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WO2025088340A1 - Nouvelles cellules et procédés - Google Patents

Nouvelles cellules et procédés Download PDF

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WO2025088340A1
WO2025088340A1 PCT/GB2024/052741 GB2024052741W WO2025088340A1 WO 2025088340 A1 WO2025088340 A1 WO 2025088340A1 GB 2024052741 W GB2024052741 W GB 2024052741W WO 2025088340 A1 WO2025088340 A1 WO 2025088340A1
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cell
ageing
expression
ipsc
sequence
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Koby BARANES
Mark Kotter
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Clock Bio Ltd
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Clock Bio Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0696—Artificially induced pluripotent stem cells, e.g. iPS
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells

Definitions

  • the present invention relates to an induced pluripotent stem cell (iPSC) or a somatic cell derived from an iPSC by forward programming or directed differentiation constitutively expressing an alternative splice form of Lamin A.
  • the cells comprise an expression cassette comprising a coding sequence for the alternative Lamin A splice form operably linked to a cumate operator (CuO) sequence and a constitutive promoter.
  • CuO cumate operator
  • the use of the cells described herein in a method of identifying a gene or combination of genes involved in the reversal of an ageing phenotype or in the maintenance of a non-aged phenotype.
  • Methods of rejuvenating somatic cells, treating diseases or disorders associated with ageing, and modulating ageing are also provided, said methods comprising modulating the expression and/or activity of the gene/combination of genes identified herein.
  • age-modulating methods and uses e.g. age-modulating methods and uses
  • said methods comprising modulating the expression and/or activity of the gene/combination of genes identified herein.
  • the Hallmarks of Ageing include mitochondrial changes, senescence, altered intracellular communication, genomic instability, telomere shortening, epigenetic changes, and deregulation of nutrient pathways. These may lead to changes in cellular function, such as a reduced ability of immune cells to survey cancerogenic events, which ultimately contribute to the plethora of age-related diseases.
  • Stem cells provide scalable model systems to study the biology of a species.
  • pluripotent stem cells such as embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) technology holds a challenge due to the CLO-C-P3598PCT embryonic nature of pluripotent stem cells and in particular the cellular rejuvenation during iPSC reprogramming (Lapasset et al. (2011) Genes Dev.; Maherali et al. (2007) Cell Stem Cell; Studer et al. (2015) Stem Cell, 16:591-600).
  • hallmarks of ageing are often related to disease associated degenerative processes. Independently, they have been associated with a gradual deterioration in structure and function.
  • the first approach involves manipulating the cellular environment by introducing toxic stressors such as reactive oxygen species (Davalli et al. (2016) Oxid. Med. Cell. Longev.), or by inflammatory cytokines to mimic the general overall inflammatory state associated with ageing.
  • toxic stressors such as reactive oxygen species (Davalli et al. (2016) Oxid. Med. Cell. Longev.)
  • inflammatory cytokines to mimic the general overall inflammatory state associated with ageing.
  • the second approach involves introducing intrinsic changes to the cells to induce ageing. That was accomplished by promoting telomere shortening through pharmacological inhibition of telomerase, the enzyme involved in maintaining telomere length (Vera et al. (2016) Cell Rep.).
  • an induced pluripotent stem cell iPSC
  • stem cell or a somatic cell derived from an iPSC by forward programming or directed differentiation comprising an expression cassette, said expression cassette comprising a coding sequence for an alternative splice form of Lamin A operably linked to a cumate operator CLO-C-P3598PCT (CuO) sequence and a constitutive promoter, thereby yielding constitutive expression of the alternative splice form of Lamin A in the cell.
  • the cell does not comprise a sequence coding for a cumate repressor protein (CymR).
  • the alternative splice form of Lamin A is progerin or a progerin-like truncated form of Lamin A.
  • the presence of the expression cassette and absence of a CymR coding sequence yields constitutive expression of the alternative splice form of Lamin A in the cell.
  • the cell chronically displays an ageing phenotype.
  • the iPSC, stem cell or somatic cell described herein in a method of identifying a gene or a combination of genes involved in the reversal of an ageing phenotype or in the maintenance of a non-aged phenotype in the cell.
  • the method of identifying a gene or combination of genes comprises the steps of: (i) culturing the iPSC, stem cell or somatic cell as described herein; (ii) optionally performing a loss-of-function, inhibitory, knock-out, gain-of-function or combinatorial screen in the cell; (iii) measuring an ageing phenotype in the cell; and (iv) identifying a gene or combination of genes as being involved in the reversal of an ageing phenotype or maintenance of a non-aged phenotype when the ageing phenotype is altered when the loss-of-function, inhibitory, knock-out, gain-of-function or combinatorial screen is performed compared to when the cell is not subjected to the screen.
  • the ageing phenotype measured in step (iii) is the same ageing phenotype chronically displayed by the cell.
  • the cell is an iPSC or stem cell and is forward programmed or differentiated to a somatic cell, or the cell is a somatic cell derived from an iPSC or a stem cell by forward programming or directed differentiation, and the forward programming/directed differentiation is performed before and/or after the identification of the gene or combination of genes, such as wherein forward programming/directed differentiation is performed in culturing step (i) and/or after performing the screen of step (ii).
  • a method of rejuvenating a somatic cell comprising modulating the expression and/or activity of one or more gene or combination of genes identified by the use described herein.
  • CLO-C-P3598PCT in another aspect, there is provided an age-modulating method for use in a method of treating a disease or disorder associated with ageing in a subject, said age-modulating method comprising modulating the expression and/or activity of a gene or a combination of genes identified by the use as described herein.
  • a method of modulating ageing in vivo said method comprising the age-modulating method described herein.
  • a method of treating a disease or disorder associated with ageing comprising the age-modulating method described herein.
  • BRIEF DESCRIPTION OF THE FIGURES Figure 1 TetOn inducible system to overexpress Progerin.
  • the rtTA cassette was introduced into the ROSA26 genomic safe harbour (GSH) site while the expression cassette was introduced into the AAVS1 GSH, to allow a stable expression of progerin.
  • Progerin was linked to GFP to allow visualisation of expression.
  • B) The GFP-progerin expression cassette was introduced into BobC and KOLF2 human iPSCs backgrounds.
  • KOLF2-GFP-progerin human iPSCs were treated with ranging dox concentrations (0-2 ⁇ g/mL). Cell viability was compromised when doxycycline concentration was above 0.05 ⁇ g/mL. Scale bars, 250 ⁇ m.
  • A-B Fluorescence microscopy images for the peripheral heterochromatin marker H3K9me3 (green) and the nucleic acid marker DAPI (blue) in both KOLF2 (A) and BOBC (B) iPSCs treated (aged) or not treated (non-aged) with doxycycline. H3K9ME3 intensity was significantly reduced in the aged group. Scale bars, 25 ⁇ m. Data is presented as means ⁇ SEM, student t-test, ****p ⁇ 0.0001.
  • Figure 6 Loss of proteostasis is shown by activation of stress response pathways in aged iPSCs.
  • A-C Volcano plots analysis, based on bulk RNA sequencing data, demonstrating activation (upregulation) of genes involved in the main stress response pathways involved with proteostasis: oxidative stress response (ROS), unfolded protein response (UPR) and heat shock response (HSR).
  • ROS oxidative stress response
  • URR unfolded protein response
  • HSR heat shock response
  • Figure 7 Disabled macroautophagy is shown by changes in autophagic flux in aged iPSCs.
  • Figure 11 Stem cell exhaustion is shown by reduced proliferation rate in aged iPSCs.
  • Figure 12 Design of a chronic ageing model in human iNeurons. A) A dual inducible system to overexpress NGN2 via the TetOn inducible system and GFP-progerin via the Cumate switch system.
  • the rtTA cassette was introduced into the ROSA26 genomic safe harbour (GSH) site while the reprogramming cassette was introduced into the AAVS1 GSH, to allow a stable expression of both NGN2 and Progerin.
  • the cumate system was modified to exclude the cumate repressor (CymR), allowing a constitutive expression of GFP-progerin.
  • Aged human iPSCs are treated with 2 ⁇ g/mL doxycycline for a week to overexpress NGN2, resulting in aged iNeurons (iNs).
  • Progerin is constitutively expressed in the background.
  • iNeuron induction is based on a previous protocol by Pawlowski et al. (2017).
  • Figure 13 Constitutive expression of Progerin does not affect iPSCs viability.
  • A-B mRNA levels of the pluripotency markers OCT4 and NANOG (A) are downregulated in the aged iNs while the neuronal markers, MAP2, Synapsin-1 and PSD95 (B) are upregulated compared to iPSCs.
  • Figure 15 Constitutive expression of Progerin to force age iNeurons.
  • an induced pluripotent stem cell iPSC
  • stem cell or a somatic cell derived from an iPSC by forward programming or directed differentiation comprising an expression cassette, said expression cassette comprising a coding sequence for an alternative splice form of Lamin A coupled to a weak promoter sequence, thereby yielding low level constitutive expression of the alternative splice form of Lamin A in the cell while maintaining cell viability.
  • the term “constitutive” is used herein in its normal context in the field of biology, namely that expression is continuous in the cell and is thus not inducible or regulated.
  • the expression of the alternative splice form of Lamin A e.g.
  • progerin is constitutive, i.e. the cell continuously expresses the alternative splice form.
  • expression of the Lamin A alternative splice form is not inducible in the cell.
  • the constitutive expression of the Lamin A alternative splice form e.g. progerin
  • the constitutive expression of the Lamin A alternative splice form is at a low level in the cell.
  • a “low level” of expression as demonstrated herein provides a level in the cell sufficient to provide an effect (i.e. display of an aging phenotype) but not at such a level that the viability of the cell is affected.
  • the cell expresses the Lamin A alternative splice form (e.g.
  • the low level expression of the Lamin A alternative splice form does not affect the cell function, such as the regeneration function or regenerative capacity of stem cells as described herein (i.e. when the cell is an iPSC or a stem cell).
  • the cell is an iPSC or a stem cell and expresses the Lamin A alternative splice CLO-C-P3598PCT form at a level whereby cellular regeneration functions are maintained.
  • the cell is an iPSC and it retains a regenerative capacity.
  • an ageing phenotype caused by expression of the Lamin A alternative splice form may be displayed following forward programming or directed differentiation of said iPSC or stem cell to a somatic cell, in particular only following/after said forward programming/directed differentiation (i.e. the iPSC/stem cell does not display an ageing phenotype, while the somatic cell derived therefrom does display an ageing phenotype).
  • a display of the ageing phenotype only following forward programming/directed differentiation may be due to the expression level of the Lamin A alternative splice form being such (i.e.
  • the resulting somatic cell which does not comprise regeneration functions or regenerative capacity, will display an ageing phenotype.
  • the cell may be a somatic cell forward programmed or differentiated from an iPSC or a stem cell as described herein constitutively expressing a low level of Lamin A alternative splice form which displays an ageing phenotype following said forward programming or directed differentiation.
  • the iPSC or stem cell from which the somatic cell is derived does not display the ageing phenotype.
  • a factor which causes major cellular changes i.e. an ageing- inducing factor, such as the Lamin A alternative splice form, progerin
  • a chronically displayed ageing phenotype would normally be expected to cause or be correlated with cell death, low cell viability and loss of cellular function (e.g. loss of regenerative capacity in the case of stem cells/iPSCs).
  • weak promoter includes constitutive transcriptional promoters which are known in the art to be weak, as well as strong constitutive promoters which are coupled (e.g. operably linked) to a repressor sequence.
  • Transcriptional repressor sequences referred to herein simply as “repressor sequences” for brevity, and are also known as transcriptional repressor recognition sequences by virtue of the fact they are bound by transcriptional repressor proteins
  • an exogenous substance which drives expression from an inducible promoter as described herein may be administered/provided CLO-C-P3598PCT constitutively at a low level/dose to the cell to drive low level expression of the Lamin A alternative splice form.
  • Said low dose constitutive administration/provision of the exogenous substance would achieve the same result as demonstrated herein, namely low level constitutive expression of the Lamin A alternative splice form in the cell leading to the chronic display of an ageing phenotype as described herein.
  • the low level constitutive expression of the Lamin A alternative splice form is driven by a weak constitutive promoter.
  • the weak promoter is a weak constitutive promoter. While it will be appreciated by the skilled person that the relative strength of a promoter will vary depending on the cell type and possibly the location in the genome, some examples in mammalian cells of weak constitutive promoters include, but are not limited to: the promoter from the human ubiquitin C gene (UPC), which provides ubiquitous (i.e. constitutive expression); and the promoter from phosphoglycerate kinase gene (PGK) which gives widespread expression in different mammalian cell types and has been reported to resist promoter down regulation due to methylation or deacetylation.
  • UPC human ubiquitin C gene
  • PGK phosphoglycerate kinase gene
  • the weak constitutive promoter may be selected from the UPC (SEQ ID NO: 1) or the PGK promoter (SEQ ID NO: 2).
  • the weak constitutive promoter may be a fragment of the UPC (SEQ ID NO: 1) or the PGK (SEQ ID NO: 2) promoters, such as a functional variant or fragment thereof.
  • Functional variants and fragments of promoters described herein may be portions of said sequences which make up enhancer elements, or which exclude enhancer elements, such that expression is still driven/provided by the promoter sequence (i.e. function is retained).
  • the low level constitutive expression of the Lamin A alternative splice form is driven by a strong constitutive promoter coupled to a transcriptional repressor sequence.
  • the weak promoter is a strong constitutive promoter coupled to a transcriptional repressor sequence.
  • strong constitutive promoters in mammalian cells which may be used coupled to a repressor sequence include, but are not limited to: the human elongation factor 1 ⁇ (EF1A) promoter, which has been shown to provide consistent expression regardless of cell type or physiology; the cytomegalovirus immediate-early (CMV) promoter which may contain an enhancer region but has been shown to be silenced in some cell types; the chicken ⁇ -Actin promoter coupled with CMV early enhancer (CAG/CAGG) which contains the CMV enhancer, chicken beta actin promoter, and rabbit beta-globin splice acceptor; the simian virus 40 early (SV40) promoter which may additionally include an enhancer element; the ⁇ -actine gene promoter which provides ubiquitous expression and may be either the human or chicken sequence; RNA polymerase III promoters such as H1, U6 and 7SK, which have been shown to give good expression in particular in neuronal cells (in the case of H1) and for which either the human or murine sequence can
  • promoters which may find utility herein are described in Qin CLO-C-P3598PCT et al. (2010; PLoS One, 5(5):e10611, doi: https://doi.org/10.1371%2Fjournal.pone.0010611), the promoters and sequences of which are hereby specifically incorporated by reference.
  • the weak constitutive promoter may be selected from the EF1A (SEQ ID NO: 3), CMV (SEQ ID NO: 4 or SEQ ID NO: 5), CAG/CAGG (SEQ ID NO: 6), SV40 (SEQ ID NO: 7), H1 (full: SEQ ID NO: 8; or ‘core’: SEQ ID NO: 9), U6 or 7SK promoters, or a variant or functional fragment/variant thereof.
  • the CMV promoter may additionally comprise the CMV enhancer of the sequence of SEQ ID NO: 10.
  • SEQ ID NO: 10 CGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCA TTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACG TCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATA TGCCAAGTACGCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGC CCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCG CTATTACCATG (SEQ ID NO: 10).
  • the constitutive promoter comprises the CMV promoter/ enhancer and comprises the sequence of SEQ ID NO: 11.
  • CLO-C-P3598PCT TCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATA TGCCAAGTACGCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGC CCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCG CTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGAC TCACGGGGATTTCCAAGTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCA AAATCAACGGGACTTTCCAAAATGTCG
  • the constitutive promoter comprises a sequence about 60%, about 70%, about 80%, about 90% or about 100% identical to any of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 10 or SEQ ID NO: 11.
  • the constitutive promoter comprises a sequence about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical to any of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 10 or SEQ ID NO: 11.
  • the constitutive promoter consists of SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 11.
  • Transcriptional repressor sequences are known in the art and any endogenous, exogenous or engineered repressor sequence may be used coupled to a strong constitutive promoter as described herein.
  • the term “coupled to” refers to the functional linkage of the repressor sequence and strong constitutive promoter whereby the repressor sequence affects (i.e. represses/reduces) transcription from the otherwise strong constitutive promoter.
  • the repressor sequence and strong constitutive promoter are coupled, the repressor sequence need not be in the same contiguous sequence as the strong constitutive promoter and Lamin A alternative splice form coding sequence.
  • the repressor sequence is not part of the expression cassette comprised in the cell.
  • the repressor sequence and the strong constitutive promoter become operably linked only after insertion of the expression cassette in the cell.
  • endogenous transcriptional repressor sequences include those bound and regulated by Kruppel associated box (KRAB) domain-containing proteins (e.g.
  • KRAB-zinc finger proteins have been shown to be involved in many cellular and organism-wide processes, including embryonic development, cell development and differentiation, cell proliferation, autophagy, apoptosis, neoplastic transformation and cell cycle regulation.
  • “coupled to” may also refer herein to the operable linkage of an exogenous and/or engineered repressor sequence and strong constitutive promoter.
  • the repressor sequence is exogenous.
  • the exogenous repressor sequence and strong constitutive promoter are operably CLO-C-P3598PCT linked, they are contained within the expression cassette together with the Lamin A alternative splice form coding sequence.
  • the operably linked repressor sequence and strong constitutive promoter sequence are in the same contiguous sequence of the expression cassette introduced into the cell.
  • the exogenous repressor sequence and strong constitutive promoter are operably linked in the expression cassette.
  • repressor sequences which may be introduced as exogenous repressors include, but are not limited to: the lac operon repressor (lacZYA operon); the met operon repressor; the L-arabinose operon repressor; the cumate operator (CuO) sequence; and the tet operator (TetO) sequence. In the presence of their corresponding repressor proteins which recognise and bind to these sequences, transcription is blocked.
  • the transcriptional repressor sequence is exogenous and is operably linked to the strong constitutive promoter such that the weak promoter sequence coupled to the Lamin
  • a alternative splice form coding sequence comprises a cumate operator (CuO) sequence and a strong constitutive promoter.
  • the expression cassette comprises the coding sequence for the alternative splice form of Lamin A coupled to (e.g. operably linked to) a weak constitutive promoter in the following order/orientation: 5’-weak constitutive promoter-coding sequence-3’.
  • the expression cassette comprises the coding sequence for the alternative splice form of Lamin A coupled to (e.g. operably linked to) a strong constitutive promoter in the following order/orientation: 5’-strong constitutive promoter-coding sequence-3’.
  • an endogenous transcription repressor sequence is located upstream of the genomic location where the expression cassette is inserted.
  • the expression cassette comprises the coding sequence for the alternative splice form of Lamin A coupled to (e.g. operably linked to) a transcriptional repressor sequence and a strong constitutive promoter in the following order/orientation: CLO-C-P3598PCT 5’-strong constitutive promoter-repressor sequence-coding sequence-3’.
  • the expression cassette comprises the coding sequence for the alternative splice form of Lamin A coupled to (e.g.
  • the constitutive expression of the Lamin A alternative splice form (e.g. progerin) in the cell is because the cell does not comprise a repressor protein which recognises and/or binds the transcription repressor sequence.
  • the repressor protein absent in the cell is the corresponding repressor protein for the repressor sequence.
  • the cell does not comprise said repressor protein and/or a sequence encoding it.
  • the constitutive expression of the Lamin A alternative splice form (e.g. progerin) in the cell is because the cell does not comprise a sequence encoding for the cumate repressor protein (CymR).
  • the cell does not comprise a sequence coding for a cumate repressor protein (CymR).
  • the cell does not comprise the cumate repressor protein (CymR). The absence of CymR in the cell allows expression from the expression cassette encoding the Lamin A alternative splice form and the constitutive promoter.
  • the expression cassette comprises a cumate operator (CuO) operably linked to the Lamin A alternative splice form coding sequence
  • the absence of CymR allows expression.
  • the presence of the expression cassette and absence of a CymR coding sequence yields constitutive expression of the alternative splice form of Lamin A in the cell.
  • the cumate switch was discovered in Pseudomonas putida and was developed as a method of inducible gene expression where transcription is reversibly mediated in the presence of the cumate, by virtue of binding of the CymR to CuO sequences in the absence of cumate.
  • the system comprising both CuO sequences and CymR is available in both activator and repressor configurations, where the presence of cumate leads to the repression of transcription or activation of transcription, respectively.
  • the repressor configuration is the naturally occurring configuration and regulation is mediated by the binding of the repressor (CymR) to the operator site (CuO), placed downstream of a constitutive promoter and upstream of an operably linked coding sequence. Addition of cumate, a small molecule, releases CymR from CuO sequences and relieves the repression, allowing transcription to proceed.
  • an engineered chimeric transactivator (cTA) protein formed by the fusion of CymR with the activation domain of VP16, is able to activate transcription when bound to the CuO operator site, placed upstream of the constitutive promoter. Cumate addition to the activator system abrogates DNA binding and therefore transactivation by cTA, stopping transcription.
  • CLO-C-P3598PCT According to the present invention, while CymR (or any CymR coding sequences) is not present in the cell, the CuO sequences are comprised in the expression cassette according to the repressor configuration, i.e. downstream of a constitutive promoter and upstream of the operably linked coding sequence for the Lamin A alternative splice form.
  • the expression cassette comprises the coding sequence for the alternative splice form of Lamin A operably linked to a CuO sequence and a promoter as described herein in the following order/orientation: 5’-promoter-CuO-coding sequence-3’.
  • the expression cassette comprises the coding sequence for the alternative splice form of Lamin A operably linked to a CuO sequence and a promoter as described herein in the following order/orientation: 5’-CuO-promoter-coding sequence-3’.
  • the expression cassette comprises the sequence of SEQ ID NO: 12.
  • the expression cassette comprises a sequence about 60%, about 70%, about 80%, about 90% or about 100% identical to SEQ ID NO: 12. In still further embodiments, the expression cassette comprises a sequence about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical to SEQ ID NO: 12. In another embodiment, the expression cassette consists of SEQ ID NO: 12. In a still further embodiment, the expression cassette further and/or additionally comprises a poly A sequence, such as an SV40 poly A sequence.
  • the expression cassette may comprise the poly A sequence (e.g. the SV40 poly A sequence) 3’ of the coding sequence, such that orientation of the expression cassette is as follows: 5’-weak constitutive promoter-coding sequence-poly A-3’. 5’-strong constitutive promoter-coding sequence-ploy A-3’. 5’-strong constitutive promoter-repressor sequence-coding sequence-poly A-3’. 5’-repressor sequence-strong constitutive promoter-coding sequence-poly A-3’.
  • the poly A sequence is the SV40 poly A sequence comprising the sequence of SEQ ID NO: 13.
  • AACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAA ATAAAGCATTTTTTTCACTGC SEQ ID NO: 13
  • a CuO repressor sequence is derived from the p-cmt and/or p-cym operons in Pseudomonas putida.
  • the CuO sequence is a variant and/or a functional fragment of the p-cmt and/or p-cym operons.
  • Such functional fragments include any variants or portions of the operons that may still be bound by CymR and thus function as CuO sequences.
  • functional fragments and variants still provide the level of expression of the Lamin A alternative splice form from the expression cassette described herein in the absence of CymR compared to the full p-cmt and/or p-cym operons.
  • the CuO sequence comprises the sequence of SEQ ID NO: 14.
  • the CuO sequence variant or functional fragment comprises a sequence about 60%, about 70%, about 80%, about 90% or about 100% identical to SEQ ID NO: 14.
  • the variant or functional fragment comprises a sequence about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical to SEQ ID NO: 14.
  • the CuO sequence consists of SEQ ID NO: 14.
  • iPSCs Induced pluripotent stem cells
  • Oct-3/4 and certain members of the Sox gene family have been identified as potentially crucial transcriptional regulators involved in the induction process.
  • genes which may be used as reprogramming factors to generate iPSCs include Oct3/4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15-2, Tcl1, beta-catenin, Lin28b, Sall4, Esrrb, Tbx3 and Glis1, GATA3, GATA6 and these reprogramming factors may be used singly, or in combination of two or more kinds thereof.
  • the reprogramming factors may comprise at least the Yamanaka factors, i.e. Oct3/4, Sox2, Klf4 and c-Myc.
  • any stem cell may be used in the methods described herein or may comprise the expression cassette as described herein, such as a pluripotent stem cell, an induced pluripotent stem cell (iPSC), a germline stem cell, a multipotent stem cell, an oligopotent stem cell, a unipotent stem cell or a tissue-specific/tissue- resident stem cell.
  • iPSC induced pluripotent stem cell
  • germline stem cell a multipotent stem cell
  • an oligopotent stem cell a unipotent stem cell or a tissue-specific/tissue- resident stem cell.
  • cells suitable for the disclosures herein may include any type of stem cell.
  • the stem cells may be pluripotent stem cells, for example iPSCs, embryonic stem cells or pluripotent stem cells derived by nuclear transfer or cell fusion. It may be preferred that the embryonic stem cell is derived without destruction of the embryo, particularly where the cells are human. In some embodiments, the stem cells are not derived from human or animal embryos, i.e. the invention does not extend to any methods or uses which involve the destruction of human or animal embryos.
  • the stem cells may also include multipotent stem cells, oligopotent stem cells or unipotent stem cells.
  • the CLO-C-P3598PCT stem cells may also include foetal stem cells or adult stem cells, such as hematopoietic stem cells, mesenchymal stem cells, neural stem cells, epithelial stem cells or skin stem cells.
  • the stem cells may be isolated from umbilical, placenta, amniotic fluid, chorion villi, blastocysts, bone marrow, adipose tissue, brain, peripheral blood, cord blood, menstrual blood, blood vessels, skeletal muscle, skin and liver.
  • the cell is an iPSC.
  • the cell is a somatic cell derived from a reprogrammed iPSC or a stem cell.
  • the somatic cell is a neuron (e.g. motor neuron, sensory neuron, GABAergic neuron, glutamatergic neuron or dopaminergic neuron), glia cell, blood cell (e.g. erythrocyte), immune cell (e.g. T cell, B cell, Macrophage, NK cell, neutrophil or granulocyte), liver cell (e.g. hepatocyte, Kupffer cell or stromal cell), muscle cell (e.g. smooth muscle cell), myocyte (e.g. cardiomyocyte), fibroblast, skin cell (e.g.
  • a neuron e.g. motor neuron, sensory neuron, GABAergic neuron, glutamatergic neuron or dopaminergic neuron
  • glia cell e.g. erythrocyte
  • immune cell e.g. T cell, B cell, Macrophage, NK cell, neutrophil or granulocyte
  • liver cell e.g. hepatocyte, Kupffer cell or stromal
  • the cell is a neuron derived from an iPSC or a stem cell by forward programming or directed differentiation.
  • the term “somatic cell” as used herein includes any type (i.e. lineage) of cell that makes up the body of an organism, excluding germ cells and undifferentiated stem cells. Somatic cells may therefore include, for example and without limitation, neurons, glia cells, blood cells (e.g. leucocytes), liver cells (e.g. hepatocytes), muscle cells (e.g. myocytes) or fibroblasts.
  • the somatic cell may be an adult cell or a cell derived from an adult which displays one or more detectable characteristics of an adult or non-embryonic cell.
  • References herein to a somatic cell “derived from an iPSC or a stem cell by forward programming or directed differentiation” refer to cells which comprise the phenotype and/or characteristics of a somatic cell as defined herein (e.g. the surface phenotype and/or functional characteristics associated with a particular lineage) and have been forward programmed or differentiated from a pluripotent stem cell which has previously been reprogrammed as described herein, i.e. an iPSC.
  • Forward programming of an iPSC or a stem cell to a somatic cell comprises the introduction of lineage-specific factors, such as transcription factors, or nucleic acids which encode said lineage-specific factors, for example in the form of mRNA or expression cassettes.
  • forward programming may comprise increasing the expression of lineage- specific factors (e.g. lineage-specific transcription factors), such as by increasing the expression of said lineage-specific factor genes and/or their protein expression.
  • lineage-specific factors e.g. lineage-specific transcription factors
  • the expression of an exogenous or endogenous (in particular an exogenous) transcription factor may be increased.
  • forward programming comprises introducing into the CLO-C-P3598PCT iPSC/stem cell a nucleic acid or protein preparation which encodes or provides a lineage- specific transcription factor or combinations thereof, and culturing the cell under conditions suitable for reprogramming the cell into a somatic cell.
  • Directed differentiation comprises culturing the iPSC/stem cell in conditions to direct differentiation towards a particular somatic cell fate, such as by using exogenous factors to mimic developmental signals which would be encountered by the cell during physiological development.
  • Directed differentiation factors may include signalling molecules and/or extracellular structures, scaffolds and/or matrices which promote cell adhesion and tissue-like structures. Such factors may be altered over time (e.g.
  • the iPSC, stem cell or somatic cell is from a mammal.
  • the mammal is a human.
  • the iPSC, stem cell or somatic cell is from a human and is a human iPSC, stem cell or a human somatic cell, such as a somatic cell forward programmed or differentiated from a re-programmed human iPSC or stem cell.
  • the mammal is a mouse, optionally such that the iPSC, stem cell or somatic cell is a mouse iPSC, stem cell or a mouse somatic cell, such as a somatic cell forward programmed or differentiated from a re-programmed mouse iPSC/stem cell.
  • iPSC iPSC
  • stem cell a mouse somatic cell
  • somatic cell forward programmed or differentiated from a re-programmed mouse iPSC/stem cell.
  • lineage-specific factors such as transcription factors
  • the iPSCs or stem cells to be forward programmed or differentiated into somatic cells may include any method known in the art, for example, by induction of expression of one or more expression cassettes previously introduced into the cells, or by introduction of nucleic acids (such as DNA or RNA), polypeptides or small molecules to the cells.
  • methods of the invention may involve culturing the cell population under conditions to artificially increase the expression level of one or more lineage-specific transcription factors.
  • the lineage-specific transcription factors are provided by an expression cassette.
  • the cell comprises a further expression cassette comprising one or more coding sequences for one or more lineage-specific transcription factors operably linked to an inducible promoter.
  • the cell comprising this further expression cassette may be forward programmed by inducing expression of the one or more lineage-specific transcription factors.
  • Suitable inducible promoters are known in the art and include, without limitation the Tet system. However, as will be readily appreciated by the presence of a CuO sequence in the expression cassette encoding the Lamin A alternative splice form, any further expression cassette (including comprising lineage-specific transcription factor coding sequences) will not use a cumate inducible promoter. Thus, in certain embodiments the inducible promoter comprised in the further expression cassette is different to that in the expression cassette encoding the Lamin A alternative splice form. This inducible promoter imparts the ability to control expression of the one or more lineage-specific transcription factors.
  • the “inducible promoter” herein comprises an operator sequence for the transcriptional regulator protein (e.g. a tetracycline operator (TetO) sequence) and a constitutive promoter.
  • the activity of the constitutive promoter is supressed until transcription is allowed/activated from the operator sequence.
  • Controlled Expression expression of the lineage-specific transcription factors is under controlled transcription.
  • the transcription and translation (expression) of the transcription factors may be controlled within the cell. This permits overexpression of the transcription factors, if required.
  • the further exogenous expression cassette carrying the lineage-specific transcription factors may comprise an externally inducible transcriptional regulatory element (i.e. an inducible promoter) for inducible expression of the transcription factors.
  • Said inducible expression cassette may be controlled by addition of an exogenous substance. Whatever culturing conditions are used, the exogenous substance will control expression of the genetic sequence within the inducible expression cassette; and may either be supplied continuously and then withdrawn in order to induce transcription or supplied as transcription is required, dependent upon its mode of action.
  • the inducible promoter is regulated by a transcriptional regulator protein and said transcriptional regulator protein is controlled by an exogenously supplied substance.
  • the exogenously supplied substance e.g. tetracycline or a derivate thereof
  • the one or more lineage-specific transcription factors are expressed upon exposure of the cell to the exogenously supplied substance (e.g. tetracycline or a derivate thereof).
  • a transcriptional regulator protein is a protein that binds to DNA, preferably sequence- specifically to a DNA site located in or near a promoter, and either facilitating the binding of the transcription machinery to the promoter, and thus transcription of the DNA sequence (a transcriptional activator) or blocks this process (a transcriptional repressor).
  • a transcriptional activator a transcriptional activator
  • CLO-C-P3598PCT The DNA sequence that a transcriptional regulator protein binds to is called a transcription factor-binding site or response element, and these are found in or near the promoter of the regulated DNA sequence.
  • Transcriptional activator proteins bind to the response element and promote gene expression. Such activator proteins are preferred in the methods of the present invention for controlling inducible cassette expression.
  • Transcriptional repressor proteins bind to the response element and prevent gene expression.
  • Transcriptional regulator proteins may be activated or deactivated by a number of mechanisms including binding of a substance, interaction with other transcription factors (e.g. homo- or hetero-dimerisation) or coregulatory proteins, phosphorylation, and/or methylation.
  • the transcriptional regulator protein may be controlled by activation or deactivation. If the transcriptional regulator protein is a transcriptional activator protein, it is preferred that the transcriptional activator protein requires activation. This activation may be through any suitable means, but it is preferred that the transcriptional regulator protein is activated through the addition of an exogenous substance to the cell. The supply of an exogenous substance to the cell can be controlled, and thus the activation of the transcriptional regulator protein can be controlled.
  • an exogenous substance can be supplied in order to deactivate a transcriptional regulator protein, and then supply withdrawn in order to activate the transcriptional regulator protein.
  • the transcriptional regulator protein is a transcriptional repressor protein, it is preferred that the transcriptional repressor protein requires deactivation.
  • a substance is supplied to prevent the transcriptional repressor protein repressing transcription, and thus transcription is permitted.
  • Any suitable transcriptional regulator protein may be used, preferably one that may be activated or deactivated. It is preferred that an exogenous substance may be supplied to control the transcriptional regulator protein.
  • Such transcriptional regulator proteins are also called inducible transcriptional regulator proteins.
  • Tetracycline-Controlled Transcriptional Activation is a method of inducible gene expression where transcription is reversibly turned on or off in the presence of the antibiotic tetracycline or one of its derivatives (e.g. doxycycline which is more stable).
  • the transcriptional activator protein is tetracycline-responsive transcriptional activator protein (rtTa) or a derivative thereof.
  • the rtTA protein is able to bind to DNA at specific TetO operator CLO-C-P3598PCT sequences. Several repeats of such TetO sequences are placed upstream of a minimal constitutive promoter (such as the CMV promoter), which together form a tetracycline response element (TRE).
  • a minimal constitutive promoter such as the CMV promoter
  • Tet-On tetracycline or a derivative activates
  • Tet-Off tetracycline or a derivative thereof binds rtTA and deactivates the rtTA, rendering it incapable of binding to TRE sequences, thereby preventing transcription of TRE- controlled genes.
  • rtTa constitutively expressed tetracycline-responsive transcriptional activator protein
  • TRE rtTa-sensitive inducible promoter
  • the transcriptional regulator protein may be a tetracycline-responsive transcriptional activator (rtTa) protein, which can be activated or deactivated by tetracycline or one of its derivatives, which are supplied exogenously.
  • rtTa tetracycline-responsive transcriptional activator
  • the expression cassette comprising an inducible promoter includes a tetracycline operator (TetO) sequence and a constitutive promoter, such as to make up a tetracycline response element (TRE).
  • TetO tetracycline operator
  • TRE tetracycline response element
  • the exogenously supplied substance is tetracycline or one of its derivatives.
  • Variants and modified rtTa proteins may also be used in the methods of the invention, these include Tet-On Advanced transactivator (also known as rtTA2S-M2) and Tet- On 3G (also known as rtTA-V16, derived from rtTA2S-S2).
  • the transcriptional regulator protein is an rtTa and the exogenously supplied substance is tetracycline or a derivative thereof, such as doxycycline.
  • the tetracycline response element generally consists of 7 repeats of the 19bp bacterial TetO sequence separated by spacer sequences, together with a minimal promoter. Variants and modifications of the TRE sequence are possible, since the minimal promoter can be any suitable promoter. Preferably the minimal promoter shows no or minimal expression levels in the absence of rtTa binding, but upon rtTa binding provides constitutive expression.
  • the inducible promoter may thus comprise a TRE.
  • the expression from the expression cassette comprising the inducible promoter occurs upon exposure of the cell to the exogenously supplied substance expression from the expression cassette comprising the inducible promoter.
  • the exogenously supplied substance e.g. tetracycline or a derivate thereof
  • the one or more lineage-specific transcription factors are expressed upon exposure of the CLO-C-P3598PCT cell to the exogenously supplied substance (e.g. tetracycline or a derivate thereof).
  • the transcriptional regulator protein is a transcriptional repressor protein, TetR.
  • the components of this system include an inducible promoter comprising a strong human cytomegalovirus immediate-early (CMV) promoter and two tetracycline operator 2 (TetO2) sites, and a Tet repressor (TetR).
  • CMV human cytomegalovirus immediate-early
  • TetO2 tetracycline operator 2
  • TetR Tet repressor
  • the Tet repressor:tetracycline complex then dissociates from the Tet operator and allows induction of expression.
  • the transcriptional regulator protein is TetR and the inducible promoter comprises two TetO2 sites.
  • the exogenously supplied substance is tetracycline or a derivative thereof (e.g. doxycycline).
  • the cumate switch is another method of inducible gene expression where transcription is reversibly turned on or off in the presence of the cumate. This system is available in both activator and repressor configurations, where the presence of cumate leads to the repression of transcription or activation of transcription, respectively.
  • repressor In the repressor configuration, regulation is mediated by the binding of the repressor (CymR) to the operator site (CuO), placed downstream of a constitutive promoter. Addition of cumate, a small molecule, relieves the repression and allows transcription to proceed.
  • a chimeric transactivator (cTA) protein In the activator configuration, a chimeric transactivator (cTA) protein, formed by the fusion of CymR with the activation domain of VP16, is able to activate transcription when bound to the CuO operator site, placed upstream of the constitutive promoter. Cumate addition abrogates DNA binding and therefore transactivation by cTA, stopping transcription.
  • the transcriptional regulator protein may be a Tet-responsive transcriptional activator protein (rtTA).
  • expression of the lineage-specific transcription factors is under the control of a Tet-responsive element.
  • the expression cassette comprising the coding sequence for the one or more lineage-specific transcription factors comprises a Tet-response element (TRE).
  • the Tet-response element comprises the sequence of SEQ ID NO: 15.
  • the TRE consists of a sequence of SEQ ID NO: 15.
  • the TRE comprises a sequence with about 60%, about 70%, about 80%, about 90% or about 100% identity to SEQ ID NO: 15.
  • Other inducible expression systems are known and can be used in the present invention. These include the Complete Control Inducible system from Agilent Technologies. This is based upon the insect hormone ecdysone or its analogue ponasterone A (ponA) which can activate transcription in mammalian cells which are transfected with both the gene for the Drosophila melanogaster ecdysone receptor (EcR) and an inducible promoter comprising a binding site for the ecdysone receptor.
  • ponA insect hormone ecdysone or its analogue ponasterone A
  • the EcR is a member of the retinoid-X-receptor (RXR) family of nuclear receptors. In humans, EcR forms a heterodimer with RXR that binds to the ecdysone-responsive element (EcRE). In the absence of PonA, transcription is repressed by the heterodimer.
  • RXR retinoid-X-receptor
  • EcRE ecdysone-responsive element
  • Other examples of repressor protein-comprising transcriptional regulator systems are also known and can include those using an ecdysone receptor or a derivative thereof. Particular examples include the VgEcR synthetic receptor from Agilent technologies which is a fusion of EcR, the DNA binding domain of the glucocorticoid receptor and the transcriptional activation domain of Herpes Simplex Virus VP16.
  • the inducible promoter comprises the EcRE sequence or modified versions thereof together with a minimal promoter.
  • Modified versions include the E/GRE recognition sequence of Agilent Technologies, in which mutations to the sequence have been made.
  • the E/GRE recognition sequence comprises inverted half-site recognition elements for the retinoid-X-receptor (RXR) and GR binding domains.
  • RXR retinoid-X-receptor
  • GR binding domains GR binding domains.
  • the exogenously supplied substance is ponasterone A, which CLO-C-P3598PCT removes the repressive effect of EcR or derivatives thereof on the inducible promoter, and allows transcription to take place.
  • Expression of the one or more lineage-specific transcription factors described herein may be increased using the dual cassette expression system described in WO 2018/096343, which is hereby specifically incorporated herein by reference in its entirety.
  • This system targets genetic safe harbour (GSH) sites which provides a reduced risk of epigenetic silencing of the inserted genetic material.
  • an expression cassette comprising one or more sequences coding for lineage-specific transcription factors as described herein are introduced into the iPSC, stem cell or somatic cell using a method comprising: - targeted insertion of a gene encoding a transcriptional regulator protein into a genetic safe harbour (GSH) site of the cell; and - targeted insertion of an inducible cassette into a further GSH of the cell, wherein said inducible cassette comprises said transcription factor sequences operably linked to an inducible promoter, and said promoter is regulated by the transcriptional regulator protein.
  • GSH genetic safe harbour
  • the insertion of the gene encoding a transcriptional regulator protein into one GSH provides the control mechanism for the expression of the inducible cassette which is operably linked to the inducible promoter and inserted into a further GSH site.
  • the GSH are different.
  • the lineage-specific transcription factor encoding expression cassette is inserted into the same GSH as the expression cassette encoding the Lamin A alternative splice form, with the transcriptional regulator protein encoding sequence inserted into a further GSH.
  • the Lamin A alternative splice form and lineage-specific transcription factor encoding expression cassettes may be inserted into a first GSH, and the transcriptional regulator protein is inserted into a second GSH.
  • the lineage-specific transcription factor encoding expression cassette is inserted into a different GSH as the expression cassette encoding the Lamin A alternative splice form, with the transcriptional regulator protein encoding sequence inserted into a yet further GSH.
  • the Lamin A alternative splice form encoding expression cassette may be inserted into a first GSH, the lineage-specific transcription factor encoding expression cassette inserted into a second GSH, and the transcriptional regulator protein is inserted into a third GSH.
  • a GSH site is a locus within the genome wherein a gene or other genetic material may be inserted without any deleterious effects on the cell or on the inserted genetic material.
  • GSH site in which expression of the inserted gene sequence is not perturbed by any read-through expression from neighbouring genes and expression of the inducible cassette minimises interference with the endogenous transcription programme.
  • More formal criteria have been proposed that assist in the determination of whether a particular locus is a GSH site in future (Papapetrou et al. (2011)). These criteria include a site that is: (i) 50 kb or more from the 5’ end of any gene; (ii) 300 kb or more from any gene related to cancer; (iii) 300 kb or more from any microRNA (miRNA); (iv) located outside a transcription unit; and (v) located outside ultraconserved regions (UCR).
  • miRNA microRNA
  • the genetic safe harbour sites are selected from (in particular any two) of the hROSA26 locus, the AAVS1 locus, the DBI locus, the CLYBL gene, the CCR5 gene or the HPRT gene.
  • the GSHs may be the hROSA26 locus, the AAVS1 locus and the DBI locus. Insertions specifically within genetic safe harbour sites is preferred over random genome integration, since this is expected to be a safer modification of the genome, and is less likely to lead to unwanted side effects such as silencing natural gene expression or causing mutations that lead to cancerous cell types.
  • the single genetic safe harbour site is selected from any GSH described herein.
  • the adeno-associated virus integration site 1 locus (AAVS1) is located within the protein phosphatase 1, regulatory subunit 12C (PPP1R12C) gene on human chromosome 19, which is expressed uniformly and ubiquitously in human tissues.
  • AAVS1 has been shown to be a favourable environment for transcription, since it comprises an open chromatin structure and native chromosomal insulators that enable resistance of the inducible cassettes against silencing. There are no known adverse effects on the cell resulting from disruption of the CLO-C-P3598PCT PPP1R12C gene. Moreover, an inducible cassette inserted into this site remains transcriptionally active in many diverse cell types.
  • the hROSA26 site has been identified on the basis of sequence analogy with a GSH from mice (ROSA26 – reverse oriented splice acceptor site #26). The hROSA26 locus is on chromosome 3 (3p25.3) and can be found within the Ensembl database (GenBank: CR624523).
  • the integration site lies within the open reading frame (ORF) of the THUMPD3 long non-coding RNA (reverse strand). Since the hROSA26 site has an endogenous promoter, the inserted genetic material may take advantage of that endogenous promoter, or alternatively may be inserted operably linked to a promoter.
  • CLYBL thus provides a GSH which may be suitable for use in the present invention.
  • CCR5 which is located on chromosome 3 (position 3p21.31) is a gene which codes for HIV-1 major co-receptor. Interest in the use of this site as a GSH arises from the null mutation in this gene that appears to have no adverse effects, but predisposes to HIV-1 infection resistance. Zinc-finger nucleases that target the third exon have been developed, thus allowing for insertion of genetic material at this locus.
  • the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene encodes a transferase enzyme that plays a central role in the generation of purine nucleotides through the purine salvage pathway.
  • GSH in other organisms have been identified and include ROSA26, HRPT and Hipp11 (H11) loci in mice. Mammalian genomes may include GSH sites based upon pseudo attP sites. For such sites, hiC31 integrase, the Streptomyces phage-derived recombinase, has been developed as a non-viral insertion tool, because it has the ability to integrate an inducible cassette-containing plasmid carrying an attB site into pseudo attP sites.
  • the insertions into the first and/or second GSH may occur on one chromosome, or on both chromosomes.
  • the GSH exists at the same genetic loci on both chromosomes of diploid organisms. Insertion within both chromosomes is advantageous since it may enable CLO-C-P3598PCT an increase in the level of transcription from the inserted genetic material within the inducible cassette, thus achieving particularly high levels of transcription.
  • Specific insertion of genetic material into the particular GSH based upon customised site- specific generation of DNA double-strand breaks at the GSH may be achieved.
  • the genetic material may then be introduced using any suitable mechanism, such as homologous recombination.
  • any method of making a specific DSB in the genome may be used, but preferred systems include CRISPR/Cas9 and modified versions thereof, zinc finger nucleases and the TALEN system.
  • One or more genetic sequences may be controllably transcribed from within the second and/or further GSH or from within the single GSH.
  • the inducible cassette may contain 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 genetic sequences which it is desired to insert into the GSH and the transcription of which be controllably induced. Therefore, the several (e.g. two) lineage- specific transcription factors may be included within the same cassette introduced into the second/further genetic safe harbour site.
  • the several lineage-specific transcription factors may be included in several mono-cistronic constructs or one or more bi- cistronic or tri-cistronic construct as required. It will be understood that similar combinations of constructs may be used to achieve higher orders of expression.
  • the lineage-specific transcription factors and the one or more ageing-inducing factor are included in one bi-cistronic construct.
  • Site-Specific Delivery & Targeting Any suitable technique for insertion of a nucleic acid sequence (i.e. an expression cassette) into a specific sequence may be used, and several are described in the art. Suitable techniques include any method which introduces a break at the desired location and permits recombination of the vector into the gap.
  • DSB double-strand DNA break
  • Distinct cellular repair mechanisms can be exploited to repair the DSB and to introduce the desired sequence, and these are non-homologous end joining repair (NHEJ), which is more prone to error; and homologous recombination repair (HR) mediated by a donor DNA template, that can be used to insert inducible cassettes.
  • NHEJ non-homologous end joining repair
  • HR homologous recombination repair
  • CRISPR/Cas9 CLO-C-P3598PCT
  • Zinc finger nucleases are artificial enzymes which are generated by fusion of a zinc-finger DNA-binding domain to the nuclease domain of the restriction enzyme FokI. The latter has a non-specific cleavage domain which must dimerise in order to cleave DNA. This means that two zinc finger nuclease monomers are required to allow dimerisation of the FokI domains and to cleave the DNA.
  • the DNA binding domain may be designed to target any genomic sequence of interest, and is a tandem array of Cys2His2 zinc fingers, each of which recognises three contiguous nucleotides in the target sequence.
  • the two binding sites are separated by 5-7bp to allow optimal dimerization of the FokI domains.
  • the enzyme thus is able to cleave DNA at a specific site, and target specificity is increased by ensuring that two proximal DNA- binding events must occur to achieve a double-strand break.
  • Transcription activator-like effector nucleases, or TALENs are dimeric transcription factor/nucleases. They are made by fusing a TAL effector DNA-binding domain to a DNA cleavage domain (a nuclease).
  • Transcription activator-like effectors can be engineered to bind practically any desired DNA sequence, so when combined with a nuclease, DNA can be cut at specific locations.
  • TAL effectors are proteins that are secreted by Xanthomonas bacteria, the DNA binding domain of which contains a repeated highly conserved 33–34 amino acid sequence with divergent 12th and 13th amino acids. These two positions are highly variable and show a strong correlation with specific nucleotide recognition. This straightforward relationship between amino acid sequence and DNA recognition has allowed for the engineering of specific DNA-binding domains by selecting a combination of repeat segments containing appropriate residues at the two variable positions.
  • TALENs are thus built from arrays of 33 to 35 amino acid modules, each of which targets a single nucleotide. By selecting the array of the modules, almost any sequence may be targeted.
  • the nuclease used may be FokI or a derivative thereof.
  • Three types of CRISPR mechanisms have been identified, of which type II is the most studied.
  • the CRISPR/Cas9 system (type II) utilises the Cas9 nuclease to make a double-stranded break in DNA at a site determined by a short guide RNA.
  • the CRISPR/Cas system is a prokaryotic immune system that confers resistance to foreign genetic elements.
  • CRISPR are segments of prokaryotic DNA containing short repetitions of base sequences.
  • CRISPR spacers recognise and cut the exogenous genetic elements using RNA interference.
  • the CRISPR immune response occurs through two steps: CRISPR-RNA (crRNA) biogenesis and crRNA-guided interference.
  • CrRNA molecules are composed of a variable sequence transcribed from the protospacer DNA and a CRISPR repeat.
  • Each crRNA CLO-C-P3598PCT molecule then hybridizes with a second RNA, known as the trans-activating CRISPR RNA (tracrRNA) and together these two eventually form a complex with the nuclease Cas9.
  • the protospacer DNA encoded section of the crRNA directs Cas9 to cleave complementary target DNA sequences, if they are adjacent to short sequences known as protospacer adjacent motifs (PAMs).
  • PAMs protospacer adjacent motifs
  • This natural system has been engineered and exploited to introduce DSB breaks in specific sites in genomic DNA, amongst many other applications.
  • the CRISPR type II system from Streptococcus pyogenes may be used.
  • the CRISPR/Cas9 system comprises two components that are delivered to the cell to provide genome editing: the Cas9 nuclease itself and a guide RNA (gRNA).
  • the gRNA is a fusion of a customised, site-specific crRNA (directed to the target sequence) and a standardised tracrRNA.
  • a donor template with homology to the targeted locus is supplied; the DSB may be repaired by the homology-directed repair (HDR) pathway allowing for precise insertions to be made.
  • HDR homology-directed repair
  • Mutant forms of Cas9 are available, such as Cas9D10A with only nickase activity. This means it cleaves only one DNA strand and does not activate NHEJ. Instead, when provided with a homologous repair template, DNA repairs are conducted via the high-fidelity HDR pathway only.
  • Cas9D10A may be used in paired Cas9 complexes designed to generate adjacent DNA nicks in conjunction with two sgRNAs complementary to the adjacent area on opposite strands of the target site, which may be particularly advantageous.
  • the elements for making the double-strand DNA break may be introduced in one or more vectors, such as plasmids, for expression in the stem or somatic cell described herein.
  • any method of making specific, targeted double strand breaks in the genome in order to affect the insertion of a gene/inducible cassette and/or expression cassettes described herein may be used in the present invention.
  • the method for inserting the gene/inducible cassette utilises any one or more of zinc finger nucleases, TALENs and/or CRISPR/Cas9 systems or any derivative thereof.
  • the gene/inducible cassette and/or expression cassettes for insertion may be supplied in any suitable fashion as described herein.
  • the cassette and associated genetic material form the donor DNA for repair of the DNA at the CLO-C-P3598PCT DSB and are inserted using standard cellular repair machinery/pathways. How the break is initiated will alter which pathway is used to repair the damage, as noted above.
  • Ageing Factors & Ageing Phenotypes As will be appreciated, the constitutive expression of the ageing-inducing factor, an alternative splice form of Lamin A, in the iPSC, stem cell or somatic cell derived from an iPSC or a stem cell by forward programming or directed differentiation leads to the chronic display of an ageing phenotype by said cell.
  • the cell i.e. the iPSC, stem cell or somatic cell derived from an iPSC/stem cell by forward programming or directed differentiation
  • chronically displays an ageing phenotype chronically displays an ageing phenotype.
  • chronic is used herein in its normal meaning and context, namely that that the cell displays or has an ageing phenotype for a prolonged period of time.
  • the ageing phenotype may also be described as persistent or permanent. Such prolonged period of time/persistence is relative to the life-time of the cell and to any active steps which may be taken to change the phenotype of the cell, including its ageing phenotype or lineage identity. It is also relative to the time in culture of said cell, such as the ageing phenotype is chronically displayed for the duration that the cell is in culture.
  • display of the ageing phenotype is caused by constitutive expression of the Lamin A alternative splice form, any alteration in this expression will result in a change in the ageing phenotype displayed.
  • ageing phenotype For example, if a CymR is expressed or otherwise provided to the cell, expression of the Lamin A alternative splice form will be reduced and thus so will the ageing phenotype.
  • ageing and ageing refer to the phenotypic changes associated with age which restrict functionality and can be considered to be a consequence of ageing. These ageing phenotypes can be distinct from ageing DNA correlates such as epigenetic changes and double strand breaks for which the causality with ageing has not yet been established and reversal of which may not lead to a reversal of ageing or maintenance of a non-aged phenotype.
  • cellular phenotypes associated with ageing are: mitochondrial dysfunction, senescence, altered intracellular communication, genomic instability, telomere shortening, epigenetic changes, and deregulation of nutrient pathways (Otin et al. (2013) Cell “The Hallmarks of Ageing”).
  • Other ageing phenotypes include: reduced proliferation, changes in cell and/or nuclear morphology, changes in gene expression, and alterations in Lamin-A and/or Lamin-C nuclear protein expression.
  • ageing and “ageing” may refer to any ageing phenotype herein and in certain embodiments refer to the phenotype of the cell (e.g. the ageing phenotype), such as the CLO-C-P3598PCT phenotype of the cell observed upon constitutive expression of the Lamin A alternative splice form and/or measured as part of a method of identifying a gene or combination of genes involved in the reversal or maintenance of a non-aged phenotype as described herein.
  • the ageing phenotype such as the CLO-C-P3598PCT phenotype of the cell observed upon constitutive expression of the Lamin A alternative splice form and/or measured as part of a method of identifying a gene or combination of genes involved in the reversal or maintenance of a non-aged phenotype as described herein.
  • the ageing phenotype may be selected from one or more of: proliferation, senescence, loss of proteostasis, changes in cell and/or nuclear morphology, mitochondrial function, changes in gene expression, altered expression of Lamin-A and/or Lamin-C nuclear protein, in particular upregulation, epigenetic marks associated with ageing, altered DNA or histone methylation, altered methylation entropy, DNA double strand breaks, telomere length, and a transcriptomic and/or epigenetic clock.
  • the ageing phenotype is a transcriptomic signature associated with an aged phenotype (e.g. as described in US 63/545,973).
  • the ageing phenotype is reduced proliferation and/or increased senescence.
  • the changes in nuclear morphology are folding abnormalities, blebbing and/or loss of nuclear circularity.
  • the mitochondrial function is reduced oxygen consumption and/or increased mitochondrial reactive oxygen species (ROS).
  • the changes in gene expression are selected from one or more of: differential expression of somatic cell lineage identity genes, differential expression of mitochondrial genes, differential expression of apoptosis- and/or senescence-related genes, differential expression of autophagy-associated genes, differential expression of inflammation response-related genes, differential expression of altered intracellular communication-related genes, deregulated nutrient-sensing-related genes, and differential expression of DNA damage-related genes.
  • the changes in gene expression are selected from one or more of: downregulation of somatic cell lineage identity genes, downregulation of mitochondrial genes, upregulation of apoptosis- and/or senescence-related genes, downregulation/upregulation of autophagy-associated genes, differential expression of inflammation response-related genes, differential expression of altered intracellular communication-related genes, deregulated nutrient-sensing-related genes, and upregulation of DNA damage-related genes.
  • the epigenetic marks associated with ageing are selected from one or more of: reduced heterochromatin trimethylated H3K9 (H3K9me3), reduced heterochromatin trimethylated H3K27 (H3K27me3), reduced HP1 ⁇ , and increased ⁇ H2AX.
  • the telomere length is shortened.
  • the transcriptomic and/or epigenetic clock is a single cell transcriptomic and/or epigenetic clock.
  • the ageing phenotype is a transcriptomic clock, such as a transcriptomic signature associated with an aged phenotype.
  • the ageing phenotype is a single cell transcriptomic clock, such as a transcriptomic signature associated with an aged phenotype.
  • the ageing phenotype such as the ageing CLO-C-P3598PCT phenotype of the cell, is a combination of one or more of the ageing phenotypes described herein.
  • DNA methylation age (further known as a “predicted age”) is characterised by the following properties: it is close to zero for ES and iPS cells; it correlates with cell passage number; it gives rise to a highly heritable measure of age acceleration; and it is applicable to chimpanzee tissues as well as to tissue from other species.
  • the DNA methylation age of blood has been shown to predict all-cause mortality in later life, even after adjusting for known risk factors, suggesting that it is related to processes that cause ageing.
  • markers of physical and mental fitness have been associated with the epigenetic clock.
  • One particular feature of the Horvath epigenetic clock is its high accuracy and applicability to a broad spectrum of tissues and cell types.
  • the Horvath epigenetic clock may be used to identify any change in DNA methylation age caused by treatment, such as reprogramming or forward programming/directed differentiation.
  • “age” is determined using a transcriptomic clock, such as a single cell transcriptomic clock.
  • the ageing phenotype is age according to a transcriptomic clock.
  • the transcriptomic clock comprises a transcriptomic signature associated with an aged phenotype of the cell.
  • a transcriptomic clock is described in Fleischer et al. (2016) Genome Biol. 19, 221 (doi: https://doi.org/10.1186/s13059-018-1599-6).
  • Previously known ageing clocks trained on single cell RNA sequencing signatures typically suffer from: i) restriction to particular cell types; ii) lower accuracy than epigenetic-based ageing clocks; and iii) high variance of age measurements.
  • a novel bioinformatic approach which yields single cell transcriptomic clocks that: i) are cell type independent; ii) exhibit accuracy rivalling epigenetic ageing clocks; and iii) have minimal variance in age measurements.
  • Said bioinformatic approach may also be applied to multiomic data where data sets such as genomic, proteomic and epigenomic data sets are combined.
  • CLO-C-P3598PCT In a further embodiment, a transcriptomic clock, such as a single cell transcriptomic clock, may be combined with any ageing phenotype described herein.
  • age is determined using a combination of a single cell transcriptomic clock with changes in cell and/or nuclear morphology, epigenetic clock measurements, mitochondrial function, epigenetic marks associated with ageing, altered methylation entropy, gene expression or any combination thereof.
  • An ageing phenotype according to an epigenetic and/or transcriptomic clock as described herein comprises an “older” age as determined by said epigenetic and/or transcriptomic clock (i.e. so that a quantifiable change can be detected) upon constitutive expression of the Lamin A alternative splice form, compared to the cell prior to said constitutive expression or compared to a cell not constitutively expressing the Lamin A alternative splice form (i.e.
  • telomere length the proteomic clock
  • metabolomic clock the metabolomic clock
  • ribosomal clock which measures the methylation status of CpG sites within ribosomal DNA
  • any of these and other molecular signatures/biological clocks may be used independently or in combination (e.g. the epigenetic and transcriptomic clocks may be used together in combination to determine age, or either/both the epigenetic and transcriptomic clocks may be used together with a further biological clock, such as the ribosomal clock).
  • making a cell “older” will therefore comprise a detectable and quantifiable change in the age determined by these clocks, such as an rDNA methylation (rDNAm) age which is older, upon constitutive expression of the Lamin A alternative splice form, compared to the cell prior to said constitutive expression or compared to a cell not constitutively expressing the Lamin A alternative splice form.
  • the ageing phenotype is specific to the cell, such as to the cell type and/or lineage.
  • the ageing phenotype may be reduced proliferation.
  • the ageing phenotype may be increased senescence.
  • CLO-C-P3598PCT If the cell is an immune cell, such as a T cell forward programmed or differentiated from an iPSC/stem cell, the ageing phenotype may be increased senescence.
  • the ageing phenotype may be a phenotype of a neurological degenerative disease, such as reduced process density and/or connectivity, reduced average dendritic length, reduced neurite diameter, downregulation of neuronal marker genes, loss of cell identity or reduced electrophysiological activity.
  • a neurological degenerative disease such as reduced process density and/or connectivity, reduced average dendritic length, reduced neurite diameter, downregulation of neuronal marker genes, loss of cell identity or reduced electrophysiological activity.
  • the ageing phenotype is a phenotype of a neurological degenerative disease, such as those described herein.
  • the ageing phenotype may be reduced electrophysiological activity or reduced mitochondrial function.
  • the Lamin A alternative splice form is herein used as an “ageing- inducing factor”.
  • the Lamin A alternative splice form is progerin or a progerin-like truncated form of Lamin A.
  • progerin-like protein may alter nuclear morphology, in particular loss of nuclear circularity, in a similar manner to progerin.
  • Progerin is a nuclear lamina protein associated with Hutchinson-Gilford Progeria Syndrome (HGPS) and is a mutant form of Lamin A lacking 50 amino acids of the C-terminus which prevents removal of a farnesyl group and leads to accumulation of the protein at the nuclear rim/lamina.
  • HGPS Hutchinson-Gilford Progeria Syndrome
  • an example of a progerin-like protein is a truncated form of Lamin A which may comprise a deletion of the C-terminal truncation site leading to the improper processing of the protein and failure to integrate into the nuclear lamina, causing morphological alterations in the cell which are similar to those caused by progerin.
  • the Lamin A alternative splice form is progerin and comprises an amino acid sequence of SEQ ID NO: 16.
  • METPSQRRATRSGAQASSTPLSPTRITRLQEKEDLQELNDRLAVYIDRVRSLETENAGLRLR ITESEEVVSREVSGIKAAYEAELGDARKTLDSVAKERARLQLELSKVREEFKELKARNTKKE GDLIAAQARLKDLEALLNSKEAALSTALSEKRTLEGELHDLRGQVAKLEAALGEAKKQLQDE MLRRVDAENRLQTMKEELDFQKNIYSEELRETKRRHETRLVEIDNGKQREFESRLADALQE LRAQHEDQVEQYKKELEKTYSAKLDNARQSAERNSNLVGAAHEELQQSRIRIDSLSAQLSQ LQKQLAAKEAKLRDLEDSLARERDTSRRLLAEKEREMAEMRARMQQQLDEYQELLDIKLAL DMEIHAYRKLLEGEEER
  • the Lamin A alternative splice form is a variant and/or functional fragment of the amino acid sequence of SEQ ID NO: 16, such as a sequence about 60%, about 70%, about 80%, about 90% or about 100% identical to SEQ ID NO: 16.
  • the term “functional fragment” as used herein refer to fragment of the specified sequence that still retain a substantial amount of the function of the full length sequence.
  • a functional fragment of SEQ ID NO: 16 e.g. a progerin-like protein
  • the Lamin A alternative splice form comprises a sequence about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical to SEQ ID NO: 16.
  • the Lamin A alternative splice form consists of SEQ ID NO: 16.
  • the Lamin A alternative splice form comprises an amino acid sequence encoded by SEQ ID NO: 17.
  • the Lamin A alternative splice form is a variant or functional fragment of the amino acid sequence encoded by SEQ ID NO: 17, such as a variant sequence encoded by a sequence about 60%, about 70%, about 80%, about 90% or about 100% identical to SEQ ID NO: 17.
  • the Lamin A alternative splice form comprises a sequence about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical to an amino acid encoded by SEQ ID NO: 17.
  • the Lamin A alternative splice form consists of an amino acid sequence encoded by SEQ ID NO: 17.
  • iPSC iPSC, stem cell or somatic cell derived from an iPSC or a stem cell by forward programming or directed differentiation described herein in a method of identifying a gene or a combination of genes involved in the reversal of an ageing phenotype or in the maintenance of a non-aged phenotype in the cell.
  • Such uses are based on the previous observations by the inventors that artificially ageing iPSCs and then optionally reversing said ageing can be used with loss-of-function, inhibitory, knock-out, activatory, gain-of-function or combinatorial screens to identify genes/combinations of genes involved in the regenerative capacity of iPSCs and stem cell.
  • an iPSC is aged and then subjected to the screen (or vice versa) and by measuring the changes in an ageing phenotype or the progression of said ageing phenotype compared to in an aged iPSC CLO-C-P3598PCT not subjected to the screen, the involvement of the knocked out, inhibited or activated gene/combination in the reversal of the ageing phenotype or the maintenance of the non-aged state can be identified.
  • the reversal of ageing can be used to identify genes/combinations of genes by subsequently introducing the repressor protein which recognises the transcriptional repressor sequence described hereinbefore (e.g.
  • the changes of ageing phenotype are the ability of the cell (in particular an iPSC) to reverse the ageing phenotype following the loss-of- function, inhibitory, knock-out, activatory, gain-of-function or combinatorial screen.
  • the cell subjected to the screen and in which the ageing phenotype is measured is compared to a cell in which the screen has not been performed, i.e. a control cell.
  • the control cell will be subjected to the same ageing factor and process as described herein (i.e. it is a cell constitutively expressing the Lamin A alternative splice form as described herein), such that the ageing phenotype can be measured and compared to that displayed by the cell in which the screen has been performed.
  • screening step (ii) of the identification method described herein may be described as optional (i.e. it is performed on one cell and not on another, control, cell).
  • genes or combinations of genes involved in reversal of an ageing phenotype or maintenance of a non-aged phenotype which have been targeted may be identified by transcriptome sequencing to determine the expression, such as the expression level, following targeting.
  • said genes or combinations of genes may be identified by detecting the agent used to target said gene(s), e.g. the guide RNA that was present in a cell in which the ageing phenotype has been altered.
  • identification of genes or combinations of genes involved in reversal of an ageing phenotype or maintenance of a non-aged phenotype is by detection of the guide RNA used to target said gene or detection of the multiple guide RNAs used to target said combination of genes.
  • the detection of the guide RNA is by sequencing.
  • the guide RNA comprises a barcode sequence and detection of the guide RNA is by sequencing using said barcode sequence.
  • the method of identifying a gene or combination of genes comprises the steps of: CLO-C-P3598PCT (i) culturing the iPSC, stem cell or somatic cell as described herein; (ii) performing a loss-of-function, inhibitory, knock-out, activatory, gain-of-function or combinatorial screen in the cell; (iii) measuring an ageing phenotype in the cell; and (iv) identifying a gene or combination of genes as being involved in the reversal of an ageing phenotype or maintenance of a non-aged phenotype when the ageing phenotype is altered when the loss-of-function, inhibitory, knock-out, activatory, gain-of-function or combinatorial screen is performed.
  • step (iv) may comprise comparing the cell in which the screen has been performed to a cell not subjected to the screen (a control cell).
  • step (ii) is optional (i.e. it is performed in one cell and not in another, control, cell).
  • the reversal of an ageing phenotype in the cell following the loss-of- function, inhibitory, knock-out, activatory, gain-of-function or combinatorial screen is measured.
  • the method of identifying a gene/combination of genes comprises additional step (iib) of introducing into the cell a repressor protein which recognises the transcriptional repressor sequence described hereinbefore (e.g.
  • control cell may be a cell in which no repressor protein has been introduced/expressed and thus in which no reversal of the ageing phenotype occurs.
  • This control cell may be in addition to or an alternative to the cell in which no loss-of-function, inhibitory, knock-out, activatory, gain-of-function or combinatorial screen has been performed.
  • the method of identifying a gene or combination of genes comprises the steps of: (i) culturing the iPSC, stem cell or somatic cell as described herein; (ii) performing a loss-of-function, inhibitory, knock-out, activatory, gain-of-function or combinatorial screen in the cell; (iib) introducing into the cell or expressing in the cell a transcription repressor protein, thereby downregulating expression of the Lamin A alternative splice form; (iii) measuring an ageing phenotype in the cell; and (iv) identifying a gene or combination of genes as being involved in the reversal of an ageing phenotype or maintenance of a non-aged phenotype when the ageing phenotype is altered when the loss-of-function, inhibitory, knock-out, activatory, gain-of-function or combinatorial screen is performed.
  • transcription repressor protein e.g. CymR
  • expression of the repressor protein may be from a transiently transfected plasmid or from an expression cassette introduced into the cell. If expression is from an introduced expression cassette, said expression could be controlled by an exogenous substance as described hereinbefore.
  • Genes/combinations of genes may be identified as having a “pro-ageing” effect when the loss- of-function, knock-out or inhibition of such genes/combinations leads to a reduction of the ageing phenotype, such as the reduction in its severity and/or a reduction in the effects of the ageing phenotype seen in the cell. Conversely, a gain-of-function or activation of these pro- ageing genes/combinations will lead to the increase of the ageing phenotype, such as increased/enhanced severity. As will be appreciated, since the cells of the present invention chronically display an ageing phenotype, no alteration in the progression or development of said phenotype is likely to be observed as it is already present/developed.
  • the pro-ageing gene/combination may have a strong, direct or active effect in driving ageing (e.g. the ageing phenotype), or may have a weak, indirect or passive effect in ageing/on the ageing phenotype, respectively.
  • Genes/combinations of genes may alternatively be identified as having an “anti-ageing” effect when the loss-of-function, knock-out or inhibition of such genes/combinations leads to an increase of the ageing phenotype, such as an increase in its severity and/or an increase in the effects of the ageing phenotype in the cell.
  • a gain-of-function or activation of anti- ageing genes/combinations will lead to a decrease of the ageing phenotype, such as decreased/reduced severity.
  • Genes/combinations may also be identified as having anti- ageing effects if their knock-out, loss-of-function or inhibition leads to a decrease in the reversal of an ageing phenotype, or their gain-of-function or activation leads to an increase in the reversal of an ageing phenotype. As described hereinbefore, such reversal may be in the presence of the Lamin A alternative splice form (i.e.
  • the anti-ageing gene/combination may have a strong, direct or weak effect in preventing ageing, or may have a weak, indirect or passive effect in ageing/on the ageing phenotype, respectively.
  • the activity and/or expression of pro- and anti-ageing genes/combinations may therefore be modulated as appropriate to alter/modulate ageing, in particular to reduce and/or slow ageing including the reduction and/or slowing of an ageing phenotype. Progression of an ageing phenotype may also be reduced and/or slowed.
  • the activity and/or expression of pro-ageing genes/combinations can be reduced and/or inhibited.
  • the activity and/or expression of anti- ageing genes/combinations can be increased and/or enhanced to reduce and/or slow ageing.
  • ageing is to be increased and/or accelerated the activity and/or expression of pro-ageing genes/combinations can be increased/enhanced and/or the activity/expression of anti-ageing genes/combinations can be decreased/reduced. Any method, compound or substance known in the art may be used to alter the activity and/or expression of the identified pro- or anti-ageing genes/combinations described herein.
  • small molecule inhibitors or activators for example and without limitation, small molecule inhibitors or activators, antagonists/blocking agents or agonists/trans- activators (e.g. co-factors), post-transcriptional inhibition or enhancement (e.g. RNAi or saRNA).
  • RNAi or saRNA post-transcriptional inhibition or enhancement
  • Further methods for altering the activity and/or expression of genes are known, and include gene editing methods, such as CRISPR (e.g. CRISPR-ko, CRISPRi and/or CRISPRa).
  • CRISPR e.g. CRISPR-ko, CRISPRi and/or CRISPRa
  • activity and/or expression of one gene or set of genes may be increased/enhanced/promoted while the activity/expression of another gene or set of genes is inhibited/reduced.
  • the ageing phenotype measured in the identification method is the same ageing phenotype as chronically displayed by the cell.
  • the ageing phenotype measured is the transcriptomic clock of the cell, optionally in combination with one or more further biological clock as described hereinbefore.
  • the ageing phenotype measured is the morphology of the cell, optionally in combination with the transcriptomic clock as described hereinbefore.
  • the loss-of-function, inhibitory, knock-out, gain-of-function, activatory or combinatorial screen comprises nucleic acids, polypeptides (e.g. proteins, such as antibodies), aptamers and/or small molecules (e.g. small molecule compounds).
  • nucleic acids, polypeptides, aptamers and small molecules include those which act as activators/agonists as well as those which act as inhibitors/antagonists.
  • the methods described herein find utility in screening any modulator (e.g. CLO-C-P3598PCT modulators of expression or activity) of a gene or a combination of genes involved in the reversal of an ageing phenotype or in the maintenance of a non-aged phenotype in a cell.
  • the loss-of-function, inhibitory, knock-out or combinatorial screen comprises a whole-genome screen.
  • Such whole-genome screens also known as genome- wide screens, aim to elucidate the relationship between genotype and phenotype by altering the expression or activity of a gene or gene product on a genome-wide scale and studying the resulting phenotypic alterations, such as in steps (ii) and (iii) of the method described herein.
  • loss-of-function, inhibitory or knock-out screens perturb the expression of genes, preventing them from functioning as normal.
  • a gene or combination of genes will be identified as being involved in the reversal of an ageing-phenotype when said ageing- phenotype persists for longer or is not reversed in an artificially aged cell following induction of ageing compared to an artificially aged cell in which no loss-of-function, inhibition or knock- out of the gene or combination of genes has occurred.
  • a gene or combination of genes will be identified as being involved in the maintenance of a non-aged phenotype when said phenotype is lost in an artificially aged cell, i.e.
  • the cell displays an aged-phenotype, following induction of ageing compared to an artificially aged cell in which no loss-of-function, inhibition or knock- out of the gene or combination of genes has occurred.
  • Gain-of-function screens enhance the activity or expression of a gene or gene product. If gain-of-function screens are used, a gene or combination of genes may be identified as being involved in the reversal of an ageing- phenotype when said ageing-phenotype is reversed more quickly or persists for a shorter period of time in an artificially aged cell following induction of ageing compared to an artificially aged cell in which no gain-of-function of the gene or combination of genes has occurred.
  • a gene or combination of genes may be identified as being involved in the maintenance of a non-aged phenotype when said phenotype persists in an artificially aged cell, i.e. the cell does not display an aged phenotype, following induction of ageing compared to an artificially aged cell in which no gain-of-function of the gene or combination of genes has occurred.
  • a combinatorial screen comprises a combination of one or more of loss-of-function, inhibitory and knock-out as described herein. Such combinatorial screens allow the accurate identification of combinations of genes involved in the reversal of an ageing phenotype or maintenance of a non-aged phenotype.
  • a combinatorial screen may also comprise a combination of a loss-of-function/inhibitory and an activatory/gain-of-function screen, e.g. by combining CRISPR-ko or CRISPRi with CRISPRa.
  • CLO-C-P3598PCT In a specific embodiment, a CRISPR screen is combined with single-cell RNA sequencing to read out an mRNA signature that captures ageing or age reversal.
  • CROP-seq which reads out the sgRNA directly (Datlinger et al. (2017) Nat. Methods, 14(3):297-301, doi: seq (Dixit et al.
  • the loss- of-function, inhibitory, knock-out, activatory or combinatorial screen is performed using CRISPR.
  • the CRISPR is CRISPRi.
  • CRISPRi can sterically repress transcription by blocking either transcriptional initiation or elongation. This is accomplished by designing single guide RNA (sgRNA) complementary to the promoter, the transcriptional start site (TSS) or exonic sequences of the gene to be transcriptionally inhibited or repressed. Depending on the nature of the CRISPR effector, targeting either the template or non-template strand leads to stronger repression.
  • sgRNA single guide RNA
  • TSS transcriptional start site
  • dCas9 repression is stronger when the guide RNA (gRNA) is complementary to the non-template strand.
  • gRNA guide RNA
  • inhibition/repression by CRISPRi is independent of the targeted DNA strand when targeting the transcriptional start site.
  • the dCas9 may be fused to a transcriptional repressor, such as a KRAB domain.
  • KRAB domains have been used in the published literature (Alerasool et al. (2020) Nat. Methods, 17(11):1093-1096, doi: https://doi.org/10.1038/s41592-020-0966-x).
  • the CRISPR is CRISPR-ko and uses the active Cas9 nuclease.
  • CRISPR-ko involves the introduction of a DNA double strand break (DSB) at the target site and the subsequent insertion or deletion of bases upon repair of DSBs created by the Cas9 nuclease in genes to which it has been targeted by a gRNA.
  • DSB DNA double strand break
  • NHEJ imprecise non-homologous end joining repair
  • Gain-of-function screens may use CRISPR activation.
  • CRISPR activation uses modified versions of CRISPR effectors which do not have endonuclease activity but comprise added transcriptional activators, such as VP64 or VPR, on dCas9 and/or the gRNAs.
  • the transcriptional activators fused to the CRISPRa components therefore increase expression of CLO-C-P3598PCT genes of interest following targeting to the gene by the gRNA.
  • the screen is a combinatorial screen
  • the CRISPR is a combination of: CRISPRi, and CRISPR-ko, i.e. a combination of two or more of any of the above mentioned CRISPR techniques.
  • Cas9 protein may be used in the CRISPR methods.
  • Cas9 protein is introduced into the iPSC, stem cell or somatic cell using an expression cassette comprising a sequence coding for the Cas9 protein.
  • the expression cassette may be as described hereinbefore, and when other expression cassettes are used for integration of the ageing-inducing factor and/or the transcriptional regulator, may be referred to as a third expression cassette.
  • the Cas9 coding sequence is operably linked to a ubiquitous promoter.
  • the promoter is the CAG promoter as described hereinbefore.
  • the expression cassette is integrated into the genome of the iPSC or stem cell at a GSH site, examples of which are described herein.
  • the expression cassette comprising the Cas9 coding sequence is integrated at a third GSH site.
  • the use of the term “third” is not intended to be limiting but merely to distinguish the GSH site for integration of the Cas9 coding expression cassette from the GSH sites optionally used for integration of the Lamin A alternative splice form coding expression cassette and/or the transcriptional regulator coding expression cassette, as well as to distinguish from said expression cassettes.
  • the third GSH is the CYBL locus.
  • the expression cassette comprising the Cas9 coding sequence is integrated into the CYBL locus.
  • the use to identify genes/combinations of genes involved in ageing comprises forward programming or directed differentiation of the iPSC or stem cell to a somatic cell.
  • Such forward programming or directed differentiation may be performed as described hereinbefore, e.g. using lineage-specific transcription factors.
  • said forward programming or directed differentiation is performed before identification of the gene or combination of genes.
  • forward programming or directed differentiation is performed after identification.
  • forward programming or directed differentiation may be performed in culturing step (i) and/or may be performed after the screen of step (ii). Additionally or alternatively, forward programming or directed differentiation can be performed concurrently with measuring step (iii).
  • the identification of the gene/combination of genes comprises the steps of: CLO-C-P3598PCT (i) culturing the iPSC or stem cell as described herein and forward programming or differentiating said iPSC or stem cell to a somatic cell; (ii) performing a a loss-of-function, inhibitory, knock-out, activatory, gain-of-function or combinatorial screen in the cell; (iii) measuring an ageing phenotype in the cell; and (iv) identifying a gene or combination of genes as being involved in the reversal of an ageing phenotype or maintenance of a non-aged phenotype when the ageing phenotype is altered when the loss-of-function, inhibitory, knock-out, activatory, gain-of-function or combinatorial screen is performed.
  • identification comprises the steps of: (i) culturing the iPSC or stem cell as described herein; (ii) performing a loss-of-function, inhibitory, knock-out, activatory, gain-of-function or combinatorial screen in the cell, followed by forward programming or differentiating the iPSC or stem cell to a somatic cell; (iii) measuring an ageing phenotype in the cell; and (iv) identifying a gene or combination of genes as being involved in the reversal of an ageing phenotype or maintenance of a non-aged phenotype when the ageing phenotype is altered when the loss-of-function, inhibitory, knock-out, activatory, gain-of-function or combinatorial screen is performed.
  • identification comprises the steps of: (i) culturing the iPSC or stem cell as described herein; (ii) performing a loss-of-function, inhibitory, knock-out, activatory, gain-of-function or combinatorial screen in the cell; (iii) forward programming or differentiating the iPSC or stem cell to a somatic cell and measuring an ageing phenotype in the cell; and (iv) identifying a gene or combination of genes as being involved in the reversal of an ageing phenotype or maintenance of a non-aged phenotype when the ageing phenotype is altered when the loss-of-function, inhibitory, knock-out, activatory, gain-of-function or combinatorial screen is performed.
  • identification comprises the steps of: (i) culturing the iPSC or stem cell as described herein and forward programming or differentiating said iPSC or stem cell to a somatic cell; (ii) performing a loss-of-function, inhibitory, knock-out, activatory, gain-of-function or combinatorial screen in the cell; CLO-C-P3598PCT (iib) introducing into the cell or expressing in the cell a transcription repressor protein, thereby downregulating expression of the Lamin A alternative splice form; (iii) measuring an ageing phenotype in the cell; and (iv) identifying a gene or combination of genes as being involved in the reversal of an ageing phenotype or maintenance of a non-aged phenotype when the ageing phenotype is altered when the loss-of-function, inhibitory, knock-out, activatory, gain-of-function or combinatorial screen is performed.
  • identification comprises the steps of: (i) culturing the iPSC or stem cell as described herein; (ii) performing a loss-of-function, inhibitory, knock-out, activatory, gain-of-function or combinatorial screen in the cell, followed by forward programming or differentiating the iPSC or stem cell to a somatic cell; (iib) introducing into the cell or expressing in the cell a transcription repressor protein, thereby downregulating expression of the Lamin A alternative splice form; (iii) measuring an ageing phenotype in the cell; and (iv) identifying a gene or combination of genes as being involved in the reversal of an ageing phenotype or maintenance of a non-aged phenotype when the ageing phenotype is altered when the loss-of-function, inhibitory, knock-out, activatory, gain-of-function or combinatorial screen is performed.
  • identification comprises the steps of: (i) culturing the iPSC or stem cell as described herein; (ii) performing a loss-of-function, inhibitory, knock-out, activatory, gain-of-function or combinatorial screen in the cell; (iib) introducing into the cell or expressing in the cell a transcription repressor protein, thereby downregulating expression of the Lamin A alternative splice form; (iii) forward programming or differentiating the iPSC or stem cell to a somatic cell and measuring an ageing phenotype in the cell; and (iv) identifying a gene or combination of genes as being involved in the reversal of an ageing phenotype or maintenance of a non-aged phenotype when the ageing phenotype is altered when the loss-of-function, inhibitory, knock-out, activatory, gain-of-function or combinatorial screen is performed.
  • identification is performed in an iPSC, stem cell or a somatic cell derived from an iPSC or a stem cell by forward programming or directed differentiation as described herein.
  • the use of the iPSC, stem cell or somatic cell CLO-C-P3598PCT as described herein is in vitro, in vivo or ex vivo.
  • In vitro includes in culture, such as in an in vitro cell culture of, for example primary cells obtained from a subject or cell lines as may be known in the art. The terms in vivo and ex vivo are used according to their normal meaning and context.
  • identifying a gene or combination of genes is performed in vitro.
  • the iPSC or stem cell is derived without destruction of an embryo, particularly where the cells are human.
  • the iPSCs or stem cells are not derived from human or animal embryos, i.e. the invention does not extend to any methods or uses which involve the destruction of human or animal embryos. Methods of Rejuvenation, Therapeutic Uses & Methods of Treatment
  • a method of rejuvenating a cell comprising altering the expression and/or activity of one or more gene or combination of genes identified by the methods/use described herein.
  • the cell to be rejuvenated is a somatic cell.
  • the cell is a tissue-specific stem cell.
  • references herein to “a” or “the” cell include a single or small number of cells, as well as to a population of cells, which may be large in number.
  • any references herein, including any aspect or embodiment, to singular include plural and vice versa, unless explicitly stated otherwise.
  • the term “rejuvenating” as used herein refers to the reversal or maintenance of an ageing phenotype, such as those described herein.
  • the rejuvenated cell comprises a reduced and/or slowed ageing phenotype, such as reduced and/or slowed progression of the ageing phenotype.
  • Whether a cell is “younger”, “non-aged” or “less-aged” may be determined using any ageing phenotype described herein, in particular using an epigenetic and/or transcriptomic clock.
  • a rejuvenated cell may display fewer alterations in cellular and/or nuclear morphology compared to cell in a non-rejuvenated state. Therefore, in one embodiment the rejuvenated cell comprises a methylation age (e.g. as determined using an epigenetic clock) of younger or less than prior to rejuvenation or than a cell in a non-rejuvenated state (i.e. a non-rejuvenated cell).
  • the rejuvenated cell comprises a transcriptomic age (e.g.
  • the younger or less-aged methylation age corresponds to that of a cell from an CLO-C-P3598PCT earlier point in the life cycle of the tissue or organism from which the iPSC or stem cell was obtained.
  • the younger or less-aged transcriptomic age corresponds to that of a cell from an earlier point in the life cycle of the tissue or organism from which the iPSC or stem cell was obtained.
  • the rejuvenated cell comprises, with respect to an ageing phenotype as described herein, the phenotype of a reprogrammed stem cell, such as a pluripotent stem cell and/or an iPSC.
  • the rejuvenated cell comprises a methylation age or a transcriptomic age corresponding to that of a reprogrammed stem cell, such as a pluripotent stem cell and/or an iPSC.
  • the rejuvenated cell comprises a methylation age or a transcriptomic age of a pluripotent stem cell.
  • the rejuvenated cell comprises a methylation age or a transcriptomic age of an iPSC or a stem cell.
  • the rejuvenated cell comprises a methylation age and a transcriptomic age of pluripotent stem cell. In a still further embodiment, the rejuvenated cell comprises a methylation age and a transcriptomic age of an iPSC or a stem cell. Any method of altering the activity and/or expression of identified genes as described hereinbefore may be used in the rejuvenation methods. In particular, in order to reduce and/or slow ageing, the activity and/or expression of pro-ageing genes/combinations can be reduced and/or inhibited. Alternatively or in addition/combination, the activity and/or expression of anti- ageing genes/combinations can be increased and/or enhanced to reduce and/or slow ageing.
  • genes and combinations of genes identified in iPSCs as pro- or anti- ageing using the methods described herein may have the reverse effects in somatic cells, such as pro-ageing genes identified in iPSCs may have anti-ageing effects in somatic cells and/or anti-ageing genes identified in iPSCs may have pro-ageing effects in somatic cells.
  • pro-ageing genes identified in iPSCs may have anti-ageing effects in somatic cells and/or anti-ageing genes identified in iPSCs may have pro-ageing effects in somatic cells.
  • the activity and/or expression of pro-ageing genes/combinations may be reduced and/or inhibited, while the activity and/or expression of anti-ageing genes/combinations may be increased and/or enhanced to reduce and/or slow ageing in somatic cells.
  • any method, compound or substance known in the art and as described hereinbefore may be used to alter the activity and/or expression of the identified pro- or anti-ageing genes/combinations described herein.
  • the rejuvenation methods and methods of altering the activity and/or expression of the identified genes/combinations of genes as described herein will be appreciated to find utility in the reduction and/or slowing of ageing, such as in an age-modulating method, in particular the slowing and/or reduction of ageing in a subject (e.g. an aged subject).
  • CLO-C-P3598PCT The terms “modulating ageing”, “age-modulating/modulation methods” and “rejuvenation” may therefore be interchangeably and refer to the alteration (e.g.
  • age- modulating methods may maintain a cell in a younger or non-aged state compared to a non- rejuvenated cell, or will alter the age of a cell so that it is younger or in a less-aged state than prior to rejuvenation.
  • Subjects suffering from age-related diseases or disorders will also benefit since it would be expected that by reducing and/or slowing ageing, the disease or disorder, its effects and/or its progression would also be reduced and/or slowed.
  • an age-modulating and/or rejuvenation method for use in a method of treating a disease or disorder associated with ageing in a subject, said age-modulating/rejuvenating method comprising modulating the expression and/or activity of a gene or a combination of genes identified as described herein.
  • said alteration of the activity and/or expression of the genes/combinations of genes identified herein for use in a method of treating a disease or disorder associated with ageing in a subject.
  • the term “in a subject” herein may be used interchangeably with or in addition to “in vivo”.
  • a method of modulating ageing in vivo and/or an in vivo rejuvenation method comprising the age-modulating method described herein (i.e. comprising modulating the activity and/or expression of the gene/combinations of genes identified herein).
  • a method of treating a disease or disorder associated with ageing comprising the age-modulating method described herein (i.e. comprising modulating the activity and/or expression of the gene/combinations of genes identified herein).
  • the in vivo age-modulating/rejuvenation methods and methods of treatment herein may comprise administration of a modulator of the activity and/or expression of a gene or combination of genes identified herein.
  • an inhibitor, an antagonist, a blocking agent and/or negative post-transcriptional regulator of a pro-ageing gene or combination of pro-ageing genes may be administered.
  • An inhibitor may include a small-molecule inhibitor, an siRNA or an antibody targeting the gene product of interest as described hereinbefore.
  • an agonist, a trans-activator and/or a post-transcriptional enhancer of an anti-ageing gene or combination of anti-ageing genes may be administered. Such administration may be systemically, e.g.
  • the modulator of the activity and/or expression of a gene or combination of genes identified herein is comprised in a pharmaceutical composition, optionally further comprising one or more pharmaceutically acceptable carriers, diluents and/or excipients.
  • a pharmaceutical composition comprising the modulator of the activity and/or expression of a gene or combination of genes identified herein for use in the age-modulating/rejuvenation methods and/or in a method of treating a disease or disorder associated with ageing in a subject.
  • the disease or disorder associated with ageing may be a degenerative disease or disorder, including without limitation of the pancreas (e.g. type 2 diabetes), of the blood and/or bone marrow, of the heart (e.g. cardiovascular disease, cardiomyopathy, ischaemic heart disease, cardiac arrhythmia or heart failure), of the skin, or a neurological disease or disorder (e.g. a neurodegenerative disease/disorder).
  • the age-modulating/rejuvenation method is performed in the skin, liver, pancreas, heart, brain, central nervous system, peripheral nervous system, blood and/or bone marrow, such as in a cell comprised in any of said tissues or organs.
  • the age-modulating method can be considered to rejuvenate said tissue, organ and/or cell in which the method is performed.
  • references herein to a patient or subject relate equally to animals and humans and that the invention finds particular utility in veterinary treatment of any of the above mentioned diseases, disorders and conditions which are also present in said animals.
  • references herein to “treatment” include such terms as “amelioration”, “prevention”, “reversal”, “suppression” and the like.
  • references herein to “administration” include such terms as “amelioration”, “prevention”, “reversal”, “suppression” and the like.
  • references include administration of the modulator of the activity and/or expression of a gene or combination of genes as described herein or composition comprising the modulator as defined herein prior to the onset of the disease or disorder. Administration may also be anticipated after the induction event of the disease or disorder, either before clinical presentation of said disease or disorder, or after symptoms manifest.
  • iPSC induced pluripotent stem cell
  • stem cell or a somatic cell derived from an iPSC or stem cell by forward programming or directed differentiation comprising an expression cassette, said expression cassette comprising a coding sequence for an alternative splice form of Lamin A coupled to a weak promoter sequence, thereby yielding low level constitutive expression of the alternative splice form of Lamin A in the cell while maintaining cell viability.
  • progerin comprises the amino acid sequence of SEQ ID NO: 16, or a variant or functional fragment thereof, and/or comprises an amino acid sequence encoded by SEQ ID NO: 17, or a variant or functional fragment thereof.
  • the ageing phenotype is selected from any one or more of: proliferation, senescence, changes in cell and/or nuclear CLO-C-P3598PCT morphology, mitochondrial function, changes in gene expression, altered expression of Lamin- A and/or Lamin-C
  • the inducible promoter comprises a tetracycline operator (TetO) sequence and a constitutive promoter.
  • TetO tetracycline operator
  • CLO-C-P3598PCT 18.
  • the transcriptional regulator protein is a tetracycline-responsive transcriptional activator protein (rtTa) and the exogenously supplied substance is tetracycline or a derivate thereof, such as doxycycline.
  • rtTa tetracycline-responsive transcriptional activator protein
  • iPSC iPSC, stem cell or somatic cell of any one of clauses 1 to 20 in a method of identifying a gene or a combination of genes involved in the reversal of an ageing phenotype or in the maintenance of a non-aged phenotype in the cell. 22.
  • the method of identifying a gene or combination of genes comprises the steps of: (i) culturing the iPSC, stem cell or somatic cell of any one of clauses 1 to 20; (ii) performing a loss-of-function, inhibitory, knock-out, activatory, gain-of-function or combinatorial screen in the cell; (iii) measuring an ageing phenotype in the cell; and (iv) identifying a gene or combination of genes as being involved in the reversal of an ageing phenotype or maintenance of a non-aged phenotype when the ageing phenotype is altered when the loss-of-function, inhibitory, knock-out, activatory, gain-of-function or combinatorial screen is performed.
  • the method of identifying a gene or combination of genes comprises the steps of: (i) culturing the iPSC, stem cell or somatic cell as described herein; (ii) performing a loss-of-function, inhibitory, knock-out, activatory, gain-of-function or combinatorial screen in the cell; (iib) introducing into the cell or expressing in the cell a transcription repressor protein, thereby downregulating expression of the Lamin A alternative splice form; (iii) measuring an ageing phenotype in the cell; and (iv) identifying a gene or combination of genes as being involved in the reversal of an ageing phenotype or maintenance of a non-aged phenotype when the ageing phenotype is CLO-C-P3598PCT altered when the loss-of-function, inhibitory, knock-out, activatory, gain-of-function or combinatorial screen is performed.
  • the method of identifying the gene/combination of genes comprises the steps of: (i) culturing the iPSC or stem cell as described herein and forward programming or differentiating said iPSC or stem cell to a somatic cell; (ii) performing a a loss-of-function, inhibitory, knock-out, activatory, gain-of-function or combinatorial screen in the cell; (iii) measuring an ageing phenotype in the cell; and (iv) identifying a gene or combination of genes as being involved in the reversal of an ageing phenotype or maintenance of a non-aged phenotype when the ageing phenotype is altered when the loss-of-function, inhibitory, knock-out, activatory, gain-of-function or combinatorial screen is performed.
  • the method of identifying the gene/combination of genes comprises the steps of: (i) culturing the iPSC or stem cell as described herein; (ii) performing a loss-of-function, inhibitory, knock-out, activatory, gain-of-function or combinatorial screen in the cell, followed by forward programming or differentiating the iPSC or stem cell to a somatic cell; (iii) measuring an ageing phenotype in the cell; and (iv) identifying a gene or combination of genes as being involved in the reversal of an ageing phenotype or maintenance of a non-aged phenotype when the ageing phenotype is CLO-C-P3598PCT altered when the loss-of-function, inhibitory, knock-out, activatory, gain-of-function or combinatorial screen is performed.
  • the method of identifying the gene/combination of genes comprises the steps of: (i) culturing the iPSC or stem cell as described herein; (ii) performing a loss-of-function, inhibitory, knock-out, activatory, gain-of-function or combinatorial screen in the cell; (iii) forward programming or differentiating the iPSC or stem cell to a somatic cell and measuring an ageing phenotype in the cell; and (iv) identifying a gene or combination of genes as being involved in the reversal of an ageing phenotype or maintenance of a non-aged phenotype when the ageing phenotype is altered when the loss-of-function, inhibitory, knock-out, activatory, gain-of-function or combinatorial screen is performed.
  • the method of identifying the gene/combination of genes comprises the steps of: (i) culturing the iPSC or stem cell as described herein and forward programming or differentiating said iPSC or stem cell to a somatic cell; (ii) performing a loss-of-function, inhibitory, knock-out, activatory, gain-of-function or combinatorial screen in the cell; (iib) introducing into the cell or expressing in the cell a transcription repressor protein, thereby downregulating expression of the Lamin A alternative splice form; (iii) measuring an ageing phenotype in the cell; and (iv) identifying a gene or combination of genes as being involved in the reversal of an ageing phenotype or maintenance of a non-aged phenotype when the ageing phenotype is altered when the loss-of-function, inhibitory, knock-out, activatory, gain-of-function or combinatorial screen is performed.
  • clause 24 or clause 25, wherein the method of identifying the gene/combination of genes comprises the steps of: (i) culturing the iPSC or stem cell as described herein; (ii) performing a loss-of-function, inhibitory, knock-out, activatory, gain-of-function or combinatorial screen in the cell, followed by forward programming or differentiating the iPSC or stem cell to a somatic cell; (iib) introducing into the cell or expressing in the cell a transcription repressor protein, thereby downregulating expression of the Lamin A alternative splice form; CLO-C-P3598PCT (iii) measuring an ageing phenotype in the cell; and (iv) identifying a gene or combination of genes as being involved in the reversal of an ageing phenotype or maintenance of a non-aged phenotype when the ageing phenotype is altered when the loss-of-function, inhibitory, knock-out, activatory, gain-of-function
  • clause 24 or clause 25, wherein the method of identifying the gene/combination of genes comprises the steps of: (i) culturing the iPSC or stem cell as described herein; (ii) performing a loss-of-function, inhibitory, knock-out, activatory, gain-of-function or combinatorial screen in the cell; (iib) introducing into the cell or expressing in the cell a transcription repressor protein, thereby downregulating expression of the Lamin A alternative splice form; (iii) forward programming or differentiating the iPSC or stem cell to a somatic cell and measuring an ageing phenotype in the cell; and (iv) identifying a gene or combination of genes as being involved in the reversal of an ageing phenotype or maintenance of a non-aged phenotype when the ageing phenotype is altered when the loss-of-function, inhibitory, knock-out, activatory, gain-of-function or combinatorial screen is performed.
  • An age-modulating method for use in a method of treating a disease or disorder associated with ageing in a subject comprising modulating the expression and/or activity of a gene or a combination of genes identified by the use of any one of clauses 21 to 32.
  • CLO-C-P3598PCT 36. A method of modulating ageing in vivo, said method comprising the age-modulating method of clause 35. 37.
  • a method of treating a disease or disorder associated with ageing said method comprising the age-modulating method of clause 35. 38.
  • the method of clause 36 or clause 37, wherein the method comprises administering a modulator of the expression and/or activity of the gene or combination of genes identified by the use of any one of clauses 21 to 32.
  • Ageing of hiPSCs expressing progerin under control of the tetracycline system was introduced on day 2 post splitting for 5 days using 0.075 ⁇ g/mL doxycycline.
  • Generation of iNeurons from hiPSCs NGN2-iPSCs and NGN2-GFP-Progerin-iPSCs were dissociated into single cells using Accutase and plated onto Geltrex coated dishes at a density of 50,000 cells per cm 2 . Forward programming was initiated 24 hours post splitting.
  • the induction was performed in DMEM/F- 12 supplemented with Glutamax (100x), Non-Essential Amino Acids (100x), 50 ⁇ M 2- Mercaptoethanol (Gibco), 1% Penicillin/Streptomycin, 1 ⁇ g/mL Doxycycline.
  • the medium was switched to Neurobasal-medium supplemented with Glutamax (100x), B27 (50x), 10ng/mL BDNF (Peprotech), 10ng/mL NT3 (R&D Systems), 1% Penicillin/Streptomycin, and 1 ⁇ g/mL Doxycycline.
  • CLO-C-P3598PCT Multi-Electrode Array (MEA) Recordings Non-aged iNs (NGN2 iNs), and chronically aged iNs (N2P caiNs) were dissociated into single cells at day 3 post induction and replated on PDL/Geltrex coated wells of CytoView MEA 48 (Axion Biosystems) with or without rat cortical astrocytes, at a ratio of 1:1.
  • Co-cultures were maintained in Neurobasal medium for the duration of the recordings. Recordings were performed using the Maestro Pro MEA system (Axion Biosystems).
  • Dose Response and Proliferation Assays GFP-Progerin targeted iPSCs were dissociated into single cells using Accutase and plated at a density of 10,000 cells per cm 2 in StemFlex medium. For dose response assay, cells were incubated each day with Hoechst (Thermo Fisher Scientific) for 10 minutes and imaged using Evos M5000 microscope system (Thermo Fisher Scientific). For proliferation assay, cells were dissociated each day and counted using Countess II (Thermo Fisher Scientific). Image analysis and cell counting was conducted using Image J.
  • Samples were prepared for Western Blot analysis by adding Laemmli buffer (final concentration of 30mM Tris-HCl pH 6.8, 6% glycerol, 2% sodium dodecyl sulphate/SDS, 0.02% bromophenol blue, and 0.25% ⁇ -mercaptoethanol), and were denatured at 95 o C for 5 minutes. 20 ⁇ g of proteins were loaded and run on 4-12% NuPAGE Bis-Tris Precast Gels (Invitrogen), then transferred to polyvinylidene fluoride (PVDF) membranes by liquid transfer using NuPAGE Transfer buffer (Invitrogen).
  • Laemmli buffer final concentration of 30mM Tris-HCl pH 6.8, 6% glycerol, 2% sodium dodecyl sulphate/SDS, 0.02% bromophenol blue, and 0.25% ⁇ -mercaptoethanol
  • Membranes were blocked for 1h at RT in PBS 0.05% Tween-20 (PBST) supplemented with 4% non-fat dried milk and incubated overnight at 4 o C on a rotating wheel with the primary antibody diluted in the same blocking buffer. After three washes in PBST, membranes were incubated for 1h at RT with horseradish peroxidase (HRP)-conjugated secondary antibodies diluted in blocking buffer, then further washed three times with PBST before being incubated with Pierce ECL2 Western Blot Substrate (Thermo Fisher) and exposed to Amersham HyperfilmTM (GE Healthcare). Total protein was measured using Licor REVERT total protein stain and wash solution kit.
  • HRP horseradish peroxidase
  • CLO-C-P3598PCT Mitochondrial Assays Measurement of cellular oxygen consumption rate (OCR): Cellular oxygen consumption rate was measured using a XF24 extracellular flux analyser (Seahorse Biosciences, Denmark). Human iPSCs at a density of 2,000 cells/well or human iNeurons (aged and non- aged) at a density of 30,000 cells/well were seeded in XFp miniplates (Agilent Technologies) and were cultured under the conditions described above for ageing or non-ageing conditions. Cells were incubated in assay medium (DMEM without sodium bicarbonate and phenol red) for at least 30 min prior to the assay.
  • assay medium DMEM without sodium bicarbonate and phenol red
  • OCR was measured every 5 minutes for three times under basal conditions and then after the sequential injection of 0.5 ⁇ M oligomycin (inhibitor of ATP synthase), 8.1 ⁇ M carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP; uncoupler of mitochondrial inner membrane allowing maximum electron flux through the ETC), and 10 ⁇ M rotenone + 10 ⁇ M antimycin A (inhibitors of mitochondrial complex I and III, respectively). At least 3 biological replicates were performed, with at least 3 technical replicates per sample. Basal respiration was measured by subtracting non-mitochondrial respiration from the last rate measurement before Oligomycin injection.
  • Cells were washed twice with FluoroBrite DMEM before being scanned. Cells were stained with Hoechst for cell density normalization, according to the manufacturer’s instructions. Fluorescence was measured using the CLARIOstar microplate reader at 510/610 nm and 644/665 nm, and the DAPI filter at 340/480nm. At least 3 biological replicates were performed, with at least 3 technical replicates per sample. Data analysis was performed by division of the dye signal by Hoechst signal of each well, to normalise to cell count. Afterwards, the dye signal/DAPI ratio of all samples was divided by the average of the untreated group, to normalise samples against it. Data were exported to GraphPad Prism and plotted.
  • Autophagy Assay Autophagic activity of iPSCs was assessed on day 5 of ageing. Briefly, hiPSCs were plated on 96 well plates at a density of 2,000 cells/well and were cultured under the conditions CLO-C-P3598PCT described above for ageing or non-ageing conditions. Live cells were stained using the Autophagy assay kit (ab139484, Abcam) to quantify autophagic vacuoles and monitor autophagic flux. The dye used leads to minimal staining of lysosomes, and bright fluorescence upon incorporation into pre-autophagosomes, autophagosomes, and autolysosomes (autophagolysosomes).
  • Cells were stained with Hoechst for cell density normalization, according to the manufacturer’s instructions. Fluorescence was measured using the CLARIOstar microplate reader with the FITC filter (480/530nm), and the DAPI filter (340/480nm). At least 3 biological replicates were performed, with at least 3 technical replicates per sample. Data analysis was performed by division of FITC signal by Hoechst signal of each well, to normalise to cell count. Afterwards, the FITC/DAPI ratio of all samples was divided by the average FITC/DAPI of the untreated group, to normalise samples against it. Data were exported to GraphPad Prism and plotted.
  • SA- ⁇ -gal Staining SA- ⁇ -gal staining was performed using a Cell Signaling Technology kit according to the manufacturer’s protocol. Colour images of SA- ⁇ -gal- stained cells were captured using an Evos microscope (Life Technologies). SA- ⁇ -gal intensity was measured using ImageJ software. Immunocytochemistry Cells were fixed in 4% paraformaldehyde (Alfa Aesar) for 15 minutes at room temperature and subsequently washed three times with PBS. The cells were then permeabilized with 0.1% Triton-X-100 for 15 minutes at room temperature.
  • DNA damage foci analysis (based on ⁇ H2AX staining) was conducted using Image J (version 2.00, NIH). Number of ⁇ H2Ax foci was calculated manually and was plotted into a bar graph. H3K9me3 intensity was measured by quantifying cellular fluorescence intensity. Images were processed using ImageJ software. Corrected total cell fluorescence (CTCF) was assessed from cells for each of the tested markers and presented in bar graphs.
  • CTCF Corrected total cell fluorescence
  • Quantitative Real-Time PCR cDNA synthesis was performed with the Maxima First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). PowerUp SYBR Green Master Mix (Thermo Fisher Scientific) was used for RT-PCR. Samples were run on the QuantStudio 6 Flex Real-Timer PCR System machine (Applied Biosystems). All samples were analysed in technical duplicates or triplicates and normalized to the house-keeping gene Porphobilinogen Deaminase 1 (PBGD1). Results were analysed with the ⁇ Ct method. See Table 2 for a full list of primer sequences. Table 2: List of Primers Used for qRT-PCR Analysis.
  • telomere length measurement was based on previously published protocol with some Front. Physiol., 13:1007418, doi: https://doi.org/ .
  • qPCR was performed in 20 ⁇ L final volume reactions including 12ng per reaction genomic DNA.
  • Reaction mixture consisted of Fast SYBR Green master mix (Thermo Fisher Scientific), 300nM of primer Tel forward (CGGTTTGTTTGGGTTTGGGTTTGGGTTTGGGTTTGGGTT (SEQ ID NO: 32)), 900nM of primer Tel reverse (GGCTTGCCTTACCCTTACCCTTACCCTT ACCCTTACCCT (SEQ ID NO: 33)) or 500nM of primer 36B4 single gene forward (CAGCAA GTGGGAAGGTGTAATCC (SEQ ID NO: 34)) and 500nM of primer 36B4 single gene reverse (CCCATTCTATCATCAACGGGTACAA (SEQ ID NO: 35)).
  • telomere length was calculated as telomere to single copy gene ratio (T/S ratio) and based on the calculation of the ⁇ Ct [Ct (telomeres)/Ct (single gene)].
  • Bulk RNA Sequencing Prior to differential expression analysis, raw reads were evaluated for quality check in terms of sequencing quality and contamination. Paired-end bulk RNA-seq data were obtained in FASTQ format generated from GFP-Progerin aged and non-aged iPSCs samples, 3 biological replicates each. Reads initial quality assessment was done by using the FQSTQC and MultiQC tools. Adapter sequences and low-quality bases from reads were trimmed using Trimmomatic (v.0.39).
  • Trimmed reads were mapped to the Homo sapiens genome (GRCh38.105 assembly) using STAR (2.7.10b) mapper. With uniquely mapped read, read counts for features (exons) were generated using the featureCounts function from the Subread package (v 1.6.1) using the Ensembl Known Gene models (version GRCh38.105) as reference annotations.
  • CLO-C-P3598PCT The differentially expressed genes (DEGs) (at P value (FDR) ⁇ 0.05) obtained by “Aged” and “Non-aged” control from GFP-Progerin iPSCs. Next, genes involved in the pathways of interest were obtained from the KEGG pathways database.
  • Example 2 Phenotypic Changes Associated with Ageing in hiPSCs Overexpressing Progerin Using the TetOn System Multiple ageing phenotypes were assessed in the hiPSCs which had been force-aged by progerin overexpression under the control of doxycycline (Example 1).
  • the reprogramming cassette included both NGN2 under doxycycline control, and a constitutive GFP-progerin expression under the cumate operator (CuO).
  • This new system allowed: 1) to remove the necessity/dependency of the system in the cumate molecule; 2) a constitutive expression of progerin to force age the cells; 3) to allow a stable and a low expression of progerin due to a weak promoter; and 4) to prevent cell death due to progerin toxicity.
  • This new line of aged iPSCs/iNs was referred to as chronically aged.
  • CLO-C-P3598PCT To assess whether the viability of the resulting aged hiPSCs were affected by the constitutive overexpression of progerin, a proliferation assay was conducted.
  • the aged iNs showed to downregulate pluripotency markers (OCT4 and NANOG) compared to iPSCs, while upregulating neuronal markers such as the dendritic marker MAP2 and the pre- and post- synaptic markers Synapsin-1 and PSD95, respectively (Figure 14). Additionally, both ICC staining and qRT-PCR analysis confirmed expression of GFP in the aged cells ( Figures 15A- B). Although, the aged iNs developed neuronal network similarly as was shown before for NGN2-iNs, the dendritic architecture was shown to be less complex with thinner processes and less neurite bundles (Figures 15C-D).
  • the energetic profile of the aged and non-aged iNeurons was assessed by measuring mitochondrial respiration and glycolysis under baseline and stressed conditions (Oligomycin + FCCP injections) (Figure 17D).
  • non-aged iNeurons exhibited a more energetic and aerobic baseline metabolic profile.
  • the aged cells displayed a comparatively quiescent metabolism.
  • both non-aged and aged iNeurons exhibited an upregulation of the glycolytic pathway, potentially compensating for a decline in mitochondrial respiration.
  • Example 4 Epigenetic Alterations in caiNs
  • Epigenetic alterations were assessed using established epigenetic clock models, including the DNAmAge prediction clock developed by Steve Horvath (Horvath 2013 Genome Biol 14, 3156, doi: https://doi.org/10.1186/gb-2013-14-10-r115).
  • the analysis revealed an increase in the predicted biological age of caiNs compared to non-aged iNs ( Figure 18).
  • the level of methylated CpG sites was analyzed to measure Shannon entropy. This analysis demonstrated higher entropy in caiNs relative to non-aged iNs, indicating that caiNs exhibit a more advanced biological age (Figure 19).

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Abstract

La présente invention concerne une cellule souche pluripotente induite (iPSC) ou une cellule somatique issue d'une iPSC par programmation sens ou différenciation dirigée exprimant de manière constitutive une forme d'épissage alternative de Lamine A. Les cellules comportent une cassette d'expression comportant une séquence de codage pour la forme d'épissage alternative de Lamine A liée de manière fonctionnelle à une séquence d'opérateur de cumate (CuO) et à un promoteur constitutif. La présente invention concerne également l'utilisation des cellules selon l'invention dans le cadre d'un procédé d'identification d'un gène ou d'une combinaison de gènes intervenant dans l'inversion d'un phénotype de vieillissement ou dans le maintien d'un phénotype de non-vieillissement. La présente invention concerne également des procédés de rajeunissement des cellules somatiques, de traitement des maladies ou des troubles associés au vieillissement et de modulation du vieillissement (par exemple, des procédés et des utilisations de modulation du vieillissement), y compris in vivo, ledit procédé comportant la modulation de l'expression et/ou de l'activité du gène/de la combinaison de gènes identifié(e) selon la présente invention.
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