OF A POPULATION OF MAMMALIAN STEM CELLS AFTER GENOME EDITING

The present invention relates to a method of reducing the tumorigenic potential of a population of mammalian stem cells, particularly induced pluripotent stem cells (iPSCs), which have undergone genome-editing using a CRISPR-based editing system. The editing step is carried out in the presence of a p53 inhibitor, e.g. pifithrin-a.

Induced pluripotent stem cells (also known iPSCs) are a type of pluripotent stem cell that can be generated directly from adult cells. The iPSC technology was pioneered by Shinya Yamanaka’s lab in Kyoto, Japan, who showed in 2006 that the introduction of four specific genes (named Myc, Oct3/4, Sox2 and Klf-4) encoding transcription factors could convert adult cells into pluripotent stem cells (Takahashi K, Yamanaka S (August 2006), "Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors", Cell. 126 (4): 663-76).

Pluripotent stem cells hold promise in the field of regenerative medicine because they can propagate indefinitely, as well as give rise to every other cell type in the body (such as neurons, heart, pancreatic and liver cells). Consequently, they represent a single source of cells that can be used to replace cells which have been lost due to damage or disease.

The development of CRISPR/Cas9 nucleases has enormously expanded our ability to engineer genetic changes in human cells. However, while immortalized human tumour cell lines have been edited with almost complete efficiency, gene-editing in human stem cells has proved more challenging. Although tumour cell lines are favoured for their robustness, they also possess unchecked proliferation, gross mutations and abnormal responses to DNA damage, and hence cannot be used in a therapeutic context. Genome engineering in human induced pluripotent stem cells (hiPSCs) is therefore desired for developing scientific disease models and for potential gene therapies. Their self-renewing capability allows hiPSCs to be gene-targeted, cloned, genotyped, and expanded, unlike primary tissues with limited growth potential. Edited pluripotent clones can then be differentiated into a variety of other cell types to analyse the phenotypic effect of the engineered mutations or employ reporter constructs.

However, genome-editing methods such as CRISPR/Cas9 involve the transient production of double-stranded breaks in the genome to be edited and such breaks are known to trigger apoptotic pathways though activation of p53. Consequently, the use of such methods on a population of iPSCs often results in a significant reduction in the number of cells in that population due to apoptosis. This has previously not been considered to be a problem due to the fact that clonal populations from each edited iPSCs can subsequently be established to provide adequate number of cells.

The inventors have now discovered, however, a significant detrimental issue regarding the iPSCs which survive (i.e. which are not lost due to apoptosis) in such genomeediting methods. The inventors have discovered that such cells have significantly- reduced levels of p53 polypeptide. In particular, the genome-editing process and the apoptosis which is induced thereby detrimentally select for cells which express significantly-reduced levels of p53 polypeptide.

The gene encoding p53 (TP53) is the most frequently-mutated gene (>50%) in human cancer, indicating that the TP53 gene plays a crucial role in preventing cancer formation. The p53 polypeptide binds to DNA and regulates gene expression to prevent mutations of the genome. As such, the p53 polypeptide plays a crucial role in multicellular vertebrates, where it prevents cancer formation, and thus acts as a tumour suppressor.

The inventors have now recognised that the fact that iPSCs which survive genomeediting have significantly-reduced levels of p53 polypeptide means that such cells will have an enhanced tumorigenic potential; this is clearly undesirable. The inventors have discovered that this issue may be overcome by using a p53 inhibitor transiently during and/or following the genome-editing step. The p53 inhibitor prevents the apoptotic cascade which is usually initiated by the double-stranded breaks which are made in the genomic DNA during the genome-editing. Hence the cells in the population of genome-edited cells which express wild-type or high levels of p53 do not become depleted, thus ensuring that such cells do not have an enhanced tumorigenic potential. Such cells may therefore be used with an enhanced degree of safety for therapeutic applications, including in-human applications.

Whilst p53 inhibitors have previously been used in the context of genome editing of hiPSCs (e.g. WO2017/184674), these inhibitors were used in an attempt to promote gene targeting. Furthermore, pifithrin-a was said in WO2017/184674 (see Example 4 and Figures 9A-9B) to "not enhance hiPSC gene targeting".

Additionally, pifithrin-a has been said to have "no or only a very minor inhibitory effect on etoposide-induced hES apoptosis" (Grandela et al., Stem Cell Research (2008) 1 , 116-128).

It is an object of the invention therefore to provide a method of reducing the tumorigenic potential of a population of mammalian stem cells which have undergone genome editing.

In one embodiment, the invention provides a method of reducing the tumorigenic potential of a population of mammalian stem cells which have undergone genomeediting, the method comprising:

(a) an editing step which comprises editing the genomes of a population of mammalian stem cells using:

(i) a CRISPR enzyme,

(ii) a CRISPR gRNA, and

(iii) optionally, a double-stranded or single-stranded donor DNA, wherein the editing step involves the production of double-stranded breaks or pairs of staggered single-stranded breaks in the genomes of the population of mammalian stem cells, wherein the editing step is carried out in the presence of a p53 inhibitor, and optionally;

(b) isolating and/or selecting a subpopulation of cells from the population of mammalian stem cells, wherein the subpopulation of cells comprises or consists of cells which have the desired edit in their genomes.

In another embodiment, the invention provides a method of producing mammalian stem cells which have a low tumorigenic potential following a genome-editing step, the method comprising Step (a) and optionally Step (b) as defined herein.

In another embodiment, the invention provides a method of maintaining p53 polypeptide expression levels in a population of mammalian stem cells following a genome-editing step, the method comprising Step (a) and optionally Step (b) as defined herein.

In another embodiment, the invention provides a method of editing the genomes of a population of mammalian stem cells, the method comprising Step (a) and optionally Step (b) as defined herein.

The invention also provides the use of a p53 inhibitor in the genome-editing of a population of mammalian stem cells to increase the proportion of cells which have substantially wild-type levels of expression of p53 polypeptide following the genomeediting method.

In one embodiment, the invention relates to a method of reducing the tumorigenic potential of a population of mammalian stem cells which have undergone genome editing.

As used herein, the terms "cells" and "stem cells" refer to the mammalian stem cells. Preferably, the mammalian stem cells are human, mouse, rat, monkey, pig, cow, sheep, horse or goat stem cells. Most preferably, the stem cells are human stem cells. Examples of suitable stem cells include embryonic stem cells, foetal stem cells, pluripotent stem cells, adult stem cells and iPS cells. In some preferred embodiments, the stem cells are iPS cells.

The term “iPS” cells refers to “induced pluripotent stem” cells. Induced pluripotent stem cells (also known iPSCs) are a type of pluripotent stem cell that can be generated directly from adult cells. The iPSC technology was pioneered by Shinya Yamanaka’s lab in Kyoto, Japan, who showed in 2006 that the introduction of four specific genes (named Myc, Oct3/4, Sox2 and Klf-4) encoding transcription factors could convert adult cells into pluripotent stem cells (Takahashi K, Yamanaka S (August 2006), "Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors", Cell. 126 (4): 663-76). Most preferably, the iPS cells are human iPS cells. The iPS cells may be derived or obtained from any suitable cells including any somatic cell type. In one embodiment, the iPS cells are derived from umbilical cord blood cells. More preferably, the iPS cells are derived from umbilical cord blood CD34 ⁺ progenitor cells. In another embodiment, the iPS cells are derived from peripheral blood mononuclear cells (PBMCs) or from fibroblasts, preferably from healthy donors. In some embodiments, the iPS cells are derived from a disease-specific cell line.

Preferably, the iPS cells are a homogeneous population or a population of substantially homogeneous iPS cells. In some embodiments, the population consists of or consists substantially of a population of the desired iPS cells. More preferably, at least 70%, 80%, 90% or 95% of the population of cells are iPS cells.

In some embodiments, the stem cells (e.g. iPS cells) are nerve stem cells, brain stem cells or cardiac stem cells. In other embodiments, the stem cells are haematopoietic or mesenchymal stem cells. iPS cells are typically derived by introducing products of specific sets of pluripotency- associated genes, or "reprogramming factors", into a given cell type. The original set of reprogramming factors (also dubbed "Yamanaka factors") are the transcription factors Oct4 (Pou5f1), Sox2, cMyc, and Klf4. While this combination is most conventional in producing iPSs, each of the factors can be functionally replaced by related transcription factors, miRNAs, small molecules, or even non-related genes such as lineage specifiers (Guo XL, Chen JS (2015), "Research on induced pluripotent stem cells and the application in ocular tissues", International Journal of Ophthalmology. 8 (4): 818-25).

Methods of producing iPS cells are now well known the art (e.g. Shi Y, Inoue H, Wu JC, Yamanaka S. “Induced pluripotent stem cell technology: a decade of progress”. Nat.

Rev. Drug Discov. 2017 Feb;16(2):115-130. doi: 10.1038/nrd.2016.245. Epub 2016 Dec 16; Sayed N, Liu C, Wu JC. “Translation of Human-Induced Pluripotent Stem Cells: From Clinical Trial in a Dish to Precision Medicine”. J. Am. Coll. Cardiol. 2016 May 10;67(18):2161-2176. doi: 10.1016/j.jacc.2O16.01.083; and Matsa E, Burridge PW, Wu JC. “Human stem cells for modeling heart disease and for drug discovery”, _Sci. Transl. Med. 2014 Jun 4;6(239):239ps6. doi: 10.1126/scitranslmed.3008921).

In one preferred method, cord blood-derived CD34+ progenitor cells are reprogrammed using OCT4, Sox2, Myc, Klf4, Nanog, SV40LT and Lin28 antigen. Such cells may be purchased from Gibco® Life Technology (Carlsbad, USA). iPS cell lines may be characterized by numerous methods including immuno-staining, qPCR, tri-lineage differentiation, teratoma assay and/or karyotyping.

In some embodiments, the cells are ones which do not have a CRISPR enzyme (e.g. a Cas9 gene) integrated into the genomes of the cells.

In some embodiments, the cells are ones which do not have a recombinant gene encoding a p53 inhibitor integrated into the genomes of the cells.

Step (a) of the method of the invention relates to (a) an editing step which comprises editing the genomes of a population of mammalian stem cells using:

(i) a CRISPR enzyme,

(ii) a CRISPR gRNA, and

(iii) optionally, a double or single-stranded donor DNA, wherein the editing step involves the production of double-stranded breaks or pairs of staggered single-stranded breaks in the genomes of the population of mammalian stem cells.

CRISPR is an acronym for Clustered, Regularly Interspaced, Short, Palindromic Repeats. The CRISPR enzyme is one which has endonuclease activity and which is capable of producing double-stranded breaks or single-stranded breaks in the genomes of the population of mammalian stem cells. In some embodiments, the CRISPR enzyme is a Type II CRISPR system enzyme. In some embodiments, the CRISPR enzyme is Cas9 or a Cas9-like polypeptide. In some embodiments, the Cas9 enzyme is obtained from S. pneumoniae, S. pyogenes, or S. thermophilus Cas9, or a variant thereof. In some embodiments, the CRISPR enzyme is codon-optimized for expression in a mammalian cell.

Examples of CRISPR enzymes which may be used in this regard include SpCas9, FnCas9, St1 Cas9, St3Cas9, NmCas9, SaCas9, AsCpfl , LbCpfl , VQR SpCas9, EQR SpCas9, VRER SpCas9, RHA FnCas9 and KKH SaCas9 (see Komor et al., CRISPR- Based Technologies for the Manipulation of Eukaryotic Genomes, Cell (2017), http://dx.doi.Org/10.1016/j.cell.2016.10.044).

The term "CRISPR gRNA” refers to a guide RNA suitable for use with CRISPR enzymes. Preferably, the gRNA is a sgRNA, i.e. a single-guide RNA. A sgRNA is a chimeric RNA which replaces the crRNA/tracrRNA which are used in the native CRISPR/Cas systems (e.g. Jinek, M. et al. (2012), “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity”, Science 337, 816-821). The term sgRNA is well accepted in the art.

One or more CRISPR gRNAs may be used in the method of the invention (e.g. 1 , 2, 3, or 4, preferably 1 or 2).

The sgRNA comprises a spacer element. The spacer element is also known as a spacer segment or guide sequence. The terms spacer element, spacer segment and guide sequence are used interchangeably. The sgRNA comprises a region which is capable of forming a complex with a CRISPR enzyme, e.g. a CRISPR endonuclease, e.g. Cas9. The sgRNA comprises, from 5' to 3', a spacer element which is programmable (i.e. the sequence may be changed to target a complementary DNA target), followed by the sgRNA scaffold. The sgRNA scaffold may technically be divided further into modules whose names and coordinates are well known in the art (e.g. Briner, A. E. et al. (2014). “Guide RNA functional modules direct cas9 activity and orthogonality”. Molecular Cell, 56(2), 333-339). The spacer element is a stretch of contiguous ribonucleotides whose sequence is fully or partially complementary to the target locus in the genomes of the mammalian cells.

In embodiments of the invention in which it is desired to edit the genomes by insertion of a stretch of DNA (i.e. by homology-directed repair with a donor template), a donor dsDNA is also used during the editing step. The ends of the donor dsDNA will have sequence homology to the target locus in the cell genome in order to promote homology-directed repair and the insertion of the donor dsDNA into the genomes of the mammalian stem cells. In other embodiments, a single-stranded donor DNA may be used instead. As used herein, the term "genome" preferably refers to the nuclear genome of the cells.

The target locus may, for example, be a gene (e.g. a mutant or defective gene) in the genome of the mammalian stem cells. In some embodiments, the target may be a regulatory element, e.g. an enhancer, promoter, or terminator sequence. In other embodiments, the target DNA is an intron or exon in a polypeptide-coding sequence.

In some embodiments, the target locus is not a Thy1 gene. In particular, in some embodiments, the or a CRISPR gRNA which is used in the method of the invention is one which does not target the Thy1 gene (e.g. the or a gRNA does not bind to intronl of Thy1 or after the polyA sequence of the Thy1 gene).

The CRISPR enzyme is one which is capable of forming a complex with the CRISPR gRNA in order to edit the genomes of the cells. The CRISPR/gRNA complex is capable of targeting a locus in the genomes of the mammalian stem cells which has a nucleotide sequence which is complementary to that of the spacer element in the gRNA.

The editing step may involve the production of double-stranded breaks in the genomes of the population of mammalian stem cells. Pairs of staggered single-stranded breaks in close vicinity are treated intracellularly in the same way as a double-stranded break and hence the production of such pairs of staggered single-stranded breaks also fall within the scope of the invention.

For D10A variants, it has been reported more robust editing is obtained when the two cleavage sites are 40-70 bp apart (in a PAM-out orientation), while the original nCas9 paper mentions an optimum range of 4 to 20 bp (Ran et al., “Double nicking by RNA- guided CRISPR Cas9 for enhanced genome editing specificity”, Cell. 2013 Sep 12; 154(6): 1380-9).

Preferably, the pairs of staggered single-stranded breaks are less than 200 bp apart, more preferably less than 100 bp apart. In some embodiments, the two staggered single-stranded breaks are 30-80 bp, more preferably 40-70 bp apart. In other embodiments, the two staggered single-stranded breaks are 2-30 bp, more preferably 4-20 bp apart.

Preferably, at least 50%, 60%, 70% or 80% of the genomes in the population of cells will be edited in the desired manner.

The CRISPR enzyme, CRISPR gRNA, and double-stranded donor DNA may be made by any suitable technique. Recombinant methods for the production of the nucleic acid molecules and packaging cells of the invention are well known in the art (e.g. “Molecular Cloning: A Laboratory Manual” (Fourth Edition), Green, MR and Sambrook, J., (updated 2014)). Genome-editing methods are well known in the art (e.g. Ran et al., “Genome engineering using the CRISPR-Cas9 system”, Nat. Protoc. 2013 Nov;8(11):2281-2308; and Petazzi et al., “CRISPR/Cas9-Mediated Gene Knockout and Knockin Human iPSCs”, Methods Mol. Biol. 2020, Nov 15). In some embodiments, the CRISPR enzyme (e.g. Cas9) is not encoded by a gene which is integrated into the genomes of the cells.

The editing step is carried out in the presence of a p53 inhibitor. The genomes of the mammalian cells comprise a gene encoding p53 from which a p53 polypeptide is expressible. As used herein, p53, also known as tumour protein p53, cellular tumour antigen p53 (UniProt name), the Guardian of the Genome, phosphoprotein p53, tumour suppressor p53, antigen NY-CO-13, or transformation-related protein 53 (TRP53), is any isoform of a protein encoded by homologous genes in various organisms, such as TP53 (humans) and Trp53 (mice). This homolog is crucial in multicellular vertebrates, where it prevents cancer formation, and thus functions as a tumour suppressor.

The actual mass of the full-length p53 protein (p53a) based on the sum of masses of the amino acid residues is only 43.7 kDa. In addition to the full-length protein, the human TP53 gene encodes at least 15 protein isoforms, ranging in size from 3.5 to 43.7 kDa. All these p53 proteins are called the p53 isoforms. The TP53 gene is the most frequently mutated gene (>50%) in human cancer, indicating that the TP53 gene plays a crucial role in preventing cancer formation. TP53 gene encodes proteins that bind to DNA and regulate gene expression to prevent mutations of the genome.

Preferably, the p53 polypeptide is a human p53 polypeptide. In humans, the TP53 gene is located on the short arm of chromosome 17 (17p13.1). The gene spans 20 kb, with a non-coding exon 1 and a very long first intron of 10 kb. The coding sequence contains five regions showing a high degree of conservation in vertebrates, predominantly in exons 2, 5, 6, 7 and 8, but the sequences found in invertebrates show only distant resemblance to mammalian TP53. The amino acid sequence of the human p53 polypeptide is given herein as SEQ ID NO: 1.

As used herein, the term "p53 polypeptide" preferably refers to a polypeptide whose amino acid sequence comprises or consists of the amino sequence as given in SEQ ID NO: 1 , or variant thereof having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto. Preferably, the variant is a tumour suppressor. The editing step is carried out in the presence of a p53 inhibitor. As used herein, the term "p53 inhibitor" relates to a moiety which may be used to transiently inhibit the production of or function of the p53 polypeptide in the cells in a specific or substantially- specific manner. The p53 inhibitor is not one which has a permanent or irreversible effect on the expression of p53 polypeptide in the cell; the inhibition is reversible. In particular, the term "p53 inhibitor" is not one which mutates the nucleotide sequence of or deletes all or part of the gene encoding p53 in the genomes of the cells.

The p53 inhibitor may, for example, be one which:

(i) affects the transcription of the p53 gene;

(ii) affects the translation of the p53 mRNA;

(iii) affects the function of the p53 polypeptide;

(iv) affects the half-life of the p53 polypeptide; or

(v) prevents binding of the p53 polypeptide to a pro-apoptotic member of the Bcl-2 family.

For example, the p53 inhibitor may be one which specifically or substantially specifically binds to the p53 polypeptide. In one embodiment, the p53 inhibitor is an anti-p53 shRNA. In other embodiments, the p53 inhibitor is a non-nucleic acid moiety, e.g. a non-nucleic acid organic compound, e.g. a tetrahydrobenzothiazolyl derivative.

In one preferred embodiment, the p53 inhibitor is pifithrin-a (PFT-a). Pifithrin-a (2-(2- imino-4,5,6,7-tetrahydrobenzothiazol-3-yl)-1 -p-tolylethanone hydrobromide) has been shown to be a specific p53 inhibitor using p53-dependent LacZ/p-Gal reporter assay for screening of chemical library (Komarov, P. G. "A Chemical Inhibitor of p53 That Protects Mice from the Side Effects of Cancer Therapy." Science 285, 1733-1737 (1999)).

In other embodiments, the p53 inhibitor is a polypeptide, e.g. a MAP kinase inhibitor (e.g. U0126); this is known to also suppress p53 expression.

The editing step may be carried out in the presence of one or more apoptosis inhibitors, e.g. an inhibitor of a pro-apoptotic member of the Bcl-2 family. The p53 inhibitor is provided in the cells such that it inhibits the production of or function of the p53 polypeptide in the cells.

In some embodiments, the p53 inhibitor is one which does not enhance the editing step (compared to a control method which is carried out on the same type of cells in the absence of the p53 inhibitor).

In some embodiments, the p53 inhibitor may be applied to a media (e.g. a physiologically-acceptable media, tissue culture media, etc.) which surrounds the cells.

In some embodiments, the p53 inhibitor is not encoded by a gene which is integrated into the genomes of the cells.

The p53 inhibitor may be present or introduced into the cells before, at the same time or after the start of the editing step. If the p53 inhibitor is introduced into the cells after the start of the editing step, it is preferably introduced less than 48 hours, more preferably less than 24, 12, 6, 3 or 1 hours after the start of the editing step. Preferably, the p53 inhibitor is introduced into the cells or the cell media immediately after the introduction of the CRISPR enzyme and gRNA. The p53 inhibitor must be in contact with the cells at the time at which any apoptosis might be initiated by the production of double-stranded or single stranded breaks in the cell genomes (which are produced by the gene-editing CRISPR enzyme). Preferably, the p53 inhibitor is present at the start of the editing step.

In some embodiments, the cells in the population of cells (before editing) are ones which would, if the editing step was carried out in the absence of the p53 inhibitor, express p53 polypeptide at a level such that at least 80% (preferably, at least 50%, 60%, 70%, 80%, 85%, 90%, 95% or 99%) of the cells in the population of cells would undergo apoptosis during or following the editing step. The end of the editing step may be defined as the point at which no detectable CRISPR enzyme or gRNA is present in the population of cells. Generally, this will be about 72 hours after the introduction of the CRISPR enzyme and gRNA into the population of cells. Preferably, the p53 inhibitor is present in the cells for all or substantially all of the duration of the editing step. In some embodiments, the p53 inhibitor is present in the cells for a time period which extends beyond the end of the editing step, e.g. for 1-5 or 5-10 hours after the end of the editing step.

In some embodiments, the cells in the population of cells (before editing) are ones which express wild-type levels or base-line levels of p53 polypeptide. Wild-type levels or base-line levels of p53 polypeptide may be determined by western blotting, e.g. using beta-actin as a reference polypeptide.

In some embodiments, the editing step is carried out in the presence of a concentration of the p53 inhibitor, wherein the concentration is such that, at least 80% (preferably at least 85%, 90%, 95% or 99%) of the cells in the population of cells do not undergo apoptosis during or following the editing step.

The desired concentration of p53 inhibitor will vary depending on the nature of the inhibitor. In some embodiments, the concentration of p53 inhibitor is 10 pM to 100 pM.

The method of the invention may additionally comprise the following optional step: (b) isolating and/or selecting a subpopulation of cells from the population of mammalian stem cells, wherein the subpopulation of cells are cells which have the desired edit in their genomes.

The presence or absence of the desired genome edit(s) may be determined by amplifying DNA regions containing the gRNA targeted locus by PCR. The generated amplicons may then be sequenced.

The p53 inhibitor may be removed from the cells by any suitable means. For example, if the p53 inhibitor is an anti-p53 shRNA, it will readily be lost from cells during subsequent growth and passaging of the cells. For example, if the p53 inhibitor is a non-nucleic acid organic compound, (e.g. a tetrahydrobenzothiazolyl derivative) which was added to the cell culture medium surrounding the cells, the cells may be centrifuged and then resuspended in fresh culture medium without the p53 inhibitor.

The expression levels of p53 polypeptide in the edited cells may subsequently be determined, e.g. using western blot with beta-actin as a reference standard. In particular, the expression levels of p53 polypeptide in the cells after the editing step may be compared to corresponding expression levels before the editing step. Cells with wild-type or substantially wild-type expression levels (i.e. expression levels before the editing step) of p53 polypeptide may then be selected.

The method steps are carried out in the order given.

The resultant population of cells (or subpopulation of cells) will be ones which express p53 polypeptide at a level which will protect or substantially protect the cells from becoming tumorigenic during the subsequent differentiation of those cells. The edited cells will inherently have a low tumorigenic potential.

In particular, the resultant population of cells (or subpopulation of cells) will have a reduced potential (e.g. at least 10%, 20%, 30%, 40% or 50% lower) to become tumorigenic compared to a population of cells (or subpopulation of cells) which were produced by the method of invention but without the presence of p53 inhibitor during the editing step.

The tumorigenic potential may be determined by whole exome or whole genome sequencing followed by determining the tumour mutational burden (TMB) or copy number variations of tumour-associated genes over time. Alternatively, the genomewide DNA methylation patterns of the cells’ genomes may be profiled and the number of DNA methylation patterns which are associated with tumours may be determined.

Furthermore, in differentiated cells, the growth of differentiated cells may be monitored and the degree to which they overcome senescence within a set time period may be determined. This may be determined by measuring senescence-associated betagalactosidase (SA-pgal) activity and hTERT expression.

Preferably, the average expression level of p53 polypeptide in the population of cells (or subpopulation of cells) after the editing step is at least 50% (preferably at least 60%, 70%, 80%, 85%, 90% or 95%) of the average expression level of p53 polypeptide in the population of cells before the editing step.

The edited stem cells may be cultured under conditions which promote the differentiation of the cells. Such differentiated (mature) cells may be used for any desired use, including for disease modelling, drug testing and cell therapy.

The invention also provides a population of cells or subpopulation of cells, or a cell line obtained therefrom, which was obtained or is obtainable by a method of the invention.

In a further embodiment, the invention provides a kit comprising:

(i) a CRISPR enzyme,

(ii) optionally a CRISPR gRNA,

(iii) optionally, a double-stranded or single-stranded donor DNA, and

(iv) a p53 inhibitor (preferably PTF-a).

The invention also provides the use of such a kit in a method of the invention.

The disclosure of each reference set forth herein is specifically incorporated herein by reference in its entirety.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the effect of PFT-a on gene-editing efficiency at two target loci.

Figure 2 shows a western blot of the levels of levels of p53 polypeptide expression before and after gene-editing iPS cells. Figure 3 shows a western blot of the levels of levels of p53 polypeptide expression before and after gene-editing iPS cells in the presence of the p53 inhibitor, PFT-a.

EXAMPLES

The present invention is further illustrated by the following Examples, in which parts and percentages are by weight and degrees are Celsius, unless otherwise stated. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

Example 1 : Effect of PFT-a on gene-editing efficiency

To investigate whether treatment of iPSCs with PFT-a would affect gene-editing efficiencies, we co-transfected iPSCs with CRISPR/Cas9 components with and without PFT-a; two different genomic loci (Target 1 and Target 2) were targeted. Briefly, PFT-a (10 pM) was added to the iPSCs following introduction of the CRISPR/Cas9 components. After 48 hours, DNA was extracted and Sanger sequencing was used to determine the gene-editing efficiencies.

Expt no. PFT-a DMSO qRNA

1 No Yes Target 1

2 Yes No Target 1

3 No Yes Target 2

4 Yes No Target 2

The results given in Figure 1 show that PFT-a had no effect on the gene-editing efficiency at either loci. Example 2: Gene editing of iPS cells

CRISPR/Cas9 components were introduced into iPSCs by nucleofection. The STAG2 gene was targeted to introduce INDELs in the genomic locus, generating STAG2 iPSC knock-out cells. Seventy-two hours post-transfection, cells were single cell sorted into 384 well plates and allowed to recover for two weeks; they were monitored over time. Automated transfer of 24 selected clones was then facilitated using Hamilton-robotics for effective expansion. Finally, genotyping was used to screen the clones. STAG2 and p53 protein expression was assessed for five clones by western blotting.

The results are shown in Figure 2. These results show that the levels of p53 expression in the edited cells were significantly reduced compared to the levels in the unedited cells.

Example 3: Gene editing of iPS cells in the presence of a p53 inhibitor

CRISPR/Cas9 components were introduced into iPSCs by nucleofection, followed by the addition of 10 pM PFTa to the medium. After 24 hours, PFT-a was removed by medium replacement. Seventy-two hours post-transfection, cells were single cell sorted into 384 well plates with medium containing 10 pM PFT-a. Twenty-four hours post-sort, PFT-a was diluted to 5 pM PFT-a by medium top-up. The following day (48 hours postsort), PFT-a was removed by medium replacement. Cells were allowed to recover for two weeks and monitored over time. Automated transfer of 24 selected clones was then facilitated using Hamilton-robotics for effective expansion. Finally, genotyping was used to screen the clones. STAG2 and p53 protein expression was assessed for six clones by western blotting. The results are shown in Figure 3. These results show that the levels of p53 expression in the edited cells were maintained at levels which were similar to the levels in the unedited cells.

SEQUENCES

The Sequence Listing filed with this patent application is fully incorporated herein as part of the description.