 40°C. Suitably, at a temperature of about 25°C to 37°C. Suitably, the cell may be cultured at a temperature of about 37°C. Suitably, the cells may be cultured under ambient CO2 concentration. Suitably, the cells may be cultured at 5% CO2 concentration. The CO2 concentration may be selected based on the culturing temperature used.

[0137] Suitably during the culture, the cell is exposed to an inducer. Suitably by introducing the inducer into the culture media. Suitably to an inducer which is capable of inducing transcription from the inducible promoter of the expression construct. Suitable inducers may include any of those listed above in relation to the possible inducible promoters which may be used in the expression construct. In one embodiment, the inducer is tetracycline or doxycycline.

[0138] Suitably the cell is exposed to the inducer in an intermittent or continuous manner during the culture. Suitably the cell may be exposed to the inducer for a part of the period of cell culture or for the entire period of the cell culture. Suitably the cell is exposed to the inducer for the entire period of the cell culture, therefore the cell is exposed to the inducer for a period of 1 day, 2, days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 5 weeks, 6 weeks, 7 weeks, 2 months, during which the cell may be exposed to the inducer intermittently or continuously. In one embodiment, the cell is exposed to the inducer for a period of 2 to 3 weeks. Suitably intermittent exposure to the inducer comprises exposing the cell to the inducer in pulses, or in a pulsed manner. Suitably the pulses of the inducer may be spaced at even intervals during the cell culture. Suitably the pulses of the inducer may be spaced at even intervals across the entire period of the cell culture, suitably across a period of 2 to 3 weeks. Suitably the pulses of the inducer may be given at intervals of once every hour, once every two hours, once every 4 hours, once every 8 hours, once every 12 hours, once every day, once every 2 days, once every 3 days, once every 4 days, once every 5 days, once every 6 days, once every week, or once every 2 weeks. In one embodiment, the pulses of the inducer may be given at intervals of once per week. In one embodiment, the pulses of the inducer may be given at intervals of three times per week. Suitably each pulse of the inducer remains in the cell culturemedia until the media is changed. Suitably the cell media may be changed at regular intervals during the culture, suitably for example every few days, suitably every 3-4 days, suitably every week. In one embodiment, after each pulse, the inducer remains in the cell media for 2 to 3 days. Suitably wherein the inducer is tetracycline.

[0139] In one embodiment the cell is exposed continuously to tetracycline during the culture, suitably over a period of 2 to 3 weeks.

[0140] Suitably the concentration of the inducer is a suitable concentration to induce transcription of the expression construct. Suitably the concentration of the inducer is between 0.1 to 10μg / ml, suitably 0.2 to 8μg / ml, suitably 0.3 to 7μg / ml, suitably 0.4 to 6μg / ml, suitably 0.5 to 5 μg / ml. In one embodiment the concentration of the inducer is between 0.5 to 5 μg / ml. In one embodiment the concentration of the inducer is 2 μg / ml. Suitably wherein the inducer is tetracycline.

[0141] Suitably culturing the cell under conditions to induce expression of the construct, also causes an increase in the number of repeats in the short tandem repeat region of the expression construct i.e. an expansion of the short tandem repeat region. Suitably the conditions of the method are chosen to induce expression of the expression construct in the cell but also to cause the short tandem repeat region to expand; thereby mimicking somatic instability in the cell model. Suitably the inducer, by way of its concentration and delivery to the cell, is operable to drive expression of the expression construct to a level which causes an expansion of the short tandem repeat region.

[0142] Suitably therefore the expansion of the short tandem repeat region can be controlled and tailored by the concentration and duration of exposure of the cell to the inducer, to reach a desired level, suitably to reach a desired number of repeats within the region. Suitably the inducer causes an expansion of the short tandem repeat region of around 1 repeat per week of culture. Suitably, in some embodiments, the inducer causes an expansion of the short tandem repeat region of around 1 CAG repeat per week of culture. Suitably the inducer causes an expansion of the short tandem repeat region by between 1 and 10 repeats, by 2 repeats, by 3 repeats, by 4 repeats, by 5 repeats, by 6 repeats, by 7 repeats, by 8 repeats, by 9 repeats, by 10 repeats per week over the duration of the culture. Suitably the inducer causes an expansion of the short tandem repeat region by at least 2 or 3 repeats over the duration of the culture.

[0143] Suitably the expansion of the short tandem repeat region can be determined by the level of expression of the reporter protein. Suitably when using certain reporter proteins such asluciferase, the expansion of the short tandem repeat region correlates with the level of expression of the luciferase protein and of the luminescence of said protein. Suitably the level of fluorescence or luminescence of said reporter protein may be used to qualitatively determine the number of repeats i.e. the size of the short tandem repeat region and the level of expansion thereof in the cell. Suitably the level of luminescence or fluorescence of said reporter protein may be determined by optionally exposing the cell to a reagent and measuring the amount of light released from the cell using a suitable assay technique. For example, when using a luciferase reporter protein, the level of expression of the reporter protein may be determined by exposing the cell, or a cell lysate thereof, to the reagent luciferin under suitable conditions for the luciferase protein to bind to the luciferin and degrade it, releasing bioluminescence. Suitably the bioluminescence may then be measured by a luminometer. In some embodiments the level of expression of luciferase may be measured using the Nano-Glo(R) assay system available from Promega.

[0144] Suitably the expansion of the short tandem repeat region can also be determined quantitatively by PCR and capillary electrophoresis. Suitably therefore the number of repeats in the short tandem repeat region i.e. the size of said region can be accurately determined by PCR and capillary electrophoresis. Suitably the PCR is performed using suitable primers to amplify the short tandem repeat region of the expression construct. Suitable primers may be those capable of amplifying the short tandem repeat region of the construct, but which do not amplify any endogenous short tandem repeat regions of the cell. Suitable such primers are provided herein as part of the invention and defined below. Suitably the PCR may be qPCR, triplet primed PCR or repeat-flanking PCR.

[0145] Suitably the method may comprise a further step of determining the number of repeats in the short tandem repeat region prior to exposure of the cell to the candidate therapeutic agent, i.e. prior to step (c), and suitably the method may comprise a further step of determining the number of repeats in the short tandem repeat region after exposure of the cell to the candidate therapeutic agent, i.e. after step (c). Suitably such that the number of repeats before and after exposure of the cell to the candidate therapeutic agent may be compared, and the difference determined. Suitably the difference in the number of repeats in the short tandem repeat region before and after exposure of the cell to the candidate therapeutic agent will determine the effect that the candidate therapeutic agent has on cells having an unstable short tandem repeat region as described below.

[0146] Suitably the method comprises a step of exposing the cell to the candidate therapeutic agent. Suitably the cell is exposed to the candidate therapeutic agent by introducing the candidate therapeutic agent into the cell media. Suitably the cell is exposed to the candidate therapeutic agent for a suitable period of time and at a suitable concentration to inhibit MutSβ activity, suitably for a suitable period of time and at a suitable concentration for the inhibition of MutSβ activity to have an effect on the expansion of the short tandem repeat region, suitably for a suitable period of time and at a suitable concentration for the inhibition of MutSβ activity to have an effect on the number of repeats in the short tandem repeat region, suitably for a suitable period of time and at a suitable concentration for the inhibition of MutSβ activity to arrest or contract the number of repeats in the short tandem repeat region.

[0147] Suitably the cell is exposed to the candidate therapeutic agent for a period of hours, days or weeks. Suitably the cell is exposed to the candidate therapeutic agent for a period of 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 10 days, 2 weeks, or 3 weeks, 1 month, 5 weeks, 6 weeks, 7 weeks, or 2 months. Suitably the cell is exposed to the candidate therapeutic agent for a period of between 2 to 6 weeks.

[0148] Suitably the cell is exposed to the candidate therapeutic agent in an intermittent or continuous manner during the culture. Suitably the cell may be exposed to the candidate therapeutic agent for a part of the period of cell culture or for the entire period of the cell culture. Suitably the cell is exposed to the candidate therapeutic agent for the entire period of the cell culture, therefore the cell is exposed to the candidate therapeutic agent for a period of 1 day, 2, days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 5 weeks, 6 weeks, 7 weeks, 2 months, during which the cell may be exposed to the candidate therapeutic agent intermittently or continuously. Suitably intermittent exposure to the candidate therapeutic agent comprises exposing the cell to the candidate therapeutic agent in pulses, or in a pulsed manner. Suitably the pulses of the candidate therapeutic agent may be spaced at even intervals during the cell culture. Suitably the pulses of the candidate therapeutic agent may be spaced at even intervals across the entire period of the cell culture, suitably across a period of 2 to 3 weeks. Suitably the pulses of the candidate therapeutic agent may be given at intervals of once every hour, once every two hours, once every 4 hours, once every 8 hours, once every 12 hours, once every day, once every 2 days, once every 3 days, once every 4 days, once every 5 days, once every 6 days, once every week, or once every 2 weeks. In one embodiment, the pulses ofthe candidate therapeutic agent may be given at intervals of once every day, suitably wherein cell is exposed to the candidate therapeutic agent for around 2 hours. In some embodiments, the cell may be exposed to the candidate therapeutic agent continuously, suitably for the duration of the cell culture after the inducing step. Suitably the cell is exposed to the candidate therapeutic agent for continuously for a period of between 2 to 3 weeks. Suitably the cell is exposed to the candidate therapeutic agent for continuously for a period of between 2 to 4 weeks. Suitably the cell is exposed to the candidate therapeutic agent for continuously for a period of between 2 to 5 weeks. Suitably the cell is exposed to the candidate therapeutic agent for continuously for a period of between 2 to 6 weeks.

[0149] Suitably the cell is exposed to the candidate therapeutic agent at a concentration of 100 μM or less, 90 μM or less, 80 μM or less, 70 μM or less, 60 μM or less, 50 μM or less, 40 μM or less, 30 μM or less, 20 μM or less, 10μM or less, 3 μM or less, 1 μM or less, 0.5 μM or less, 0.3 μM or less, 0.1 μM or less. In one embodiment, the cell is exposed to the candidate therapeutic agent at a concentration of about 0.1 μM.

[0150] Suitably the cell is exposed to the candidate therapeutic agent after the step (b) of inducing expression of the expression construct in the cell. Suitably immediately after step (b) of inducing expression of the expression construct.

[0151] Suitably the method then comprises a step (c) of determining the effect of the candidate therapeutic agent on the short tandem repeat region. Suitably as described above, this step comprises determining the number of repeats within the short tandem repeat region and comparing this value to the number of repeats within the short tandem repeat region before exposure of the cell to the candidate therapeutic agent. Suitably the number of repeats in the short tandem repeat region may be determined qualitatively or quantitatively or both. Suitably this may be referred to as ‘sizing’ the short tandem repeat region. Suitably the number of repeats in the short tandem repeat region may be determined qualitatively by measuring the reporter protein expression as described above. Suitably the number of repeats in the short tandem repeat region may be determined qualitatively by PCR and capillary electrophoresis as described above.

[0152] Suitably the method comprises a step of selecting those candidate therapeutic agents which arrest or contract the number of short tandem repeats in the expression construct as determined above. Suitably the method comprises a step of selecting those candidate therapeutic agents which arrest the expansion of short tandem repeats in the expressionconstruct as determined above. Suitably in which case the number of repeats in the short tandem repeat region did not increase after exposure of the cell to the candidate therapeutic agent. Suitably the method comprises a step of selecting those candidate therapeutic agents which contract or reverse the expansion of short tandem repeats in the expression construct as determined above. Suitably in which case the number of repeats in the short tandem repeat region reduced after exposure of the cell to the candidate therapeutic agent. Suitably an arrest or reduction in the number of repeats in the tandem repeat region may indicate that the candidate therapeutic agent inhibits MutSβ activity. Suitably an arrest or reduction in the number of repeats in the tandem repeat region may indicate that the candidate therapeutic agent inhibits the ATP hydrolysis activity of MutSβ. Suitably, inhibition of MutSβ activity may comprise inhibition of one or more proteins comprised in MutSβ, for example inhibition of MSH2 and / or MSH3. Suitably inhibition may not comprise direct inhibition of MutSβ or its component proteins, but may comprise inhibition of proteins associated therewith, in mismatch repair pathways. Suitably therefore inhibition of MutSβ activity may be indirect inhibition, for example by inhibiting another associated protein such as MLH1, MLH3, PMS1, PMS2 or FAN1. Suitably therefore, an arrest or reduction in the number of repeats in the tandem repeat region may indicate that the candidate therapeutic agent inhibits MutSβ activity directly or indirectly by inhibiting the activity of a protein comprised in MutSβ, or a protein associated with MutSβ.

[0153] Suitably those candidate therapeutic agents which contract the short tandem repeat region in the expression construct may contract the short tandem repeat region by at least 3 repeats, at least 5 repeats, at least 7 repeats, at least 9 repeats. Suitably the method may comprise selecting those candidate therapeutic agents which arrest or contract the number of short tandem repeats in the expression construct by at least 3 repeats, at least 5 repeats, at least 7 repeats, at least 9 repeats.

[0154] Suitably the method may further comprise a step of determining whether each candidate therapeutic agent is toxic to the cell, and optionally selecting those candidate therapeutic agents which have low toxicity to the cell. Suitably low toxicity may be defined as having an IC50 of 50 µM or less. Suitably low toxicity may be defined as having an IC50 of 10 µM or less, an IC50 of 1 µM or less, an IC50 of 0.5 µM or less. Suitably, those candidate therapeutic agents are selected that are substantially non-toxic to the cell. Suitable methods of determining whether an agent is toxic to a cell are known in the art, but may include: Dyereduction assays (e.g. MTT and resazurin assays), mitochondrial membrane potential dyes (e.g. mitotracker), ATP assays (e.g. luminescence ATP assays), cell membrane damage assays (e.g. Trypan blue), cell proliferation and / or cell cycle measurements, apoptosis assays (e.g. Annexin V), glycolytic flux and oxygen consumption assays.

[0155] Suitably the method may comprise a further step of determining the minimally effective concentration of a candidate therapeutic agent, suitably a minimally effective concentration is defined as the lowest concentration for which a significant effect can be detected versus a control. Suitably the method may further comprise a step of selecting those candidate therapeutic agents which have an MEC of 0.3 μM or lower.

[0156] Suitably the method may further comprise a step of isolating said selected candidate therapeutic agent. Suitably the method may further comprise a step of identifying said selected candidate therapeutic agent. Suitably by any known technique of molecular identification such as mass spectroscopy, HPLC, LC, NMR, infrared spectroscopy, crystallography, gene or protein sequencing, and the like. Suitably identifying the selected candidate therapeutic agent may comprise determining the molecular structure of said candidate therapeutic agent.

[0157] Suitably the method may further comprise a step of formulating each of said selected candidate therapeutic agents into a composition, suitably into a pharmaceutical composition. Suitably said pharmaceutical composition may comprise one or more pharmaceutically acceptable carrier substances and / or additives, e.g., buffers, carriers, excipients, stabilisers, liposomes, etc. The compositions may be in a form suitable for oral use (for example as tablets, lozenges, hard or soft capsules, aqueous or oily suspensions, emulsions, dispersible powders or granules, syrups or elixirs), for topical use (for example as creams, ointments, gels, or aqueous or oily solutions or suspensions), for administration by inhalation (for example as a finely divided powder or a liquid aerosol), for administration by insufflation (for example as a finely divided powder) or for parenteral administration (for example as a sterile aqueous or oily solution for intravenous, subcutaneous, intramuscular ,intraperitoneal or intramuscular dosing or as a suppository for rectal dosing). The compositions may be obtained by conventional procedures using conventional pharmaceutical excipients, well known in the art. Thus, compositions intended for oral use may contain, for example, one or more colouring, sweetening, flavouring and / or preservative agents. An effective amount of a selected candidate therapeutic agent for use in therapy is an amount sufficient to treat or prevent a condition referred to herein, slow its progression and / or reduce the symptoms associated with thecondition. The amount of active ingredient that is combined with one or more excipients to produce a single dosage form will necessarily vary depending upon the individual treated and the particular route of administration. For Example, a formulation intended for oral administration to humans will generally contain, for example, from 0.5 mg to 0.5 g of active agent (more suitably from 0.5 to 100 mg, for example from 1 to 30 mg) compounded with an appropriate and convenient amount of excipients which may vary from about 5 to about 98 percent by weight of the total composition. The size of the dose for therapeutic or prophylactic purposes of a selected candidate therapeutic agent will naturally vary according to the nature and severity of the conditions, the age and sex of the animal or patient and the route of administration, according to well-known principles of medicine.

[0158] Suitably a pharmaceutical composition comprising a selected candidate therapeutic agent may be for use in therapy, suitably for use in the treatment or prevention of a disease or disorder. Suitably the disease or disorder is caused by a short tandem repeat region, suitably by a pathogenic short tandem repeat region. Suitable such diseases are described hereinabove. Suitably therefore the or each selected candidate therapeutic agent may be formulated either alone or in combinations into a pharmaceutical composition for use in the treatment or prevention of a disease or disorder associated with a short tandem repeat region, suitably the disease or disorder associated with the pathogenic short tandem repeat region contained in the expression construct which was used in the method of the invention. In one embodiment, the or each selected candidate therapeutic agent may be formulated alone or in combination into a pharmaceutical composition for use in the treatment or prevention of a trinucleotide repeat disorder. In one embodiment, the or each selected candidate therapeutic agent may be formulated alone or in combination into a pharmaceutical composition for use in the treatment or prevention of a CAG repeat disorder. In one embodiment, the or each selected candidate therapeutic agent may be formulated alone or in combination into a pharmaceutical composition for use in the treatment of Huntington's disease.

[0159] In one embodiment the method of the invention may comprise the steps as described in figures 15A, 15B, 15C, and / or 15D, or any combination thereof.

[0160] Primer Pair

[0161] The present invention further provides a pair of primers for accurately determining the number of repeats in a short tandem repeat region, suitably for ‘sizing’ such a short tandem repeat region.

[0162] Suitably, there is provided a primer pair comprising a first primer capable of annealing to a site within exon 1 of the HTT gene, and a second primer capable of annealing to a site within a transcriptional or translational split mechanism or within a nucleic acid encoding a reporter protein.

[0163] Suitably the first primer is the forward primer and the second primer is the reverse primer, but the skilled person will understand that the inverse is possible.

[0164] Suitably, the second primer is capable of annealing to a site within a nucleic acid sequence encoding a P2A self-cleaving peptide. Alternatively, the second primer is capable of annealing to a site within a nucleic acid sequence encoding a luciferase reporter protein, suitably NanoLuc reporter protein.

[0165] Suitably the first primer anneals to nucleotides corresponding to positions 29 – 51 (following from initiation codon ATG corresponding to nucleotides at positions 1-3) in exon 1 of the HTT gene, according to SEQ ID NO: 3. Suitably the second primer anneals to any site in a nucleic acid sequence encoding the P2A self-cleaving peptide. Suitably therefore the first and second primers are complementary to said sites.

[0166] In one embodiment the first primer comprises or consists of a sequence according to SEQ ID NO:9, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity thereto, or a functional fragment thereof. In one embodiment the second primer comprises or consists of a sequence according to SEQ ID NO:10 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity thereto, or a functional fragment thereof.

[0167] In some cases, one or both of the primers may comprise a probe, suitably a detectable probe. In one embodiment, the detectable probe may be FAM. In one embodiment the forward primer comprises a detectable probe, suitably FAM, suitably at the 5' end thereof. In one embodiment therefore, the first primer comprises or consists of a sequence according to SEQ ID NO:11, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity thereto, or a functional fragment thereof.

[0168] The present invention further provides the use of the primer pair for determining the number of short tandem repeats in a nucleic acid sequence comprising exon 1 of the HTT gene. Suitably therefore the primer pair of the invention may be used in the method according to the invention, suitably in any steps of the method in which the size of the short tandem repeat region is determined.

[0169] Suitably the primer pair is for use in determining the number of repeats in exon 1 of the HTT gene. In one embodiment the primer pair is for use in determining the number of short tandem repeats in an expression construct according to the first aspect, suitably in which the nucleic acid sequence comprising a pathogenic short tandem repeat region comprises exon 1 of the HTT gene. Advantageously, the primer pair will not amplify endogenous HTT sequences or other "exon 1 only" constructs, therefore the resulting PCR signal from the amplified fragments is very specific and able to be detected easily, because it does not get masked by the much stronger signal from the endogenous HTT loci within the host cells.

[0170] In another aspect, the present invention further provides a kit comprising the expression construct of the first aspect and the primer pair of the fifth aspect. Optionally the kit may further comprise one or more vectors, one or more cell culturing reagents (for example media, one or more additives, one or more selective agents such as antibiotics), a suitable cell line such as U2OS cells, one or more PCR reagents (such as Taq DNA polymerase, probes, ddNTP nucleotides, buffer), and one or more capillary electrophoresis reagents (such as electrolyte buffers). Suitably additional reagents which may be comprised within the kit include: reagents for performing a luciferase reporter assay such as luciferin, reagents for performing HTRF (Homogeneous Time Resolved Fluorescence) or another HTT detection assay such as labelled donor and acceptor antibodies capable of binding the HTT protein. . FIGURES

[0171] The invention will now be described with reference to the following figures in which:

[0172] FIG. 1A, FIG. 1B, and FIG. 1C, show diagrams of exemplary expression constructs of the invention comprising different numbers of short tandem repeats, the two tetracycline operator sites under the control of the CMV promoter are upstream of the canonical exon 1 HTT sequence with either 18, 50 or 100 CAG repeats, a P2A self-cleaving peptide and nanoluciferase sequences prior to a stop codon and HSV TK polyA sequences (not shown). Length in base pairs for each construct component is indicated.

[0173] FIG. 2 shows an exemplary plasmid vector of the invention, pcDNA4TO_18CAG, comprising an expression construct of the invention with 18 CAG repeats in exon 1 HTT sequences, and various other regulatory elements.

[0174] FIG. 3 shows canonical mismatch repair (MMR) proteins are confirmed to be robustly expressed at the RNA level in U2OS cells via the custom Quantigene Plex assay. Shown are therelative expression levels with respect to hRNA controls. Indicated are MutS, MutL and other MMR components in addition to housekeeping targets.

[0175] FIG. 4 shows western blots confirming MSH3, MLH1, EXO1 and FAN1 protein expression in U2OS cells. Bands of the expected size are highlighted in boxes while marker sizes (kDa) are indicated with lines.

[0176] FIG. 5 shows stable pools of transfected-U2OS cells; U2OS cells were transfected with a pcDNA6 / TR vector and maintained under blasticidin. Untransfected cells show death predominantly by 48 hours. TRex-U2OS cells were subsequently cultured at 1.25 ug / ml blasticidin to maintain selection pressure.

[0177] FIG. 6 shows stable pools of U2OS cells containing Exon 1 HTT expression constructs containing 100 or 18 CAG repeats with zeocin selection.

[0178] FIG. 7 shows capillary electrophoresis data FIG. 7A shows the GeneScan 1200-LIZ size standards (60-1200) as visualised in GeneMapper and FIG. 7B shows an example of PCR product trace seen with gDNA extracted from a 1C6 clone containing a Exon 1 HTT expression construct in GeneMapper. Each individual peak >10% of the highest peak is labelled according to size of the peak, outlined in large box (sizes in clear boxes under each peak). These PCR products are representative of all the integrated construct sequences in the population sampled.

[0179] FIG. 8 shows a visualisation of PCR products by agarose gel electrophoresis showing the measurement of the size of CAG repeats in cells comprising an Exon 1 HTT expression construct, the three clonal cell lines prioritised for further expansion were cultured in the presence of either no tetracycline or tetracycline pulsed once a week in medium. Cells were collected at weekly intervals (days of collection indicated). Genomic DNA was extracted and construct-specific CAG repeats were amplified prior to separation on an agarose gel. Dashed line indicates the starting population of CAG repeats for 1C6 clonal line (103 CAG) and PCR products can be seen to have retarded migration at longer time points; which was more marked in the presence of tetracycline pulses. These data were an early indication of the time- and Tet- dependent CAG expansion phenotypes seen in the three clones. 50 and 100 CAG plasmids were included as controls (right).

[0180] FIG. 9 illustrates HTRF assay confirmation of exon 1 HTT expression in lysates from stable pools with 100 CAG construct. FIG. 9A shows an assay schematic illustrating the location of epitopes that the antibodies recognise with 2B7-Tb binding to the first 17 amino acids of exon 1 HTT and MW1-AF488 binding to the glutamine repeats. The signal increaseswith increasing glutamine repeat size. FIG.9B illustrates the HTRF signal from stable pools cultured with (Tet, filled diamonds) or without tet (no Tet, open diamonds) at different seeding densities thus illustrating tetracycline based induction of exon 1 HTT.

[0181] FIG. 10 shows orthogonal confirmation of tetracycline dependency expression induction with the nano-luc reporter. FIG. 10A indicates the NanoGlo signal (relative luminescent units) for the different stable pools with and without tetracycline induction. 18 CAG containing pools yielded a higher signal than 100 CAG containing pools; and tetracycline addition increased the signal further. Included is a control line without nanoluc expression sequences to confirm low background. FIG. 10B shows the NanoGlo signal for each of the clonal lines with 100 CAG with and without tetracycline induction to enable calculation of fold-induction for each line (Table 1).

[0182] FIG. 11 shows Cell Titer Glo signals (relative luminescent units) for each of the pools. Tetracycline addition and increased expression of exon 1 HTT and nanoluc sequences did not confer additional cytotoxicity.

[0183] FIG. 12 shows further characterization of two clonal lines: 1C6 and 2E5. FIG. 12A shows the cumulative cell counts over 42 days of culture for three replicates of either 1C6 or 2E5 clonal lines. FIG. 12B shows cell doubling time in days over 42 days culture for the same three replicates with both clonal lines showing similar cell numbers and doubling rates. FIG. 12C shows the cell viability over time, which remains around 95-100% for both cell lines.

[0184] FIG. 13A shows a summary table of each 100 CAG clonal line with respect to nanoluc induction, HTT exon 1 expression and CAG repeat sizes. FIG 13B shows representative images of lines 1C6 and 2E5 at day 0, day 21 and day 42. There are no obvious changes in morphology over time.

[0185] FIG. 14A illustrates the schematic of activities for siRNA treatment in 1C6 cells over three weeks. FIG 14B shows an example 96 well plate layout for siRNA treatment in 1C6 cells over three weeks.

[0186] FIG. 15A illustrates the schematic of activities for candidate compound treatment in 1C6 cells over three weeks. FIG 15B shows compound stock preparation table. FIG 15C shows an example 96 well plate layout for compound treatment in 1C6 cells over three weeks. Fig 15D shows the processes to be employed over the candidate compound treatment time course.

[0187] FIG. 16 shows the dynamic and progressive repeat expansions in stable, clonal cell lines 1C6, 1G10 and 2E5 as determined by capillary electrophoresis data. FIG. 16A illustratesthe change in modal repeat size for each of the lines maintained without tetracycline addition or with once-weekly tetracycline pulses. For each of the three clones (1C6, 1G10 and 2E5), there is a degree of tetracyline-induced increase in repeat size over time. Furthermore, each of the three clones shows more instability in a permissive (ie tetracycline addition) environment than other published options such as iPSC with 109 repeats either as stem cells or in vitro differentiated to medium spiny like neurons (at 60 days) and U2OS cells transiently transfected (at 47 days). FIG. 16B illustrates the normalised peak height data over time for the 2E5 cell lines illustrating how the modal peak shifts to the right (i.e. increased CAG repeat number) and how the spread of distribution alters over time. These data echo those illustrated in FIG. 8.

[0188] FIG. 17 shows the results of a reproducibility study with respect to repeat expansion profiles measured at 3 weeks with once-weekly tetracycline pulsing. Capillary electrophoresis profiles for cells treated with weekly tetracycline pulses from Day 0 and Day 21 samples are shown for 1C6 (FIG. 17A) and 2E5 (FIG. 17B). Each of the three replicates shows strong concordance. Clone 1C6 (FIG. 17A) showed a lower rate of expansion than Clone 2E5 (FIG. 17B) but data are more reproducible with less variation.

[0189] FIG. 18 shows the refinement of induction of CAG repeat expansion by exposing cells comprising an Exon 1 HTT expression construct to pulsed or continuous tetracycline inducer over 3 to 6 weeks; the duration of culture has clear effect on repeat expansion profiles, and Tet addition has clear effect on repeat expansion profiles. Mean CAG repeat numbers in 1C6 clones are indicated for each experimental conditions. “D0” (Day 0) is the starting point for each experiment. “No Tet” are cultures maintained in the absence of tetracycline and “Tet” are cultures maintained with tetracycline, either pulsed once a week or added continuously. The differential mean CAG numbers for no Tet versus Tet treatments indicates a minimal assay window for responsiveness to a manipulation.

[0190] FIG. 19A shows the results of a validation experiment using 1C6 cells transduced with vectors expressing either scrambled (Scr) shRNA sequences (control) or shRNA sequences designed to reduce expression of MSH2, MSH3 or MSH6. Control conditions are shown on the left from baseline (Day 0 or D0), no tetracycline (no Tet) and tetracycline (Tet) addition together with scrambled shRNA (Scr). Tetracycline addition induced expansion of CAG repeats in the cell line with scrambled shRNA compared to no tetracycline and Day 0. Knockdown of MSH2 or MSH3 reduced the expansion rate of repeats while knockdown of MSH6 accelerated the expansion rate. FIG. 19B shows the effect of individual MSH3 shRNA sequences on repeatexpansion profiles. Control conditions are shown on the left from baseline (Day 0), no tetracycline (no Tet) and tetracycline (Tet) addition alone or together with scrambled shRNA (Scr). The number of mean CAG repeats with each individual shRNA targeting MSH3 (teal) is shown (left y-axis) versus the MSH3 expression level as measured by quantitative RT-qPCR (right y-axis). Increasing levels of knockdown of MSH3 corresponds to increasing levels of repeat stabilisation.

[0191] FIG. 20 illustrates candidate compound response curves in 1C6 stable clonal line. A candidate compound (compound 5) was titrated from an appropriate top concentration in half- log concentrations and tested for its repeat stabilization effect versus vehicle using the assay method of the invention (0.1% DMSO final assay concentrations). Shown on the left are controls for Day 0, no tetracycline and tetracycline addition, and vehicle. Compound associated data are indicated with compound 5 (a potent and selective MutSβ inhibitor) shown as an exemplar, using the assay it can clearly be detected that this compound arrests CAG repeat expansion. Significance (determined by comparison with the vehicle) is indicated by asterisks (ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001).

[0192] FIG. 21 shows a summary of additional data generated in 1C6 stable clonal line with eight candidate MutSβ inhibitory compounds. This table indicates the biochemical inhibitory activity of each compound in μM; the minimum effective concentration (MEC) in μM (defined as the lowest concentration for which a significant effect can be detected versus the vehicle control); the estimated cellular EC50 in μM and the compound concentration response data figure for reference.

[0193] FIG. 22 illustrates candidate compound response curves in 1C6 stable clonal line. A candidate compound (compound 1) was titrated from an appropriate top concentration in half- log concentrations and tested for its repeat stabilization effect versus vehicle using the assay method of the invention (0.1% DMSO final assay concentrations). Shown on the left are controls for no tetracycline and tetracycline addition, and vehicle. Compound associated data are indicated with compound 1 (a potent and selective MutSβ inhibitor) shown as an exemplar, using the assay it can clearly be detected that this compound arrests CAG repeat expansion. Significance (determined by comparison with the vehicle) is indicated by asterisks (ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001).

[0194] FIG. 23 illustrates candidate compound response curves in 1C6 stable clonal line. A candidate compound (compound 2) was titrated from an appropriate top concentration in half-log concentrations and tested for its repeat stabilization effect versus vehicle using the assay method of the invention (0.1% DMSO final assay concentrations). Shown on the left are controls for no tetracycline and tetracycline addition, and vehicle. Compound associated data are indicated with compound 2 (a potent and selective MutSβ inhibitor) shown as an exemplar, using the assay it can clearly be detected that this compound arrests CAG repeat expansion. Significance (determined by comparison with the vehicle) is indicated by asterisks (ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001).

[0195] FIG. 24 illustrates candidate compound response curves in 1C6 stable clonal line. A candidate compound (compound 3) was titrated from an appropriate top concentration in half- log concentrations and tested for its repeat stabilization effect versus vehicle using the assay method of the invention (0.1% DMSO final assay concentrations). Shown on the left are controls for Day 0, no tetracycline and tetracycline addition, and vehicle. Compound associated data are indicated with compound 3 (a potent and selective MutSβ inhibitor) shown as an exemplar, using the assay it can clearly be detected that this compound arrests CAG repeat expansion. Significance (determined by comparison with the vehicle) is indicated by asterisks (ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001).

[0196] FIG. 25 illustrates candidate compound response curves in 1C6 stable clonal line. A candidate compound (Compound 4) was titrated from an appropriate top concentration in half- log concentrations and tested for its repeat stabilization effect versus vehicle using the assay method of the invention (0.1% DMSO final assay concentrations). Shown on the left are controls for Day 0, no tetracycline and tetracycline addition, and vehicle. Compound associated data are indicated with compound 4 (a potent and selective MutSβ inhibitor) shown as an exemplar, using the assay it can clearly be detected that this compound arrests CAG repeat expansion. Significance (determined by comparison with the vehicle) is indicated by asterisks (ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001).

[0197] FIG. 26 illustrates candidate compound response curves in 1C6 stable clonal line. A candidate compound (compound 6) was titrated from an appropriate top concentration in half- log concentrations and tested for its repeat stabilization effect versus vehicle using the assay method of the invention (0.1% DMSO final assay concentrations). Shown on the left are controls for Day 0, no tetracycline and tetracycline addition, and vehicle. Compound associated data are indicated with compound 6 (a potent and selective MutSβ inhibitor) shown as an exemplar, using the assay it can clearly be detected that this compound arrests CAG repeatexpansion. Significance (determined by comparison with the vehicle) is indicated by asterisks (ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001).

[0198] FIG. 27 illustrates candidate compound response curves in 1C6 stable clonal line. A candidate compound (compound 7) was titrated from an appropriate top concentration in half- log concentrations and tested for its repeat stabilization effect versus vehicle using the assay method of the invention (0.1% DMSO final assay concentrations). Shown on the left are controls for Day 0, no tetracycline and tetracycline addition, and vehicle. Compound associated data are indicated with compound 7 (a potent and selective MutSβ inhibitor) shown as an exemplar, using the assay it can clearly be detected that this compound arrests CAG repeat expansion. Significance (determined by comparison with the vehicle) is indicated by asterisks (ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001).

[0199] FIG. 28 illustrates candidate compound response curves in 1C6 stable clonal line. A candidate compound (compound 8) was titrated from an appropriate top concentration in half- log concentrations and tested for its repeat stabilization effect versus vehicle using the assay method of the invention (0.1% DMSO final assay concentrations). Shown on the left are controls for Day 0, no tetracycline and tetracycline addition, and vehicle. Compound associated data are indicated with compound 8 (a potent and selective MutSβ inhibitor) shown as an exemplar, using the assay it can clearly be detected that this compound arrests CAG repeat expansion. Significance (determined by comparison with the vehicle) is indicated by asterisks (ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001).

[0200] FIG. 29 illustrates candidate compound response curves in 1C6 stable clonal line. A candidate compound 7 was titrated from an appropriate top concentration in half-log concentrations and tested for its repeat stabilization effect versus vehicle and no tetracycline control using the assay method of the invention (0.1% DMSO final assay concentrations). Compound associated data are indicated with compound 7 (a potent and selective MutSβ inhibitor) shown as an exemplar, using the assay it can clearly be detected that this compound inhibits CAG repeat expansion over 6 weeks. EXAMPLES

[0201] Materials and Methods

[0202] Construct generation

[0203] The pcDNA™4 / TO vector contains two tetracycline operator 2 (TetO2) sites within the human cytomegalovirus immediate-early (CMV) promoter for tetracycline regulated expression of your gene of interest (Yao et al., 1998). The TetO2 sequences serve as binding sites for four Tet repressor molecules (comprising two Tet repressor homodimers) and confer tetracycline-responsiveness to the gene of interest. In the absence of tetracycline, expression of your gene of interest is repressed by the binding of Tet repressor homodimers to the TetO2 sequences. Addition of tetracycline to the cells de-represses the hybrid CMV / TetO2 promoter in pcDNA™4 / TO and allows expression of your gene of interest. The pcDNA™6 / TR regulatory plasmid is designed for use with the T-REx™ System for tetracycline-regulated expression of the gene of interest in mammalian cells. (www.lifetechnologies.com).

[0204] 18, 50 and 100 CAG repeat containing plasmids according to the construct design (Figures 1 and 2) were custom synthesized at GeneArt. The expression sequence comprises exon 1 huntingtin with ~100 pure CAG repeats, a P2A self-cleaving peptide and a nanoluc sequence followed by a polyA signal (Seq ID NO: 18). Full plasmid sequence is indicated (Seq ID NO:7 ). The plasmid contains sequences for ampicillin (bacterial propagation) and zeocin (mammalian cell lines) resistance.

[0205] Cell culture

[0206] The U-2 OS cell line (purchased from LGC standards, ATCC) is derived from a moderately differentiated sarcoma from the tibia of an adolescent female. Karyotyping has shown that although there are a lot of chromosomal abnormalities the tumour suppressor genes p53 and pRb are still fully functional in the U2OS cell line.

[0207] Routine culture of the adherent cells in non-PDL plastic ware in a humidified 37^C incubator with 5% CO2. Cells were maintained in McCoy’s 5A (modified) medium with GlutaMAX supplement (Life Technologies 3660-021) and tetracycline-free FBS (Clontech 631106). Maintenance medium for the clonal cell lines expressing Tet repressor and exon 1 HTT / nanoluc constructs were additionally supplemented with zeocin to 100 ug / ml and blasticidin to 1.25 ug / ml. Cells were passaged at 70-80% confluence once or twice a week, either to a split ratio or defined seeding density using DPBS and TrypLE express enzyme (1x, no phenol red, Life technologies 12604-039) was used to dissociate the cells. Cells were frozen with Cell Banker (AMSBIO 11891) or with medium plus 10% DMSO and stored in liquid nitrogen vapour phase.

[0208] Cell line characterisation (U2OS background)

[0209] This included expression analyses (branched genomic DNA analyses (see Figure 3 and transcriptomic profiling) to confirm expression levels of key MMR components, whole exome sequencing to confirm which genetic variants were present and Western blotting (see Figure 4).

[0210] Branched DNA analyses

[0211] We leveraged a fast, high-throughput solution to assess gene expression levels in a multiplexed fashion. The QuantiGene Plex assay (Invitrogen) is hybridisation based and incorporates branched DNA technology which uses signal amplification as opposed to direct measurement of RNA transcripts. We generated a custom-designed Invitrogen QuantiGene plex reagent system (QGP-144-M19101702) for 76 target specific probes associated with magnetic beads. Cell lysates were prepared and assessed using manufacturer recommendations. The negative control was baseline signal while the positive control was hRNA. The geomean of housekeeping genes was employed as a normalisation strategy.

[0212] gDNA and RNA extraction and sequencing

[0213] Tissues were homogenised in Qiazol lysis reagent (Qiagen). RNA and gDNA isolation was performed using the RNeasy plus mini kit (Qiagen) following the manufacturer’s protocol.

[0214] RNA and DNA concentration, integrity and quality were measured (Qubit Fluorometer, Themo Fisher Scientific Q33238 as per manufacturers protocol).

[0215] RNA (N=3) was converted into cDNA, indexed and libraries sequenced on the Illumina NovaSeq 6000 system to generate at least 30 million paired-end reads. Sequence mapping and assembly enabled transcript identification and quantification (FPKM values). Scatter and volcano plots confirmed good agreement between samples.

[0216] gDNA (N=3) was indexed and libraries sequenced on the Illumina NovaSeq 6000 system to generate at least 30 million paired-end reads. Sequences were mapped and mapped to hg19 reference with 95% of data aligned to one site on the reference genome. GATK haplotype caller was used to identify SNPs and indels with over 25,000 functional variants identified.

[0217] Western blot analyses

[0218] For confirming MSH3 expression homogenates were prepared (10 mM HEPES pH 7.2, 250 mM sucrose, 1 mM EDTA + protease inhibitor cocktail (Roche), 1 mM NaF and 1 mM Na3VO4and sonicated for 10 seconds. Protein concentration was determined using Bradford. NuPAGE™ gels (4-12%) under reducing conditions using the MES SDS Running Buffer system. Subsequently, the proteins were transferred to PVDF membranes using the iBlot™ 2Gel Transfer system. To block non-specific binding, the membranes were incubated in 5% milk in TBS-T (TBS with 0.05% Tween) and then probed with primary antibodies, which were diluted in 5% milk in TBS-T. The primary antibodies used were MSH3 (SC-271079, 127 kDa), MLH1 (ab92312, 84 kDa), FAN1 (ab68572, 114 kDa) and EXO1 (MAS-12262, 95 kDa). Bound antibodies were detected using enhanced chemiluminescence (ECL Prime, Cytiva) with a ChemiDoc MP imaging system (BioRad).

[0219] Cell line generation

[0220] Kill curves for antibiotics were performed on U2OS cells: blasticidin = 12.5 μg / ml for cell line generation and 1.25 μg / ml for maintenance (TetR); zeocin (TetO) = 100 μg / ml; puromycin (shRNA) = 1 μg / ml and hygromycin B = 100 μg / ml.

[0221] All cell line generation transfections were performed with Lipofectamine™ 3000 Transfection Reagent according to the manufacturers protocol.

[0222] Genotyping and CAG repeat sizing

[0223] For genotyping and CAG repeat sizing, cell culture medium was removed, cells were washed twice with 1x DPBS then genomic DNA isolation using QIAGEN 96 well plate kits was performed according to the manufacturers protocol. DNA was quantified and diluted to 10 ng / μL with TE buffer. Genotyping PCRs were performed in 20 ul volume in the presence of 1x AmpliTaq Gold 360 mastermix, 1 μM each of forward and reverse primers, GC enhancer. Forward primer is upstream of the CAG repeats and labelled at 5’ site with FAM dye: 5’-FAM- CCTTCGAGTCCCTCAAGTCCTT (SEQ ID NO: 11), reverse primer is located in the P2A coding sequence: 5’-TGCTTGCTTTAGCAGAGAGAA-3’ (SEQ ID NO: 10) . Thermal cycling was performed with an initial denaturing step at 95C for 10 minutes followed by 35 cycles of 95C for 30 seconds, 58C for 30 seconds and 72C for 90 seconds. The reactions were held at 72C for 10 minutes then cooled to 15C. 1 μl of PCR product was denatured at 95C for 3 minutes in a mix of 8.75 μl HiDi Formamide (Thermo Fisher Scientific) and 0.25 μl of GeneScan 1200-LIZ size standards (total volume of 10 μl) prior to rapidly cooling and loading on an ABI3500XL Genetic Analyser. The values for injection conditions with at 36cm capillary array are: injection time = 24 seconds, injection voltage = 1.2 kV, run voltage = 15.0 kV, run temperature = 60C and run time = 2000 seconds. Samples were initially analysed using GeneMapper v6 software (Thermo Fisher Scientific). All the peaks adjacent to the main peak that are greater than 10% of the main peak height are labelled with the size and peak height prior to exporting these data (see Figures 7 and 8).

[0224] In addition, the genomic DNA extracted from cells were used as a template for Sanger sequencing (chain termination) using the forward primer 5’- ATGAAGGCCTTCGAGTCCCTCAAGTCCTTC-3’ (SEQ ID NO: 16) and the reverse primer 5’-CGGCTGAGGCAGCAGCGGCTGT-3’ (SEQ ID NO: 17) as described in Benn et al 2005 (https: / / pubmed.ncbi.nlm.nih.gov / 16183657 / ).

[0225] Homogenous time-resolved fluorescence

[0226] Homogeneous time-resolved fluorescence (HTRF) was performed as previously described (Smith et al 2023 or Landles et al 2022 refs). The antibody parings and concentrations for the soluble HTT exon1 assay were donor: 2B7-Tb (which recognises the first 17 amino acids of exon 1 HTT), 1 ng and acceptor: MW1-AF488 (which recognises polyglutamine repeat sequences), 40 ng. The donor antibody will fluoresce upon excitation and transfer the energy to the nearby acceptor. The signal is known to increase with CAG length with this assay format (see Figure 9). The donor and acceptor antibodies were added in 5 μL HTRF detection buffer (50 mM NaH2PO4, 0.2 M KF, 0.1% bovine serum albumin, 0.05% Tween-20) to give the optimized antibody concentration when added to 10 μL of a 10 % lysate concentration in a 384-well ProxiPlate (Greiner Bio-One). Following incubation for 3 h on an orbital shaker (250 rpm), plates were read using an EnVision (Perkin Elmer) plate reader.

[0227] NanoGlo

[0228] Cells were washed twice with 1x PBS, 50 ul PBS added to each well followed by 50 ul NanoGlo assay reagent according to the manufacturers protocol (Promega) and luminescence measured on a Pherastar (Figure 10).

[0229] Cell viability

[0230] The CellTiterGlo® assay from Promega was performed according to the manufacturers protocol. In addition, cell counts (with trypan blue) was leveraged to track population doubling times and cell viability (Figures 11, 12 and 13). Statistical analyses

[0231] The analysis of a set of summary statistics was generated using the raw capillary electrophoresis data using a custom R script to rapidly derive the following: mean, median and mode (as measures of central location), standard deviation, variance and interquartile range (as measures of variation), CAG repeat indices (instability index, contraction index and expansion index as described: Lee et al 2011) and also kurtosis, skewness and N (sum height, similar toarea under curve). The set of summary statistics was subjected to more detailed analyses using JMP, v17 (SAS Institute Inc., Cary, NC) including principal component analyses. Data were screened for outliers using the ROUT test (Q = 10%; GraphPad Prism v9) and outliers were removed from the analysis. Graphs were prepared using Prism v9 (GraphPad Software, California, USA). Significance testing was by Dunnett’s, one-way ANOVA, two-way ANOVA or mixed-effects model (REML) with either Tukey’s or Bonferroni post hoc tests. P-values <0.05 were considered statistically significant.

[0232] Lentivirus transduction

[0233] Transductions with lentivirus containing shRNA constructs were performed according to the manufacturer recommendations (Mission library, Merck Millipore Sigma).

[0234] siRNA transfection standard operating protocol MATERIALS Cell culture • Trypan Blue Gibco™ (Cat# 15250061) • Nexcelom counting slides Nexcelom (Cat# SD100) • Penicillin-Streptomycin (P / S) (10,000 U / mL) Gibco™ (Cat# 15140122) • McCoys 5A (Modified) medium Gibco™ (Cat# 16600082) • Tet-System Approved FBS Gibco™ (Cat# A47362-01) • Zeocin® 100 mg / mL Invivogen (Cat# ant-zn-05) • Blasticidin 10 mg / mL Invivogen (Cat# ant-bl-05) • 0.25% Trypsin EDTA Gibco™ (Cat# 15050065) • PBS -MgCl2-CaCl2Gibco™ (Cat# 10010023) • Freezing Medium Cryo-SFM Sigma Aldrich (Cat# C-29912) siRNA Transfection ^ ON-TARGETplus siRNA Horizon (Cat# N.A) ^ Lipofectamine RNAiMAX Thermofisher (Cat# 13778) ^ Opti-MEM Gibco™ (Cat# 51985034) ^ Tetracycline Gibco™ (Cat# A39246) ^ DMSO Fisher (Cat# BP231-100) 2.3 DNA extraction ^ Quick-DNA™ 96 Kit™ 10 x 96 preparations Zymo (Cat# D3012) ^ Quick-DNA Microprep Plus Kit Zymo (Cat# D4074) ^ Β-mercaptoethanol MP (Cat# 190242) 2.4 Consumables ^ Centrifuge tubes ^ 50 mL falcon tubes ^ 96-well flat bottom microplates (sterile)^ 96-well white opaque microplates ^ T75 culture flasks ^ Sterile 100 mL single use reservoirs ^ Cryovials ^ Foil Plate seals 2.5 Instrumentation ^ T4 Automated cell counter Nexcelom ^ GloMAX Explorer Promega® METHOD All work should be conducted under aseptic sterile technique, in a laminar flow hood. Methods of cell thawing, maintenance, and freezing are covered above. Lo-Q platform Assay for siRNA Knockdown

[0235] The Lo-Q platform (i.e. stable clonal cell line such as 1C6) has been optimised to run as a 3-week assay, siRNA transfections should be replenished every time the cells are passaged, or the media changed, following the standard process detailed in the schematic (Figure 14A). Thus, the cell line can be used to identify novel targets that modulate the repeat expansion behaviour. Assay initiation - Cell seeding: ^ Cell seeding for Lo-Q platform assays should be initiated on Mondays (am) to ensure Tetracycline pulsing can be maintained every other day. ^ The Friday prior to initiating any Lo-Q platform assay ensure there are sufficient cells in culture to seed the required number of assay plates. If required, passage the cells, otherwise perform a media change to McCoys 5A, 10% Tet-free FBS, 1.25 µg / mL Blasticidin®, and 100 µg / mL Zeocin®. ^ On the Monday morning, aspirate the media from the required number of culture flasks containing U20S-IC6 cells and wash with 2 mL PBS. ^ Aspirate the PBS and add 2 mL 0.25% Trypsin EDTA. Incubate the plate at 37°C for ~2 m, or until >90% of the cells have detached. ^ Resuspend the detached cells with 8 mL McCoys 5A, 10% Tet-Free FBS (total volume 10 mL), and transfer to a 50 mL Falcon tube. ^ Take a 60 µL sample of the cell suspension into a fresh 1.5 mL Eppendorf tube and add 20 µL Trypan Blue. Mix and transfer 20 µL of the solution to a clean cell counting slide. Determine the cell count per mL and the overall viability. ^ Dilute a sufficient volume of the cell suspension in McCoys 5A, 10% Tet-Free FBS, to achieve a cell density of 5,000 cells / well at a volume of 200 µL per well of a 96-well plate (25,000 cells / mL). ^ Using sterile clear flat bottom 96-well plates, fill the top row (A1-12) with 200 µL PBS. These wells must be kept empty for subsequent downstream analysis processing purposes.^ Next, transfer the diluted cell suspension to a sterile reservoir and using a 1250 µL multichannel integra, pipette 200 µL per well of the clear flat bottom 96-well plates (rows B-H). ^ Place the seeded 96-well plates in an incubator at 37°C, 5% CO2, for ~24 h. ^ At least 3 day 0 samples should be taken at this point. Using the leftover cells from the trypsinised T75 flasks, prepare 3 x 1.5 mL Eppendorf tubes containing a minimum of 3 x 105cells. ^ Centrifuge the tubes to pellet the cells and remove the supernatant. ^ Store the cell pellets at -80°C until ready to extract the DNA using a Quick-DNA Microprep Plus Kit from Zymo, according to the manufacturer’s instructions. Initial siRNA transfection: ^ Approximately 24 h after cell seeding, reconstitute siRNA (Dharmacon™) in sterile H2O to a stock concentration of 10 pmol / µL. ^ Prepare sufficient siRNA transfection reactions to treat 8 replicate wells per siRNA. ^ Briefly, in a sterile 1.5 mL Eppendorf tube add 3 µL of siRNA to 97 µL Opti-MEM media. ^ In a separate sterile 1.5 mL Eppendorf tube add 3 µL of Lipofectamine RNAiMAX to 97 µL opti-MEM media. ^ Combine the two tubes contents and incubate for 10 m at room temperature. ^ Add 20 µL of the resultant mixture to 8 wells of the previously seeded 96-well plates. See example plate layout (Figure 14B): ^ Return the plates to the incubator at 37°C, 5% CO2. Initial Tetracycline Pulse: ^ Approximately 24 h post siRNA transfection, prepare fresh siRNA transfection mixes as per 3.1.2. ^ During the 10 m incubation of the siRNA transfection mix, perform a media change on the 96-well plates, replacing all media, except for the ‘no Tet’ and ‘Non-targeting siRNA no Tet’ controls with 100 µL McCoys 5A, 10% Tet-Free FBS, supplemented with 2 x Tetracycline (2 µg / mL). For the ‘no Tet’ and ‘Non-targeting siRNA no Tet’ control wells, replace the media with 100 µL McCoys 5A, 10% Tet-Free FBS. ^ Immediately after media change, re-transfect the cells by adding 20 µL of the desired siRNA transfection mix per well and return the plate to the incubator for ~48 h. Tetracycline Pulse & stable clone maintenance: To ensure that the U20S-IC6 clonal cell line retains expression of the 2 stably transfected Lo-Q platform plasmid constructs, the cells must be exposed to the relevant section antibiotics for a 72 h period every 7 days of the assay.^ Approximately 48 h (Friday) after the initial Tetracycline pulse, perform another media change in the same manner as 3.1.3, but ensure all media is additional supplemented with 1.25 µg / mL Blasticidin®, and 100 µg / mL Zeocin®. ^ After the media change, prepare and add the siRNA transfection mixes as before and return the plate to the incubator for ~72 h. Passaging Lo-Q Platform plates: ^ Cells should be passaged every Monday at a 1 / 8 dilution unless visual observation by light microscope reveals impeded proliferation, or cytotoxicity. ^ Using a multi-channel 300 µL integra pipette, aspirate the media from each triplicate, using a custom multi aspirate and dispense function. Do not use the same pipette tips for different siRNA transfection triplicates. ^ Wash the cells once with 200 µL PBS. ^ Detach the cells by adding 25 µL of 0.25% Trypsin EDTA to each well and incubating for 2- 3 m at 37°C. Ensure the >95% of the cells have detached before quenching with media. If necessary, tap the 4 sides of the plate gentle to detach persistent cells. ^ During trypsinisation prepare fresh 96-well plates for seeding. Add 75 µL of appropriate media to each well. Adjust this volume if the passage ratio has been altered due to the confluency of the condition in question. The ‘No Tet’ and ‘Non-targeting siRNA no Tet’ controls and all other conditions should receive McCoys 5A, 10% Tet-Free FBS and McCoys 5A, 10% Tet- Free FBS, 2 x Tetracycline (2 µg / mL) respectively. Ensure the top row is filled with 100 µL PBS. ^ Quench the trypsinisation by adding 175 µL of appropriate media to the wells. ^ To perform the 1 / 8 ratio passage, using a multi-channel P300 pipette, gently mix the quenched cells and transfer 25 µL to the fresh 96-well plates prepared earlier. ^ As soon as possible, prepare the siRNA transfection mixes and add 20 µL per well to the newly passage plates. ^ Return the plates to the incubator for 48 h at 37 °C, 5% CO2. CellTiter Glo®: ^ In the first instance of passaging the cells, a CellTiter Glo® assay can be performed with the leftover unused cells to determine successful Tetracycline activation and infer compound cytotoxicity. ^ Centrifuge the 96-well plates containing the unused cells that were quenched after trysinisation at 300 x g for 5 m. ^ Aspirate the media and add 50 µL fresh McCoys 5A, 10% Tet-free FBS. ^ To each well, add 50 µL CellTiter Glo® reagent and incubate in the dark for up to 10 m shaking. ^ Transfer the contents of each well to a solid white opaque 96-well plate and measure luminescence on a compatible plate reader (GloMax Explorer). Cell processing for DNA extraction:Either when the cells are passaged at the end of week 2, or when the assay ends at the end of week 3, the cells should be retained for DNA extraction. ^ Centrifuge the 96-well plates containing the unused cells (week 2) or all cells (week 3) at 300 x g for 5 m. ^ Aspirate the media and wash the cells once with PBS. ^ Seal the plate with a foil plate seal and store at -80°C until ready to extract the DNA using the Quick-DNA™ 96 Kit™ from Zymo, according to the manufacturer’s instructions.

[0236] Compound treatment protocol

[0237] Stable, clonal cell lines (such as 1C6) are leveraged to assess the effectiveness of therapeutic compounds against a Huntington’s disease, by way of quantifying CAG repeats expansion rates. MATERIALS Cell culture • Trypan Blue Gibco™ (Cat# 15250061) • Nexcelom counting slides Nexcelom (Cat# SD100) • Penicillin-Streptomycin (P / S) (10,000 U / mL) Gibco™ (Cat# 15140122) • McCoys 5A (Modified) medium Gibco™ (Cat# 16600082) • Tet-System Approved FBS Gibco™ (Cat# A47362- 01) • Zeocin® 100 mg / mL Invivogen (Cat# ant-zn-05) • Blasticidin 10 mg / mL Invivogen (Cat# ant-bl-05) • Puromycin 10 mg / mL Invivogen (Cat# ant-pr-1) • 0.25% Trypsin EDTA Gibco™ (Cat# 15050065) • PBS -MgCl2-CaCl2Gibco™ (Cat# 10010023) • Freezing Medium Cryo-SFM Sigma Aldrich (Cat# C-29912) Compound Treatment ^ Tetracycline Gibco™ (Cat# A39246) ^ Test compounds ^ DMSO Fisher (Cat# BP231-100) DNA extraction ^ Quick-DNA™ 96 Kit™ 10 x 96 preparations Zymo (Cat# D3012) ^ Quick-DNA Microprep Plus Kit Zymo (Cat# D4074) ^ Β-mercaptoethanol MP (Cat# 190242) Consumables ^ Centrifuge tubes ^ 50 mL falcon tubes^ 96-well flat bottom microplates (sterile) ^ 96-well white opaque microplates ^ T75 culture flasks ^ Sterile 100 mL single use reservoirs ^ Cryovials ^ Foil Plate seals Instrumentation ^ T4 Automated cell counter Nexcelom ^ GloMAX Explorer Promega® METHOD All work should be conducted under aseptic sterile technique, in a laminar flow hood. Cell Thawing, Passaging, and Freezing Thawing: ^ Place a vial of the cell line U20S-IC6 (1-2x106cells / mL) in a water bath at 37°C for 30 s. Once thawed, quickly transfer the cells into a 15 mL falcon tube containing 9 mL of McCoys 5A media, supplemented with 10% Tet-free FBS. ^ Pellet the cells by centrifugation at 300 x g for 5 m and discard the supernatant. ^ Resuspend the cell pellet in 10 mL fresh media and transfer to a sterile T75 culture flask. ^ Inspect the culture after 24 h and maintain the cells below 80% confluency, typically passaging every 3-4 days. Passaging: ^ To passage cells, aspirate the media and wash once with 2 mL PBS. ^ Aspirate the PBS and add 2 mL 0.25% Trypsin EDTA for 2 m at 37°C or until the cells detach. ^ Resuspend the detached cells with 8 mL McCoys 5A, 10% Tet-Free FBS (total volume 10 mL), and transfer to a 50 mL Falcon tube. ^ Centrifuge the cell suspension at 300 x g for 5 m to pellet the cells. ^ Aspirate the supernatant and resuspend the cell pellet in 10 mL McCoys 5A, 10% Tet-Free FBS. ^ Perform a ratio split (typical 2:10) by transferring 2 mL of the cell suspension to a fresh T75 flask and add 8 mL McCoys 5A, 10% Tet-Free FBS. ^ Return the passaged culture to the incubator at 37°C, 5% CO2. Freezing: ^ Aspirate the media from a T75 flask of U20S-IC6 and wash once with 2 mL PBS. ^ Aspirate the PBS and add 2 mL 0.25% Trypsin EDTA for 2 m at 37°C or until the cells detach.^ Resuspend the detached cells with 8 mL McCoys 5A, 10% Tet-Free FBS (total volume 10 mL), and transfer to a 50 mL Falcon tube. ^ Take a 60 µL sample of the cell suspension into a fresh 1.5 mL Eppendorf tube and add 20 µL Trypan Blue. Mix and transfer 20 µL of the solution to a clean cell counting slide to determine the cell count per mL and the overall viability. ^ Centrifuge the cell suspension at 300 x g for 5 m to pellet the cells. ^ Aspirate the supernatant and resuspend the cell pellet in sufficient CryoSFM media to achieve a cell count of 1-2 x 106cells / mL. Alternatively, complete media supplemented with 10% DMSO can be used for cryopreservation. ^ Aliquot the cell suspension in 1 mL volumes into labelled cryovials and transfer to a -80°C freezer in a Mr Frosty container. Lo-Q platform Assay for Candidate Compounds The Lo-Q platform (i.e. stable clonal cell line 1C6) has been optimised to run as a 2-week assay, following the standard processes detailed in the schematic in Figure 15A. Assay initiation - Cell seeding: ^ Cell seeding for Lo-Q platform assays should be initiated on Mondays (am) to ensure Tetracycline pulsing can be maintained every other day. ^ The Friday prior to initiating any Lo-Q platform assay ensure there are sufficient cells in culture to seed the required number of assay plates. If required, passage the cells, otherwise perform a media change to McCoys 5A, 10% Tet-free FBS, 1.25 µg / mL Blasticidin®, and 100 µg / mL Zeocin®. ^ On the Monday morning, aspirate the media from the required number of culture flasks containing U20S-IC6 cells and wash with 2 mL PBS. ^ Aspirate the PBS and add 2 mL 0.25% Trypsin EDTA. Incubate the plate at 37°C for ~2 m, or until >90% of the cells have detached. ^ Resuspend the detached cells with 8 mL McCoys 5A, 10% Tet-Free FBS (total volume 10 mL), and transfer to a 50 mL Falcon tube. ^ Take a 60 µL sample of the cell suspension into a fresh 1.5 mL Eppendorf tube and add 20 µL Trypan Blue. Mix and transfer 20 µL of the solution to a clean cell counting slide. Determine the cell count per mL and the overall viability. ^ Dilute a sufficient volume of the cell suspension in McCoys 5A, 10% Tet-Free FBS, to achieve a cell density of 5,000 cells / well at a volume of 200 µL per well of a 96-well plate (25,000 cells / mL). ^ Using sterile clear flat bottom 96-well plates, fill the top row (A1-12) with 200 µL PBS. These wells must be kept empty for subsequent downstream analysis processing purposes. ^ Next, transfer the diluted cell suspension to a sterile reservoir and using a 1250 µL multichannel integra, pipette 200 µL per well of the clear flat bottom 96-well plates (rows B-H). ^ Place the seeded 96-well plates in an incubator at 37°C, 5% CO2, for ~24 h.^ At least 3 ‘day 0’ samples should be taken at this point. Using the leftover cells from the trypsinised T75 flasks, prepare 3 x 1.5 mL Eppendorf tubes containing a minimum of 3 x 105cells. ^ Centrifuge the tubes to pellet the cells and remove the supernatant. ^ Store the cell pellets at -80°C until ready to extract the DNA using a Quick-DNA Microprep Plus Kit from Zymo, according to the manufacturer’s instructions. Initial compound stocks preparation: ^ Approximately 24 h after cell seeding, reconstitute the test compounds in DMSO to a concentration of 30 mM. in this case compound 5 was used having structure:^ From this 30 mM stock, prepare ½ log serial dilutions in DMSO, typically a 5 point dilution series, (10 mM, 3 mM, 1 mM, 0.3 mM, and 0.1 mM). See table below for typical volumes for a 2-week experiment. Initial Compound Pre-treatment: The test compounds are added to the cells approximately 24 h prior to the first Tetracycline pulse. This ensures that potential effects of slower acting compounds are not obscured by the quick de- repression of the CAG expansion by the addition of the Tetracycline. For each instance of Tetracycline pulsing, the test compounds are refreshed. ^ Using a fresh sterile 96-well plate, prepare an intermediate compound dilution of each of the 6 DMSO stocks created in section 3.2.2. ^ Create the intermediate stocks by diluting 2 µL of each DMSO stock in 18 µL McCoys 5A, 10% Tet-Free FBS. See Figure 15B. ^ Next, using the repeat dispense function of a 12.5 µL integra digital pipette, dispense 2 µL of each intermediate compound dilution or DMSO control into triplicate wells of the 96- well plates containing the U20S-IC6 cells seeded 24 h prior. See example plate layout in Figure 15C. ^ Return the treated 96-well plates to the incubator for ~24 h at 37°C, 5% CO2. Initial Tetracycline Pulse: ^ Approximately 24 h post initial compound pre-treatment, prepare the intermediate compound dilution plate again, in the same manner as for 3.2.3, and set the plate aside.^ In the meantime, perform a media change on the 96-well plates pre-treated with test compound, replacing all media, except for the ‘no Tet’ control, with 198 µL McCoys 5A, 10% Tet-Free FBS, supplemented with 2 x Tetracycline (2 µg / mL). For the ‘no Tet’ control wells, replace the media with 198 µL McCoys 5A, 10% Tet-Free FBS. ^ Immediately after media change, re-add the intermediate test compound dilutions as previously described in 3.2.3 and return the plate to the incubator for ~48 h. Tetracycline Pulse & stable clone maintenance: To ensure that the U20S-IC6 clonal cell line retains expression of the 2 stably transfected Lo-Q platform plasmid constructs, the cells must be exposed to the relevant section antibiotics for a 72 h period every 7 days of the assay. ^ Approximately 48 h (Friday) after the initial Tetracycline pulse, perform another media change in the same manner as 3.2.4, but ensure all media is additionally supplemented with 1.25 µg / mL Blasticidin®, and 100 µg / mL Zeocin®. ^ After the media change, prepare and add the intermediate compound dilutions as before and return the plate to the incubator for ~72 h. Standard Lo-Q Platform Assay Process: ^ After the first week of the assay, the standard process should be followed is outlined in Figure 15D. Passaging Lo-Q Platform plates: ^ Cells should be passaged every Monday at a 1 / 8 dilution unless visual observation by light microscope reveals impeded proliferation, or cytotoxicity. ^ Prior to performing any cell passaging, prepare the intermediate compound dilution plate as per 3.2.4. Set the plate aside until required after passaging is complete. ^ Next, using a multi-channel 300 µL integra pipette, aspirate the media from each triplicate, using a custom multi aspirate and dispense function. Do not use the same pipette tips for different treatment condition triplicates. ^ Wash the cells once with 200 µL PBS. ^ Detach the cells by adding 25 µL of 0.25% Trypsin EDTA to each well and incubating for 2- 3 m at 37°C. Ensure the >95% of the cells have detached before quenching with media. If necessary, tap the 4 sides of the plate gentle to detach persistent cells. ^ During trypsinisation prepare fresh 96-well plates for seeding. Add 175 µL of appropriate media to each well. Adjust this volume if the passage ratio has been altered due to the confluency of the condition in question. The ‘No Tet’ control and all other conditions should receive McCoys 5A, 10% Tet-Free FBS and McCoys 5A, 10% Tet-Free FBS, 2 x Tetracycline (2 µg / mL) respectively. Ensure the top row is filled with 200 µL PBS. ^ Quench the trypsinisation by adding 175 µL of appropriate media to the wells. ^ To perform the 1 / 8 ratio passage, using a multi-channel P300 pipette, gently mix the quenched cells and transfer 25 µL to the fresh 96-well plates prepared earlier.^ Immediately add the intermediate dilutions of the test compounds to the newly passage plates as per 3.2.4 and return the plates to the incubator for 48 h at 37 °C, 5% CO2. NanoGlo®: ^ In the first instance of passaging the cells, a NanoGlo® assay can be performed with the leftover unused cells to determine successful Tetracycline activation and infer compound cytotoxicity. ^ Centrifuge the 96-well plates containing the unused cells that were quenched after trypsinisation at 300 x g for 5 m. ^ Aspirate the media and add 50 µL fresh McCoys 5A, 10% Tet-free FBS. ^ To each well, add 50 µL NanoGlo® reagent and incubate in the dark for up to 10 m shaking. ^ Transfer the contents of each well to a solid white opaque 96-well plate and measure luminescence on a compatible plate reader (GloMax Explorer). Cell processing for DNA extraction: At the end of the assay period, the cells should be retained for DNA extraction. ^ Centrifuge the 96-well plates of cells at 300 x g for 5 m. ^ Aspirate the media and wash the cells once with PBS. ^ Seal the plate with a foil plate seal and store at -80°C until ready to extract the DNA using the Quick-DNA™ 96 Kit™ from Zymo, according to the manufacturer’s instructions. Candidate Compound treatment plan variation: In some instances, the assay described above can be modified to achieve specific compound exposure times during treatment regimes. Compound pulsing for 2, 4, 8, and 24 h can be implemented under the existing assay format, with the following adjustments: ^ Each morning, test compounds are added in the presence of Tetracycline containing media for the desired time. ^ After which the media is removed, the wells washed 1 x with PBS, and 200 µL fresh McCoys 5A, 10% Tet-free FBS + Tetracycline is added. ^ The cells remain in this media until the following morning, when the wells are aspirated, and 200 µL fresh McCoys 5A, 10% Tet-free FBS + Tetracycline is added along with the test compounds. ^ For the 24 h compound pulse, cells will remain in constant contact with test compound and Tetracycline throughout the 2-week assay, with replenishment every morning. ^ Test compound pulse assays require treatments every day for the 2-week period, including the weekend. Therefore, during Friday, Saturday, and Sunday treatments, all media should be additionally supplemented with 1.25 µg / mL Blasticidin®, and 100 µg / mL Zeocin®.

[0238] ResultsMethods of arresting, or reversing, somatic repeat expansions is hypothesised to be an effective therapeutic approach to slow or stop repeat disease progression. Genome-wide association studies (GWAS) have identified variants in genes collectively identified as part of the mismatch repair pathway (MSH3, MLH1, MLH3, PMS1, PMS2, LIG1, FAN1), which have been experimentally shown to modulate somatic instability. Research in this field have collectively highlighted MSH3, part of the MutSβ obligate heterodimer, as being of particular interest as a therapeutic target. Furthermore, the possibility exists that other proteins modulate somatic instability which have not been identified through GWAS.

[0239] The preclinical testing of novel somatic instability targeting therapeutics is not straightforward. Somatic CAG repeat expansion is a conserved and well-documented biological process in HD mouse models but is historically challenging to assess in cell models of HD. Ideally, the chosen model will develop robust somatic expansion phenotypes within a timescale amenable to the administration and durability of the potential therapeutic agents and with sufficient throughput. Existing published options lack these key features: both shuttle vector systems and stem cell-based models are compromised with respect to throughput, timescales and are both highly labour intensive. Accordingly, we set out to develop a novel cellular assay to measure the effects of therapeutic agents on the key phenotype of interest: expansion of a pathogenic simple nucleotide repeat sequence.

[0240] Novel cellular assay design features 1. Repeat sequence context: HD was identified as a prototypic repeat expansion disease of interest. Exon 1 of human HTT with expanded CAG repeat sequence was selected as the disease-causing sequences. Flanking sequences for expanded repeats appear to influence the biology of repeat expansion. Transgenic mouse models with truncated human HTT sequences stably integrated into the host genome exhibit robust somatic repeat expansion phenotypes. Truncated HTT sequences either transiently or stably integrated into mammalian cells have previously been employed to study pathophysiological features of the disease such as aggregation formation and clearance. 2. Repeat length: Given that repeat expansion phenotypes are more pronounced with longer repeat lengths, we prioritised highly expanded (100 CAG) and expanded (50 CAG) repeat sequences for assessment versus non-pathogenic repeat lengths (18 CAG). This is further supported by multiple publications that human stem cell (hESC, hiPSC) carrying over 100 CAG repeats show reproducible instability over extended timescales (i.e. 60 days)either in the pluripotent state or after in vitro differentiation protocols to mixed neuronal- like populations. 3. Need to drive transcription across the repeats in a controllable fashion: Repeat expansion is thought to result from polymerase slippage during processes such as cell replication or transcription. The cell types with the most prominent repeat expansion phenotype are non-dividing (i.e. medium spiny neurons in the striatum) and accordingly, transcription must be key. This notion is further supported by the observation that non- expressing exon 1 HTT transgenes have no somatic repeat instability (i.e. R6 / 0 mouse line) compared to expressing transgenes (i.e. R6 / 1, R6 / 2 and R6 / 5 mouse lines). The tetracycline operon has previously been successfully leveraged as an inducible expression system for HTT exon 1 constructs. An additional potential benefit is the ability to modulate expression levels of exon 1 HTT with expanded CAG repeats to a degree that is not toxic to the cells. 4. Expression reporter: related to the above, we sought to incorporate an indirect readout for induction of expression. We prioritised a luciferase reporter as a sensitive, accurate, high-throughput, non-toxic, low-cost option. We placed the HTT exon 1 sequence upstream, followed by a P2A self-cleaving peptide (to facilitate optimal processing into two separate proteins) and then a Nano-Luc reporter downstream, followed by a stop codon and polyA sequences. Furthermore, there was published potential that luciferase expression may scale in a CAG-length dependent manner and thus indicated potential as a surrogate for repeat expansion (Scoles et al 2015, https: / / pubmed.ncbi.nlm.nih.gov / 26086378 / ; Lufino et al 2013 https: / / pubmed.ncbi.nlm.nih.gov / 23943791 / ). 5. Human cell background permissive for repeat expansion: while multiple human exon 1 HTT cell models have been generated, their propensity for repeat expansion is unknown. Repeat expansion is influenced by cellular context as evidenced by the knowledge that specific neuron subtypes show prominent expansion phenotypes while other neuronal subtypes do not. We do not know what the critical cellular determinants are for supporting these repeat expansion phenotypes. U2OS cells are an unusual cellular background to study HD pathophysiology given that U2OS cells are derived from osteosarcoma. However, U2OS cells are widely used for studies of genomic instability phenotypes including alternative lengthening of telomeres (ALT), a mechanism by which telomeres (a hexameric repetitive sequence) are re-elongated. We prioritised U2OS as a cell background of choice for integration of our novel construct. This is a robust, easy-to-culture cell line that is amenable to throughput assessments (i.e. in 96 well plates).6. Accurate measurement of expanded repeat phenotype: Tracking somatic expansion requires quantitative measurements of repeat lengths. Next-generation sequencing is constrained through the 300 bp read-length and thus cannot accurately resolve over 100 CAG. Similarly, nanopore platforms are unable to support assessment of >100 CAG containing sequences. The current genetic diagnostic test measures the length of the inherited allele through a PCR-based amplification which is biased toward expansion of shorter repeats such as the two endogenous HTT alleles in U2OS cells. A novel PCR-based assay was developed that specifically amplified the long CAG repeats in the construct to improve sensitivity and resolution of somatic instability phenotypes. Capillary electrophoresis was employed as a gold-standard approach to derive PCR product size measurement. 7. Statistical analyses: Existing approaches to quantifying CAG repeats in the literature focus on identifying the modal repeat length and / or generating a CAG indice such as an instability index or an expansion index (Lee 2010). We extended these approaches further by inclusion of additional statistical measures including (a) additional measures of central location (mean, median in addition to mode), (b) measures of variation (standard deviation, variance, interquartile range), (c) additional descriptive statistics (sum height which is similar to area under curve, kurtosis, skewness). These summary statistics can be derived from raw capillary electrophoresis profiles and can be used further in other analyses such as principal component analyses.

[0241] Construct synthesis

[0242] The pcDNA™4 / TO vector contains two tetracycline operator 2 (TetO2) sites within the human cytomegalovirus immediate-early (CMV) promoter for tetracycline regulated expression of your gene of interest (Yao et al., 1998). The TetO2 sequences serve as binding sites for four Tet repressor molecules (comprising two Tet repressor homodimers) and confer tetracycline-responsiveness to the gene of interest. In the absence of tetracycline, expression of the gene of interest is repressed by the binding of Tet repressor homodimers to the TetO2 sequences. Addition of tetracycline to the cells de-represses the hybrid CMV / TetO2 promoter in pcDNA™4 / TO and allows expression of the gene of interest. The pcDNA™6 / TR regulatory plasmid is designed for use with the T-REx™ System for tetracycline-regulated expression of the gene of interest in mammalian cells. (www.lifetechnologies.com). See Figures 1 and 2.

[0243] 18, 50 and 100 CAG repeat containing plasmids according to the construct design (Figure 1 A) were custom synthesized at GeneArt (Figure 1B). The expression sequence comprises exon 1 huntingtin with ~100 pure CAG repeats, a P2A self-cleaving peptide and a nanoluc sequence followed by a polyA signal. Full plasmid sequence is indicated (Figure 1C). The plasmid contains sequences for ampicillin (bacterial propagation) and zeocin (mammalian cell lines) resistance.

[0244] Characterisation of U2OS cells to confirm suitability for interrogating mismatch repair

[0245] The U-2 OS cell line (purchased from LGC standards, ATCC) is derived from a moderately differentiated sarcoma from the tibia of an adolescent female. Karyotyping has shown that although there are a lot of chromosomal abnormalities the tumour suppressor genes p53 and pRb are still fully functional in the U2OS cell line.

[0246] We sought to confirm expression levels of key MMR components at both RNA and protein levels. We leveraged a fast, high-throughput solution to assess gene expression levels in a multiplexed fashion. The QuantiGene Plex assay (Invitrogen) is hybridisation based and incorporates branched DNA technology which uses signal amplification as opposed to direct measurement of RNA transcripts. We generated a custom-designed Invitrogen QuantiGene plex reagent system (QGP-144-M19101702) for 76 target specific probes associated with magnetic beads. Cell lysates were prepared and assessed using manufacturer recommendations. The negative control was baseline signal while the positive control was hRNA. The geomean of housekeeping genes was employed as a normalisation strategy. These analyses confirmed that all expression products required for mismatch repair and those identified through GWAS were expressed by the cells (Figure 3).

[0247] Expression of key MMR pathway members was orthogonally confirmed by RNA Seq analyses. Sequence mapping and assembly enabled transcript identification and quantification (FPKM values). Scatter and volcano plots confirmed good agreement between samples. Interrogation of quantified transcripts confirmed that key components were present (e.g. MSH3, MSH2, MLH1, MLH3, PMS1, PMS2, FAN1, EXO1, LIG1, PCNA etc).

[0248] Whole exome sequencing was performed to confirm which genetic variants (if any) were present..

[0249] Protein expression was confirmed by Western blotting. As shown in Figure 4, bands of the appropriate size were observed for all proteins tested (MSH3, MLH1, EXO1, FAN1) in U2OS lysates.

[0250] Clonal cell line generation

[0251] The first step was to generate “TRex-U2OS cells” where the tet repressor under the control of the CMV promoter (pcDNA6 / TR) was stably integrated into parental U2OS cell (Figure 5). Stable pools were obtained via blasticidin selection and were used to generate clonal lines via single cell sorting. The culture conditions were identical to those used for parental U2OS except that cell culture medium (McCoy’s 5A (modified) medium with GlutaMAX supplement and tetracycline-free FBS) was supplemented with blasticidin at 1.25 ug / ml to maintain selection pressure.

[0252] The second step was to add the exon 1 huntingtin plus nanoluc sequences (pcDNA4 / TO backbone plus added sequences as described above). The 100 CAG containing plasmid was prioritised given the knowledge that repeat instability is a length-dependent phenomenon. Stable pools were obtained via zeocin selection (Figure 6) and used to generate clonal lines via single cell sorting. Maintenance medium for the stable pools and clonal cell lines expressing Tet repressor and exon 1 HTT / nanoluc constructs consisted of McCoy’s 5A (modified) medium with GlutaMAX supplement and tetracycline-free FBS additionally supplemented with zeocin to 100 ug / ml and blasticidin to 1.25 ug / ml to maintain selection pressure for both components.

[0253] Stable pools and clonal lines were characterised for successful construct integration through multiple complementary approaches. The CAG repeat number was measured by our proprietary, specific PCR assay (Figure 7) and confirmed through Sanger sequencing of CAG repeats from cells. We additionally visualized CAG repeat products by agarose gel electrophoresis (Figure 8). These early observations indicated the ongoing repeat instability of the expanded CAG repeats in each of the clonal lines. Furthermore, we were able to observe an indication of increased instability in the presence of tetracycline versus the absence.

[0254] Expression of the construct components were tested for tetracycline responsiveness at 1 ug / ml using a well-characterised HTRF assay for mutant huntingtin protein (Figure 9) and nano-Glo (Figure 10). We noted decreased nano-Glo signal as CAG repeats increased in the stable pools (Figure 10A). Each stable clonal line had specific levels of induction of signal in a tetracycline-dependent manner.

[0255] Cell viability was also assessed using CellTiterGlo which indicated that there was no overt cytotoxicity with 100 CAG containing construct (with or without tetracycline) compared to 18 CAG constructs or control constructs (Figure 11). In addition, cumulative cell counts was leveraged to track population doubling times and cell viability for each clonal line (Figures 12 and 13). These results show that the cells grew as expected and maintained good viability during the culture period and no toxicity was observed. There were no obvious morphological differences between clonal lines (Figure 13).

[0256] These assays collectively informed the selection of three clonal lines bearing 100 CAG-containing constructs for further assessment for biological and compound interrogations as outlined in Figures 14 and 15.

[0257] Refinement of cell lines and culture conditions for interrogations

[0258] Figures 16 to 18 show the comparison of the change in number of CAG repeats for three different stable, clonal cell lines (1C6, 1G10, and 2E5) carrying the expression construct of the invention during cell culture as described above, and the change in number of CAG repeats over time for the three prioritized stable clonal cell lines which had optimal performance characteristics. Each of the three lines have ongoing, dynamic repeat expansion trajectories with at least some degree of tetracycline sensitivity (ie expansion rates are increased further with tetracycline addition). Furthermore, each of the three lines show expansion rates that are typically faster than published alternatives (e.g. iPSC, iPSC-MSN and transient exon 1 HTT) (Figure 16A). As repeats expand over time, the profile of repeat populations shows characteristic shifts with increased modal and mean repeat number and increased range of distribution (Figure 16B).

[0259] The reproducibility of the repeat expansion trajectories was examined. Figure 17 shows a study with respect to repeat expansion profiles measured at 3 weeks with once-weekly tetracycline pulsing for 1C6 (FIG. 17A) and 2E5 (FIG. 17B). Each of the three replicates shows strong concordance. Clone 1C6 showed a lower rate of expansion than Clone 2E5 but data are more reproducible with less variation shown by the tight distribution. These data informed the selection of stable clonal line 1C6 for biological interrogation and compound treatment.

[0260] Assay window and tetracycline responsiveness were examined. Figure 18 shows the testing of this cell line with different tetracycline exposure protocols and demonstrates that the greatest increase in CAG repeat number is obtained by continuous culture in the presence ofTet (as opposed to once-weekly pulsed tetracycline) without obvious effect on cell viability (not shown).

[0261] Validation of the use of the cell based model in assays

[0262] We tested whether the stable clonal cell lines exhibited concordant response to published biological interrogations performed in vivo and in vitro. Figure 19A and 19B show the results of testing the host cells described herein containing the expression construct of the invention in an assay format using siRNA knockdown of key mismatch repair proteins (as explained above). MSH2 and MSH3 knockdown stabilizes repeat expansion while MSH6 knockdown exacerbates repeat expansions (Figure 19A). This acts as a positive control demonstration of how the assay of the invention can distinguish and clearly show when there is successful arrest or contraction of CAG repeats in response to a therapeutic agent. As shown in Figure 19B, in response to MSH3 shRNAs the expression of MSH3 in the host cells was reduced and consequently the mean number of CAG repeats in the construct was arrested to varying degrees of effect depending on the different shRNA used. The number of CAG repeat was determined accurately as explained hereinabove. Thereby demonstrating that the assay can detect such effects on CAG repeat number.

[0263] Figure 20 shows the results of testing an exemplary candidate compound 5 using the assay of the invention (performed as explained above). Much like the siRNA demonstration, the assay allows the therapeutic effect of the candidate compound to be seen in terms of its ability to arrest CAG repeat expansion in a dose dependent manner. Thereby showing that the expression construct, and host cells comprising it work in the format of an assay to provide useful means of determining and selecting therapeutic agents which could be used to treat short tandem repeat disorders as described herein.

[0264] The assay of the invention as shown in figure 20 and described hereinabove was further repeated with several different candidate therapeutic compounds 1-8, and showed the same results, demonstrating its useability and consistency across different candidate compounds as summarized in the table in Figure 21 and in subsequent figures 22-28. Exceptions for figures 22 and 23 are that they were generated without Day 0 data.

[0265] Figure 29 shows the results of testing an exemplary candidate compound 7 using the assay of the invention as described for figures 20 and 22-28 with the exception that the treatment was performed for longer periods of time in culture. The exemplary candidate compound 7 was titrated from an appropriate top concentration in half-log concentrations andtested for its repeat stabilization effect versus vehicle and no tetracycline control using the assay method of the invention (0.1% DMSO final assay concentrations). The assay was exposed to the compound for 2, 4 and 6 weeks in culture followed by an accurate assessment of the number of CAG repeats. The degree of blockade of somatic expansion in a concentration dependent manner was consistent regardless of the duration of culture or the number of repeat units gained in the time period assayed. SEQUENCES All sequences given in a 5' to 3' direction. SEQ ID NO:1 Tet Responsive Element: CMV promoter (bold) and TetO operator (underline) GCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGAC CCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGG ACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCA GTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGT AAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACT TGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGG CAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTC CACCCCATTGACGTCAATGGGAGTTTGTTTTGGAACCAAAATCAACGGGACTTT CCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTA CGGTGGGAGGTCTATATAAGCAGAGCTCTCCCTATCAGTGATAGAGATCTCCCTAT CAGTGATAG SEQ ID NO:2 TetO operator CTATCAGTGATAG SEQ ID NO: 3 Exon 1 of HTT gene with 100 CAG repeats ATGGCGACCCTGGAAAAGCTGATGAAGGCCTTCGAGTCCCTCAAGTCCTTCCA GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAG CAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAG CAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC AGCAGCAGCAGCAGCAGCAGCAGCAACAGCCGCCACCGCCGCCGCCGCCGCCGCCG CCTCCTCAGCTTCCTCAGCCGCCGCCGCAGGCACAGCCGCTGCTGCCTCAGCCGCAG CCGCCCCCGCCGCCGCCCCCGCCGCCACCCGGCCCGGCTGTGGCTGAGGAGCCGCTG CACCGA SEQ ID NO:4 P2A self-cleaving peptide ACCGGCAGCGGCGCCACAAACTTCTCTCTGCTAAAGCAAGCAGGTGATGTTG AAGAAAACCCCGGGCCT SEQ ID NO: 5 NanoLuc Luciferase Reporter Protein ATGGTCTTCACACTCGAAGATTTCGTTGGGGACTGGCGACAGACAGCCGGCTA CAACCTGGACCAAGTCCTTGAACAGGGAGGTGTGTCCAGTTTGTTTCAGAATCTCGG GGTGTCCGTAACTCCGATCCAAAGGATTGTCCTGAGCGGTGAAAATGGGCTGAAGAT CGACATCCATGTCATCATCCCGTATGAAGGTCTGAGCGGCGACCAAATGGGCCAGAT CGAAAAAATTTTTAAGGTGGTGTACCCTGTGGATGATCATCACTTTAAGGTGATCCT GCACTATGGCACACTGGTAATCGACGGGGTTACGCCGAACATGATCGACTATTTCGG ACGGCCGTATGAAGGCATCGCCGTGTTCGACGGCAAAAAGATCACTGTAACAGGGA CCCTGTGGAACGGCAACAAAATTATCGACGAGCGCCTGATCAACCCCGACGGCTCCC TGCTGTTCCGAGTAACCATCAACGGAGTGACCGGCTGGCGGCTGTGCGAACGCATTC TGGCGTAA SEQ ID NO:6 HSV TK PolyAdenylation sequence AATAAAACGCACGGTGTTGGGTCGTTTGTT SEQ ID NO:7 pcDNA4 / TO-100CAG (7160bp) Vector Key: CMV promoter (bold), TetO operator (underline), Exon 1 HTT with 100 CAG repeats (italics), P2A self-cleaving peptide (bold and underline), Nanoluc reporter protein (italics and underline), HSV TK PolyA (bold and italics)GATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTATTAATA GTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATA ACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGAC GTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACG TCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTA TCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTG GCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTA CGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGG GCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGT CAATGGGAGTTTGTTTTGGAACCAAAATCAACGGGACTTTCCAAAATGTCGTAA CAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCT ATATAAGCAGAGCTCTCCCTATCAGTGATAGAGATCTCCCTATCAGTGATAGAGAT CGTCGACGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGT TTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGGACTCTAGCGTTTAAA CTTAAGCTTACCATGGCGACCCTGGAAAAGCTGATGAAGGCCTTCGAGTCCCTCAAGTCC TTCCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAG CAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAG CAGCAGCAGCAGCAACAGCCGCCACCGCCGCCGCCGCCGCCGCCGCCTCCTCAGCTTC CTCAGCCGCCGCCGCAGGCACAGCCGCTGCTGCCTCAGCCGCAGCCGCCCCCGCCGCC GCCCCCGCCGCCACCCGGCCCGGCTGTGGCTGAGGAGCCGCTGCACCGACCAGGTAC CGGCAGCGGCGCCACAAACTTCTCTCTGCTAAAGCAAGCAGGTGATGTTGAAG AAAACCCCGGGCCTGGATCCATGGTCTTCACACTCGAAGATTTCGTTGGGGACTGGCG ACAGACAGCCGGCTACAACCTGGACCAAGTCCTTGAACAGGGAGGTGTGTCCAGTTTGTT TCAGAATCTCGGGGTGTCCGTAACTCCGATCCAAAGGATTGTCCTGAGCGGTGAAAATGG GCTGAAGATCGACATCCATGTCATCATCCCGTATGAAGGTCTGAGCGGCGACCAAATGGG CCAGATCGAAAAAATTTTTAAGGTGGTGTACCCTGTGGATGATCATCACTTTAAGGTGATCC TGCACTATGGCACACTGGTAATCGACGGGGTTACGCCGAACATGATCGACTATTTCGGAC GGCCGTATGAAGGCATCGCCGTGTTCGACGGCAAAAAGATCACTGTAACAGGGACCCTGT GGAACGGCAACAAAATTATCGACGAGCGCCTGATCAACCCCGACGGCTCCCTGCTGTTCC GAGTAACCATCAACGGAGTGACCGGCTGGCGGCTGTGCGAACGCATTCTGGCGTAAGAATTCCGGCAATAAAAAGACAGAATAAAACGCACGGTGTTGGGTCGTTTGTTCCGTTAC ATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGAC GTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCA ATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATAT GCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGC CCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATC GCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTT GACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGG CACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAA ATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTATCGATGCCAC CATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCT GGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATG CCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGC CCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACC CCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCC AGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTG AAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAA GGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACG TCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGC CACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCC CATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGC CCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGA CCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGGCTCGAGTCTAGAGG GCCCGTTTAAACCCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTT GTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTT CCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGG GGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCA TGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTC TAGGGGGTATCCCCACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGT TACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTC TTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGC TCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTA GGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAA CCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGT TAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTAATTCTGTGGAATGTGTG TCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCA TGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCA GAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTC CGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACT AATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCTGCCTCTGAGCTATTCCAGAAG TAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTCCCGGGAGCTTGT ATATCCATTTTCGGATCTGATCAGCACGTGTTGACAATTAATCATCGGCATAGTATAT CGGCATAGTATAATACGACAAGGTGAGGAACTAAACCATGGCCAAGTTGACCAGTG CCGTTCCGGTGCTCACCGCGCGCGACGTCGCCGGAGCGGTCGAGTTCTGGACCGACC GGCTCGGGTTCTCCCGGGACTTCGTGGAGGACGACTTCGCCGGTGTGGTCCGGGACG ACGTGACCCTGTTCATCAGCGCGGTCCAGGACCAGGTGGTGCCGGACAACACCCTGG CCTGGGTGTGGGTGCGCGGCCTGGACGAGCTGTACGCCGAGTGGTCGGAGGTCGTGT CCACGAACTTCCGGGACGCCTCCGGGCCGGCCATGACCGAGATCGGCGAGCAGCCG TGGGGGCGGGAGTTCGCCCTGCGCGACCCGGCCGGCAACTGCGTGCACTTCGTGGCC GAGGAGCAGGACTGACACGTGCTACGAGATTTCGATTCCACCGCCGCCTTCTATGAA AGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGG GATCTCATGCTGGAGTTCTTCGCCCACCCCAACTTGTTTATTGCAGCTTATAATGGTT ACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATT CTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGTATACCGTCGAC CTCTAGCTAGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTAT CCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGG TGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAG TCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGG CGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTC GTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACA GAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCA GGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACG AGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAA AGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGC CGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTG CACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAG TCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATT AGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTAC GGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTC GGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTTTT TTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTG ATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTG GTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGT TTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTA ATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGAC TCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTG CAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGC CAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGT CTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCA ACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTC ATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAA AAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGT GTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTA AGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATG CGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGC AGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGG ATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTT CAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATG CCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTT TTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGA ATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCC ACCTGACGTCGACGGATCGGGAGATCTCCCGATCCCCTATGGTGCACTCTCAGTACA ATCTGCTCTGATGCCGCATAGTTAAGCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGG TCGCTGAGTAGTGCGCGAGCAAAATTTAAGCTACAACAAGGCAAGGCTTGACCGAC AATTGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCGC SEQ ID NO:8 HTT exon 1-NanoLuc fusion proteinKey: HTT exon 1 protein (underline), P2A self-cleaving peptide (italics), NanoLuc Luciferase Reporter protein (bold), * stop codon MATLEKLMKAFESLKSFQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQ QQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQ QQQQQQQQQQQQQQPPPPPPPPPPPQLPQPPPQAQPLLPQPQPPPPPPPPPPGPAVAEEPL HRPGTGSGATNFSLLKQAGDVEENPGPGSMVFTLEDFVGDWRQTAGYNLDQVLEQGG VSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDD HHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERL INPDGSLLFRVTINGVTGWRLCERILA* SEQ ID NO:9 CAG repeat sizing forward primer CCTTCGAGTCCCTCAAGTCCTT SEQ ID NO:10 CAG repeat sizing reverse primer TGCTTGCTTTAGCAGAGAGAA SEQ ID NO: 11 labelled CAG repeat sizing forward primer FAM-CCTTCGAGTCCCTCAAGTCCTT SEQ ID NO:12 T3 primer ATTAACCCTCACTAAAGGGAACAAA SEQ ID NO:13 T7 primer AATACGACTCACTATAGGGCGAATT SEQ ID NO:14 HTT exon 1 forward primer ATGAAGGCCTTCGAGTCCCTCAAGTCCTTC SEQ ID NO:15 HTT exon 1 reverse primer CGGCTGAGGCAGCAGCGGCTGTSEQ ID NO:16 Sanger sequencing forward primer ATGAAGGCCTTCGAGTCCCTCAAGTCCTTC SEQ ID NO:17 Sanger sequencing reverse primer CGGCTGAGGCAGCAGCGGCTGT SEQ ID NO:18 Expression Construct Key: CMV promoter (bold), TetO operator (underline), Exon 1 HTT with 100 CAG repeats (italics), P2A self-cleaving peptide (bold and underline), Nanoluc reporter protein (italics and underline), HSV TK PolyA (bold and italics) GCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGAC CCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGG ACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCA GTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGT AAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACT TGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGG CAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTC CACCCCATTGACGTCAATGGGAGTTTGTTTTGGAACCAAAATCAACGGGACTTT CCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTA CGGTGGGAGGTCTATATAAGCAGAGCTCTCCCTATCAGTGATAGAGATCTCCCTAT CAGTGATAGAGATCGTCGACGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACG CCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGGAC TCTAGCGTTTAAACTTAAGCTTACCATGGCGACCCTGGAAAAGCTGATGAAGGCCTTCG AGTCCCTCAAGTCCTTCCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAG CAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC AGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAG CAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGC AGCAGCAGCAGCAGCAGCAGCAGCAGCAACAGCCGCCACCGCCGCCGCCGCCGCCGCC GCCTCCTCAGCTTCCTCAGCCGCCGCCGCAGGCACAGCCGCTGCTGCCTCAGCCGCAGC CGCCCCCGCCGCCGCCCCCGCCGCCACCCGGCCCGGCTGTGGCTGAGGAGCCGCTGCACCGACCAGGTACCGGCAGCGGCGCCACAAACTTCTCTCTGCTAAAGCAAGCAGG TGATGTTGAAGAAAACCCCGGGCCTGGATCCATGGTCTTCACACTCGAAGATTTCGTT GGGGACTGGCGACAGACAGCCGGCTACAACCTGGACCAAGTCCTTGAACAGGGAGGTGT GTCCAGTTTGTTTCAGAATCTCGGGGTGTCCGTAACTCCGATCCAAAGGATTGTCCTGAGC GGTGAAAATGGGCTGAAGATCGACATCCATGTCATCATCCCGTATGAAGGTCTGAGCGGC GACCAAATGGGCCAGATCGAAAAAATTTTTAAGGTGGTGTACCCTGTGGATGATCATCACT TTAAGGTGATCCTGCACTATGGCACACTGGTAATCGACGGGGTTACGCCGAACATGATCG ACTATTTCGGACGGCCGTATGAAGGCATCGCCGTGTTCGACGGCAAAAAGATCACTGTAA CAGGGACCCTGTGGAACGGCAACAAAATTATCGACGAGCGCCTGATCAACCCCGACGGCT CCCTGCTGTTCCGAGTAACCATCAACGGAGTGACCGGCTGGCGGCTGTGCGAACGCATTC TGGCGTAAGAATTCCGGCAATAAAAAGACAGAATAAAACGCACGGTGTTGGGTCGTT TGTT

Claims

CLAIMS 1. An expression construct comprising: an inducible promoter, operably linked to a nucleic acid sequence comprising a pathogenic short tandem repeat region, fused via a transcriptional or translational split mechanism to a nucleic acid sequence encoding a reporter protein.

2. The expression construct of claim 1 wherein the pathogenic short tandem repeat region is unstable and / or expandable.

3. The expression construct of claim 1 or 2 wherein the pathogenic short tandem repeat region causes or contributes to a disease in humans.

4. The expression construct of any one of claims 1 to 3 wherein the pathogenic short tandem repeat region comprises between 15 and 800 repeats, between 20 and 500 repeats, between 25 and 300 repeats, between 30 and 200 repeats, between 35 and 150 repeats, preferably between 36 and 120 repeats, most preferably around 100 repeats.

5. The expression construct of any one of claims 1 to 4 wherein the pathogenic short tandem repeat region comprises a tandem repeat of one of the following sequences: CAG, CTG, GAA, CCTG, CGG, GCN, ATTCT, GGCCTG, GCG, and GGGGCC.

6. The expression construct of any one of claims 1 to 5 wherein the pathogenic short tandem repeat region comprises a tandem trinucleotide repeat, preferably wherein the tandem trinucleotide repeat is a CAG repeat.

7. The expression construct of any one of claims 1 to 6 wherein the pathogenic short tandem region comprises exon 1 of the HTT gene.

8. The expression construct of any one of claims 1 to 7 wherein the inducible promoter is a tetracycline or doxycycline inducible promoter, preferably wherein the inducible promotor is a Tet-on promoter.

9. The expression construct of claim 8 wherein the Tet-on promoter comprises a Tet responsive element (TRE) comprising a minimal promoter and one or more Tet operator sequences, preferably wherein the minimal promoter is hCMV and preferably wherein the TRE comprises two Tet operator sequences.

10. The expression construct of any one of claims 1 to 9 wherein the transcriptional or translational split mechanism is selected from an IRES, a furin cleavage site and / or a self- cleaving peptide.

11. The expression construct of any one of claims 1-10 wherein the transcriptional or translational split mechanism is a self cleaving peptide selected from: F2A, P2A, E2A, T2A, GF2A, GP2A, GE2A and GT2A, preferably wherein the transcriptional or translational split mechanism is a P2A self cleaving peptide.

12. The expression construct of any one of claims 1-11 wherein the reporter protein is a fluorescent or luminescent protein selected from: luciferase, YFP, CFP, GFP, RFP, EBFP, ECFP, EGFP, YFP, mHoneydew, mBanana, mOrange, tdTomato,mTangerine, mStrawberry, mCherry,mGrape1, mRaspberry, mGrape2 and mPlum.

13. The expression construct of any one of claims 1-12 wherein the reporter protein is a luciferase protein.

14. The expression construct of any one of claims 1-13 further comprising one or more of the following elements: a Kozak sequence, and a polyadenylation signal.

15. The expression construct of any one of claims 1-14 comprising a sequence according to SEQ ID NO: 18 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity thereto, or a functional fragment thereof.

16. A vector comprising the expression construct of any one of claims 1 to 15.

17. A cell comprising the expression construct of any one of claims 1 to 15, or the vector of claim 16.

18. The cell of claim 17, wherein the cell is a mammalian cell, preferably wherein the cell is a human cell, more preferably wherein the cell is a U2OS cell.

19. A population of cells of claim 17 or claim 18.

20. A method of screening one or more candidate therapeutic agents for the ability to inhibit MUTSβ activity, the method comprising: (a) Providing a cell according to claim 17 or claim 18;(b) Culturing the cell under suitable conditions to induce expression of the expression construct therein, and to expand the number of short tandem repeats in the pathogenic short tandem repeat region to a desired level; (c) Exposing the cell to one or more candidate therapeutic agents under suitable conditions to inhibit MUTSβ activity; (d) Determining the effect of each candidate therapeutic agent on the pathogenic short tandem repeat region of the expression construct; and (e) Selecting those candidate therapeutic agents which cause the number of short tandem repeats in the short tandem repeat region to arrest or contract, indicating inhibition of MUTSβ activity.

21. A method according to claim 20 wherein step (b) of culturing the cell to induce expression of the expression construct therein causes expression of the reporter protein, and optionally expression of any proteins encoded by the pathogenic short tandem repeat region.

22. A method according to claim 20 or 21, wherein step (b) comprises culturing the cell in the presence of an inducer, preferably culturing the cell in the presence of a tetracycline or doxycycline inducer.

23. A method according to any one of claims 20 to 22, wherein step (b) comprises culturing the cell in the presence of an inducer continuously or intermittently, and / or over a period of 2 to 3 weeks.

24. A method according to any one of claims 20 to 23, wherein step (b) comprises culturing the cell in the presence of an inducer at a concentration of between 0.1 to 10μg / ml, 0.2 to 8μg / ml, 0.3 to 7μg / ml, 0.4 to 6μg / ml, or 0.5 to 5 μg / ml, preferably at a concentration of about 2 μg / ml.

25. A method according to any one of claims 20 to 24, wherein step (b) expands the number of short tandem repeats in the pathogenic short tandem repeat region by 2 repeats, by 3 repeats, by 4 repeats, by 5 repeats, by 6 repeats, by 7 repeats, by 8 repeats, by 9 repeats, or by 10 repeats per week over the duration of the culture, preferably by around 1 repeat per week of culture.

26. A method according to any one of claims 20 to 25, wherein step (c) comprises exposing the cell to a concentration of 100 μM or less, 90 μM or less, 80 μM or less, 70 μM or less, 60 μM or less, 50 μM or less, 40 μM or less, 30 μM or less, 20 μM or less, 10μM or less, 3 μM or less,1 μM or less, 0.5 μM or less, 0.3 μM or less, 0.1 μM or less of each candidate therapeutic agent, preferably step (c) comprises exposing the cell to a concentration of about 0.1 μM of each candidate therapeutic agent.

27. A method according to any one of claims 20 to 26, wherein step (d) comprises determining whether the number of short tandem repeats in the short tandem repeat region expands, arrests or contracts in response to the therapeutic agent.

28. A method according to any one of claims 20 to 27, wherein the method further comprises a step of determining the number of short tandem repeats in the short tandem repeat region before step (c), and after step (c).

29. A method according to any one of claims 20 to 28, wherein the method further comprises a step of determining whether each candidate therapeutic agent is toxic to the cell, and optionally selecting those candidate therapeutic agents which are substantially non-toxic to the cell.

30. A method according to claim 29, wherein candidate therapeutic agents which are substantially non-toxic to the cell have an IC50 of 10 µM or less, an IC50 of 1 µM or less, or an IC50 of 0.5 µM or less.

31. A method according to any one of claims 20 to 30, wherein the method further comprises a step of identifying the one or more selected candidate therapeutic agents.

32. A method according to any one of claims 20 to 31, wherein the method further comprises a step of formulating the one or more selected candidate therapeutic agents, preferably formulating the one or more selected candidate therapeutic agents into a pharmaceutical composition.

33. A primer pair comprising a first primer and a second primer, wherein the first primer is capable of annealing to a site in exon 1 of the HTT gene, and the second primer is capable of annealing to a site in a transcriptional or translational split mechanism or within a nucleic acid sequence encoding a reporter protein.

34. The primer pair of claim 33 wherein the first primer comprises a sequence according to SEQ ID NO:9, and / or wherein the second primer comprises or consists of a sequence according to SEQ ID NO:10.

35. Use of a primer pair according to claim 33 or claim 34 for determining the number of short tandem repeats in a nucleic acid sequence comprising exon 1 of the HTT gene.

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