WO2013117713A1 - Reversal of senescence - Google Patents

Abstract

The present invention relates to the reversal of senescence, in particular the reversal of p16-mediated replicative senescence, using p16 inhibitors. The p16 inhibitors are generally small interfering RNA (siRNA) molecules that target and silence p16 expression, or anti-micro RNA molecules that target and disrupt senescence-associated micro RNA molecules. The use of such inhibitors in ex vivo cell culture methods, treatment of age-related disorders, cell culture media, reperfusion solutions, anti-ageing compositions, anti-wrinkle compositions and wound healing promoting compositions are also provided.

Description

REVERSAL OF SENESCENCE

The present invention relates to materials and methods useful in reversing cellular senescence. The present invention also relates to methods of treating certain diseases and conditions affected by cellular senescence and compositions for use in such methods.

Since the 1960s, it has been known that explanted human cells display limited replicative life span in culture. In a suitable medium, cells initially divide rapidly, but gradually reduce the rate of expansion and finally cease division entirely, entering a state known as replicative senescence (RS). RS is widely considered to be an irreversible state, regardless of cell density and the culture medium used. This rule is universal, with the single exception of embryonic stem cells (or induced pluripotent stem cells). Extension of life span of any cell type can however be achieved by mutation, and many cancer cells can be propagated indefinitely in culture. Senescent cells are not able to progress through the cell cycle, usually with a DNA content that is typical of Gl phase, but they remain metabolically active. Senescence may be caused by a number of factors, such as dysfunctional telomeres, DNA damage, chromatin perturbation, oncogenes or stress factors.

RS has been proposed as a model of organismal ageing, which is associated with a decay in the ability of tissues to renew themselves (for example reduced wound healing, ability to mount an immune response to infection, etc). Most notably, cells taken from older people display a reduced number of divisions in culture compared to similar cells obtained from a younger person. The vast majority of cultured cells progressively accumulate the cell cycle inhibitor, pl6ESfK4a (pl6), with passage. Eventually levels of this molecule reach a point at which further cellular expansion is inhibited, pi 6 does not accumulate in cultured embryonic stem cells, and the gene is almost invariably inactivated or bypassed in immortalised cancer cells, so its activation is an important event in cancer prevention. Ageing is also associated with replicative senescence and levels of pi 6 protein increase with ageing in most mammalian tissues. In fact, the levels of pl6 in an individual can be predicted by stochastic modelling that takes into consideration the subjects' age.

The pl6 gene is contained within the locus encompassing the Ink4b/ARF/Ink4a genes on chromosome 9p21. Two distinct reading frames are used for the transcription of pl4ARF (alternate reading frame) and pl6 tumour suppressor genes. The poly comb repressor complex (PRC) 1 (PRC1) and PRC2 complexes function as transcriptional repressors of the Ink4b/ARF/Ink4a locus. Normal cell homeostasis needs to balance the expression of pi 6 between growth and differentiation and a complex mechanism for the activation and inactivation of pi 6 has therefore developed. Senescence-inducing signals (such as those that trigger a DNA- damage response and oncogene- and stress-mediated activators) activate the retinoblastoma protein (pRb) and/or the p53 tumour suppressor pathways. p53 causes a cell to enter senescence in part by inducing the expression of p21, a cyclin-dependent kinase (CDK) inhibitor that can act to suppress the inactivation of pRb via suppression of phosphorylation. pRb suppresses the activity of the transcription factor E2F, which stimulates the expression of genes required for cell-cycle progression, and in this way pRb can halt cell proliferation. E2F itself can also inhibit proliferation by inducing ARF (pl4) expression, which in turn engages the p53 pathway. The binding of pi 6 to CDK4/CDK6 inhibits the phosphorylation of pRb family proteins and enables E2F to mediate a cell cycle arrest. The pRb pathway interacts with the p53 pathway: ARF binds to and inactivates MDM2. As a consequence, p53 is stabilised resulting in p21 activation. p21 in turn can prevent the phosphorylation and inactivation of pRB (Sherr and Roberts, 1999), thereby enabling E2F to mediate a cell cycle arrest. A detailed explanation of the cellular mechanism of senescence can be found in Campisi & d'Adda di Faggana, Nature Reviews Molecular Cell Biology, 2007, 8:729-740. pi 6 also plays a role in the reduced proliferative capacity of cells in ageing animals. For example, the protein accumulates as a function of age in a very wide range of cell types in rodents and man, and it appears that senescent cells accumulate with age. These may exert an ageing effect beyond their individual existence by secreting pro-ageing factors that act upon their neighbours. Indeed, selective deletion of pi 6 in mouse tissues (for example those of the immune system) can delay the onset of ageing in that particular tissue and possibly beyond. Although the pl6 and p53 pathways of cellular senescence can interact, they are considered separate pathways that can induce cellular senescence, and the relative contribution of these pathways in humans depends on the cell strain: while some are significantly delayed in their onset of senescence upon activation of pi 6 alone, other require a deficiency in p53 or in p53 as well as pl6 for the abrogation of senescence. The p53 and pl6 pathways can independently halt cell-cycle progression. pl6-mediated senescence has long been considered to be irreversible. There have been no previous reports of reversal of senescence in human cells, with the exception of direct microinjection of antibodies against p53 into individual cells (Gire et al , Mol. Cell. Biol , 1998, 18(3): 1611-21) or the lentiviral expression of p53shRNA (short hairpin RNA, Dirac & Bernards, J. Biol. Chem. , 2003, 278(14): 11731-4), which allows a very limited number of cell divisions. Most notably, transfection of senescent cells with telomerase (Beausejour et al, EMBO J. , 2003, 22(16):4212-22) or T-antigen (Gire et al.) are without effect in senescent cells. Beausejour et al. specifically addresses the issue of pl6-mediated senescence and concluded, in line with the very well established consensus on the matter, that pl6-mediated senescence is irreversible, with pi 6 providing a dominant second barrier to the unlimited growth of human cells that cannot be reversed by p53 inactivation. Rayess et al. ("Cellular senescence and tumor suppressor gene pi 6", Int. J. Cancer, published online 5 December 2011) report that cellular senescence is an irreversible arrest of cell growth. Shay & Roninson {Oncogene, 2004, 23:2919- 2933) report that the signal transduction program leading to cellular senescence is irreversible. Wei et al. {EMBO Rep. , 2003, 4(11): 1061-1066) reported that pl6-targeted siRNA was not sufficient to prevent a cell entering senescence. Sage et al. {Nature, 2003, 424:223-228) reported that loss of Rb function in mouse embryo fibroblasts leads to a reversal of senescence. However, this occurs in the presence on unaltered levels of pl6 expression.

International patent application WO2011/028673 discloses compositions for delaying cellular senescence using a hexapeptide. Although the prior art has suggested that p53-mediated senescence might be reversible under certain conditions, there remains a need in the art for a method of reversing (and preventing) pl6-mediated senescence in order for any therapies based on the reversal or inhibition of senescence to be useful. The present inventors have introduced small interfering RNAs (siRNAs) into senescent human epithelial cells (human mammary epithelial cells, HMECs). The ability of an siRNA directed against pl6 to restore proliferative capacity to fully senescent HMECs (RS-HMECs) was examined. The siRNAs were surprisingly found to be highly effective, allowing multiple rounds of cell division. Thus the long-established notion that human cell senescence, in particular pl6-mediated senescence, is irreversible has been over-turned by the findings of the present disclosure. It was particularly surprising that this could be achieved in adult human cells and that the cells were able to re-enter and complete the cell cycle. siRNA, also known as short interfering RNA or silencing RNA, is a class of double stranded RNA molecules that can be used to interfere with the expression of specific gene. siRNA molecules occur in nature, but can also be used experimentally to suppress the expression of a gene in vitro or in vivo. This may be achieved by targeting an mRNA for degradation, preventing mRNA translation or by establishing regions of silenced chromatin. siRNA molecules are usually short with an average length of 20 to 25 residues and may be incorporated into vectors when used experimentally to aid the successful transfection of these molecules into living cells.

A number of other molecules that are implicated in RS were also investigated by the present inventors. Specifically a family of senescence-associated microRNAs (hereafter SA-miRNAs) accumulate with passage of HMECs. A microRNA (miRNA) is a short RNA molecule that binds to complementary sequences on target messenger RNA (mRNA) transcripts. This results in translational repression or target degradation and gene silencing. By interfering with the expression of Polycomb genes (PcG, which normally repress the expression of pl6), the SA-miRNAs are instrumental in causing the accumulation of pl6 with passage. Transfection of SA- miRNAs into proliferating HMEC causes increased expression of pl6 and induction of RS. By contrast, the present inventors have surprisingly found that introduction of RNA antagonists directed against SA-miRNAs (referred to as "anti-SA-miRNAs") reduces pl6 expression and enables extension of cellular life-span. Furthermore, anti-SA-miRNAs introduced into RS-HMEC faithfully mimic pl6 siRNA, and allow restoration of proliferative capacity in fully senescent cells. It was particularly surprising that this could be achieved in adult human cells and that the cells were able to re-enter and complete the cell cycle.

Multiple miRNAs have been implicated in regulating the p53 pathway of cellular senescence. Martinez et al. (AGING, 2011, 3(2):77-78) reported the up-regulation of 25 miRNAs and the downregulation of 24 miRNAs in an Rb-dependent manner during induced senescence.

Throughout this application, the terms "senescence", "cellular senescence" and "replicative senescence" are used interchangeably and are all intended to refer to the state into which cells enter after multiple rounds of division and, as a result of cellular pathways, future cell division is prevented from occurring, although the cell remains metabolically active. Generally the senescence is pl6-mediated senescence, in particular pl6-mediated replicative senescence.

Accordingly, in a first aspect of the invention there is provided a pi 6 inhibitor for use in the prevention, inhibition or reversal of senescence, in particular replicative senescence. In some embodiments, the pi 6 inhibitor is a pl6-targeted small interfering RNA (siRNA) or an anti-SA-miRNA. Such uses can be in vivo. There is therefore also provided a pl6-targeted small interfering RNA (siRNA) or an anti-SA-miRNA for use in the prevention, inhibition or reversal of senescence, in particular replicative senescence. Methods of inhibiting, preventing or reversing senescence comprising administering a pi 6 inhibitor to a cell, tissue, biopsy or patient are also provided. Such methods can be in vivo, in vitro or ex vivo.

"Targeting" or "pl6-targeted", as used herein, refers to the ability of a pi 6 inhibitor to selectively bind to nucleic acids, in particular RNA molecules, of interest. The pi 6 inhibitors may be antisense nucleotides that bind to target sequences of interest. For example, an siRNA molecule may hybridize to nucleic acid, such as an mRNA molecule that encodes pi 6 protein or even genomic DNA sequences (such as genomic DNA encoding pi 6). Alternatively, an siRNA molecule may hybridise to an mRNA molecule that encodes a protein that promotes expression of pl6. In this way, other genes can be targeted to indirectly inhibit the expression of pl6. Targeting of nucleic acids, such as mRNA, using siRNA may be achieved by exploitation of a cell's RNA-inducing silencing complex (RISC). In such an approach, without wishing to be bound by theory, double-stranded RNA is cleaved by the enzyme Dicer, an RNAase III type nuclease. The siRNAs are loaded on to pre -RISC complex and the two strands separated. The RISC complex selectively hybridises to the target RNA and can induce cleavage or inhibit translations, and hence inhibition of gene expression. The siRNAs used in the present invention may or may not require processing by Dicer before being loaded onto a pre -RISC complex. An anti-SA-miRNA may be an oligonucleotide that hybridizes to a target nucleic acid, for example an miRNA, such as miRNAs that are associated with senescence (SA-miRNAs), or genomic DNA, such as pi 6 genomic DNA. "Senescence-associated" or "associated with senescence" refers to those miRNAs that are expressed or up-regulated before or during pl6-mediated senescence and are involved in initiating or maintaining pi 6- mediated senescence. The anti-SA-miRNAs and siRNAs used in the present invention are exogenously applied artificial anti-SA-miRNAs and siRNAs.

"Target sequences" or "target nucleic acids", depending on the context, may refer to nucleic acids, such as mRNA or genomic DNA, encoding pi 6 protein, or may refer to miRNA molecules that are associated with senescence. In particular, the target sequence may be a segment of the target nucleic acid or RNA molecule. For example, in some embodiments of the invention, the pi 6 inhibitor can target any section of the sequence of the pl6 gene (or corresponding pl6 mRNA or SA-miRNA), for example a segment of the pl6 gene (or pl6 mRNA or SA-miRNA) that is 10 to 50 or 15 to 30 nucleotides in length, optionally 15 to 29 nucleotides in length, such as 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29 nucleotides in length, typically 19 to 29 nucleotides. The siRNA or anti-SA-miRNA molecules themselves can be between 15 and 30 nucleotides in length, optionally 15 and 25 nucleotides in length, such as 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29 nucleotides in length, typically 19 to 29 nucleotides. The target nucleotides and/or the nucleotides of an siRNA or anti-SA- microRNA are generally contiguous, although siRNA and anti-SA-microRNA molecules may undergo processing or chemical modification prior to delivery to increase their stability and/or efficacy. Double stranded RNA molecules may also be used that are later processed into smaller siRNA molecules in vivo, for example by action of the enzyme Dicer.

In order to hybridize to a target nucleic acid, the pi 6 inhibitor may itself be a nucleic acid (polynucleotide or oligonucleotide, single or double stranded) that is complementary, or substantially complementary, to the target sequence or segment thereof. If the pi 6 inhibitor is double stranded (for example, an siRNA), at least one of the strands may be complementary, or substantially complementary, to the target sequence or segment thereof. "Substantially complementary" refers to at least 50% homology to a sequence that is complementary to the target nucleotide or target miRNA (or segment thereof), for example at least 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% homologous. For example, a pl6-targeted siRNA may be at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% homologous to a 19 to 29 residue nucleotide sequence that is complementary to a segment of pl6 mRNA. Similarly, an anti-SA-miRNA may be at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99 % or 100%o homologous to a 19 to 29 residue nucleotide sequence that is complementary to a segment of an SA-miRNA (or indeed may be homologous to the entire SA-miRNA). Therefore, pi 6 inhibitors that are nucleic acids may have a sequence that is the reverse complement (antisense) to the target nucleic acid or segment thereof. Preferably the pi 6 inhibitor does not substantially hybridize with other nucleic acids in the cell, in particular mRNA or miRNA molecules other than the target mRNA or miRNA molecules. When an siRNA hybridizes to target mRNA, it can effectively silence the corresponding gene or reduce expression of that gene (in this case, the pl6 gene). When an anti-miRNA molecule hybridizes to its target miRNA, it can interfere with their gene modulatory functions of the miRNAs and effectively silence or reduced expression of a particular gene (in this case, the pl6 gene).

Prevention of senescence can include complete prevention of senescence. Alternatively, it can refer to a reduction in the level of senescence compared to a control in which a cell has not been exposed to a pi 6 inhibitor, such as a pl6-targeted small interfering RNA (siRNA) or an anti-SA-miRNA. For example, when compared to a control, the number of cells that senesce may be reduced by more than 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%. Alternatively stated, the present invention may achieve a reversal of senescence of more than or equal to 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of cells in a given sample or population.

The siRNA used in the present invention can be any siRNA that targets pl6. Accordingly, any siRNA designed to target pi 6 mRNA can be used in the present invention. In addition, the anti-micro RNAs used in the present invention can be any anti-microRNAs that target senescence-associated microRNAs, i.e. microRNA molecules that promote or are involved in initiating, establishing or maintaining pl6-mediated senescence. The anti- microRNAs may themselves be nucleic acids or oligonucleotides, such as RNA or DNA.

The pi 6 gene is also known as cyclin-dependent kinase inhibitor 2A (CDKN2A is the official symbol provided by the Human Genome Organisation (HUGO) Gene Nomenclature Committee (HGNC)). The NCBI database has allocated ID no. 1029 (as of 29 January 2012) to this gene. GenBank accession numbers include AB060808.1, AF044170.1, AF527803.1, AA 05391.1, AF527803.1, AAR05391.1, AL449423.14, CAH70601.1, CH471071.2, EAW58599.1, EAW58600.1, EAW58601.1, EAW58602.1, EAW58603.1, DQ325544.1, DQ406745.1, S69804.1, U12818.1, U12819.1, U12820.1, X94154.1, AF115544.1, AI859822.1, AL582909.3, BC015960.2.

Any part of this gene may be targeted in the present invention. For example, the siRNA or other pi 6 inhibitors used in the present invention may target the sequence TACCGTAAATGTCCATTTATA, or a corresponding mRNA sequence (SEQ ID NOs: 1 and 2, respectively). Such a sequence can be targeted using the commercially available siRNA #SI02664403 (Qiagen), which specifically targets the sequence NM_000077.4 (SEQ ID NO. 5) or a corresponding mRNA (SEQ ID NO. 6). Accordingly, such an siRNA can be used in methods of the present invention. However, other siRNA molecules that target the pl6 gene can be used, and it is within the ability of the skilled person to select a suitable siRNA according to known protocols and requirements. Other transcripts that may be targeted using siRNA technology include NM_001195132.1, NM_058195.3, NM_058196.1 or NM_058197.4 or corresponding mRNA sequences (SEQ ID NOs 7 to 14). siRNAs can easily be designed to target specific DNA (or mRNA) sequences according to a number of algorithms known to the skilled person. Such methods are described in, for example, Walton et ah, FEBS, 2010, 277(23):4806-13 and Pei et ah , Nat Methods, 2006, 3(9)670-6. The siRNA molecules may be chemically modified, for example to increase potency, half-life or stability (for example by optionally adding one or more thymine residues to the end of the siRNA duplex). Such chemical modification is described in, for example, Bramsen & Kjems, Methods Mol Biol, 2011, 721 :77-103.

In embodiments of the invention, the pi 6 inhibitor, such as an siRNA targets the pi 6 gene, as described above. An example nucleotide sequence that may be targeted by the pi 6 inhibitor is the pi 6 sequence (or segments thereof) provided in SEQ ID NO: 3 (taken from NM_000077.3 [gi:47132606] Homo sapiens cyclin-dependent kinase inhibitor 2A (melanoma, pi 6, inhibits CDK4) (CDKN2A), transcript variant 1), or a corresponding mRNA sequence (or segment thereof) provided in SEQ ID NO: 4. The pl6 genomic sequence is provided in SEQ ID NO. 206 and this, or segments thereof, may be targeted by pl6 inhibitors, for example antisense nucleotides such as siRNA molecules. The siRNA molecules used as pi 6 inhibitors in the present invention may therefore comprise a sequence that is the reverse complement of any one of the sequences in SEQ ID NOs 1 to 14 or 206, or the reverse complement of a segment of any one of the sequences in SEQ ID NOs 1 to 14 or 206.

In some embodiments of the invention, the pl6 inhibitor, such as a siRNA or an anti-SA-microRNA, may be modified to increase its stability and/or uptake by cells. For example, the pl6 inhibitor may be incorporated into a vector. Alternative, the pi 6 inhibitor may be incorporated into a virus, such as lentivirus or an adenovirus. There are now a number of commercially available lentivirus and retroviral constructs that enable the stable expression of, for example, anti-miRNAs in cells (for example miRZIP lentiviral system http:/ywww.systembio.conv'microrna-research/microrna-knockdown'mirzip/overview. see also, for example, Mavrakis et ah, Nat Genet., 2011, 43(7):673-8). A non-integrating derivative of such systems to enable the longer term expression of anti-miRNAs of choice could be used in the present invention. In some embodiments of the invention, the pl6 inhibitor, such as an siRNA or an anti-SA-microRNA, can target any nucleotide sequence (DNA or mRNA) or segment thereof that encodes the protein sequence of pi 6, for example the following protein sequence of pl6:

MEPAAGSSMEPSADWLATAAARGRVEEVRALLEAGALPNAPNSYGRRPIQVMMMGSARVAELLLLHGAEPNCADP ATLTRPVHDAAREGFLDTLVVLHRAGARLDVRDAWGRLPVDLAEELGHRDVARYLRAAAGGTRGSNHARIDAAEG PSDIPD

MW = 16.53264 KDa

(SEQ ID NO: 15, Taken from NP_000068.1[gi:4502749] Cyclin-dependent kinase inhibitor 2A isoform 1 [Homo sapiens]). Accordingly, the siRNA molecules used as pl6 inhibitors in the present invention may comprise a sequence that is the reverse complement of a nucleotide sequence (such as a DNA sequence of mRNA sequence) that encodes SEQ ID NO: 15, or the reverse complement of a nucleotide sequence (such as a DNA sequence of mRNA sequence) that encodes a segment of SEQ ID NO: 15.

Other genes that may be targeted to indirectly inhibit the expression of pl6 include FLJ13215 (Gene ID 80071, GenBank Accession numbers include AP003501.2 and CH471065.1), GPR147 (Gene ID 64106, GenBank Accession numbers include AB065729.1, AL355138.19 and CH471083.1), LOC93109 (Gene ID 93109, GenBank Accession numbers include AC046143.20 and CH471052.2), MSF (Gene ID 10801 GenBank Accession numbers include AC068594.15, AC111170. i l, AC111182.21 and CH471099.1), AP3B2 (Gene ID 8120 GenBank Accession numbers include AC105339.9, CH471188.1,FJ695193.1), WDR44 (Gene ID 54521 GenBank Accession numbers include AL391474.8, CAI41403.1, AL391474.8, CAI41403.1, AL391803.14, CAI41482.1, CAI41483.1, AL391830.14, CAI41513.1 and CH471161.2), SLC7A11 (Gene ID 23657 GenBank Accession numbers include AB042201.2, AC093903.3, AC110804.3, AC116610.4 and CH471056.2), MOBKL1A (Gene ID 92597 GenBank Accession numbers include AC021989.8, AC093851.5, AC108014.4 and CH471057.1), LOC388813 (Gene ID 388813 GenBank Accession numbers include AF130351.2, AF165138.1 and AF198098.1), XPA (Gene ID 7507 GenBank Accession numbers include AF503166.1, AL445531.10, CH471105.1, U10347.1 and U16815.1), ABCB5 (Gene ID 340273 GenBank Accession numbers include AC002486.1 (61203..79410), AC005060.3, CH236948.1, EAL24274.1, CH471073.1 and EAW93727.1), FLJ13231 (Gene ID 65250 GenBank Accession numbers include AC008925.4 and AC025449.6), GJB3 (Gene ID 2707 GenBank Accession numbers include AF099730.1, AJ004856.1, AL121988.10 and CH471059.2) and OR8B8 (Gene ID 26493 GenBank Accession numbers include AB065832.1, AF399510.1, BK004496.1 and CH471065.1), and their corresponding mRNAs. Therefore, in some embodiments of the invention, "pie- targeted inhibitor" can include pi 6 inhibitors (such as siRNA molecules) that inhibit the expression of pi 6 by inhibiting other genes that modulate the expression of pl6. The pl6 inhibitors that are nucleic acids and target these alternative genes may hybdrise to contiguous segments of target nucleic acids that are 15 to 30 nucleotides in length, optionally 15 to 25 nucleotides in length, such as 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29 nucleotides in length, typically 19 to 29 nucleotides. Targeting of such sequences may be achieved by using pl6 inhibitors with homologous sequences, for example at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100%o homology with the complement of the nucleic acid (such as mRNA) to be targeted, or a segment thereof.

The term "anti-SA-miRNA" as used in the present invention refers to miRNA inhibitors that target senescence associated microRNAs (senescence associated-microRNAs; SA-miRNAs). Such SA-miRNAs have been identified previously. SA-miRNAs include the miRNAs listed in Table 1. Any of these (optionally one or more) can be targeting using the anti- SA-miRNAs

Table 1

S A- miRNA Accession Number(s)

hsa-let-7b MI0000063

hsa-let-7c MI0000064

hsa-miR-1180 MI0006273

hsa-miR-1184 MI0006277, MI0015971, MI0015972

hsa-miR-1205 MI0006338

hsa-miR-122 MI0000442

hsa-miR-1253 MI0006387

hsa-miR-125a-5p MI0000469

hsa-miR-125b MI0000446, MI0000470,MIMAT0000423, MIMAT0004592,

MIMAT0004603

hsa-miR-1269 MI0006406, MI0016888, MIMAT0005923

hsa-miR-1270 MI0006407, MI0015976

hsa-miR-1271 MI0003814

hsa-miR-127-3p MI0000472

hsa-miR-128 MI0000447, MI0000727

hsa-miR-1283 MI0003832, MI0006430

hsa-miR-1288 MI0006432

hsa-miR-1301 MI0003815

hsa-miR-130b MI0000748

hsa-miR-134 MI0000474

hsa-miR-146b-5p MI0003129

hsa-miR-148a* MI0000253

hsa-miR-181a MI0000269, MI0000289, MIMAT0000256, MIMAT0000270,

MIMAT0004558

hsa-miR-181a* MIMAT0000270

hsa-miR-182 MI0000272, MIMAT0000260

hsa-miR-182* MI0000272, MIMAT0000260

hsa-miR-184 MI0000481

hsa-miR-191* MIMAT0001618

hsa-miR-193a-3p MI0000487

hsa-miR-193b MI0003137

hsa-miR-196a* MIMAT0004562

hsa-miR-197 MI0000239

hsa-miR-19b-2* MIMAT0004492

hsa-miR-200b MI0000342

hsa-miR-203 MI0000283

hsa-miR-206 MI0000490

hsa-miR-208a MI0000251

hsa-miR-208b MI0005570

hsa-miR-21 MI0000077

hsa-miR-210 MI0000286

hsa-miR-218 MI0000294, MI0000295, MIMAT0000275, MIMAT0004565,

MIMAT0004566

hsa-miR-22* MI0000078, MIMAT0004495

hsa-miR-26a MI0000083, MI0000750, MIMAT0000082, MIMAT0004499, SA-miRNA Accession Number(s)

MIMAT0004681

hsa-miR-26b MI0000084

hsa-miR-30a MI0000088

hsa-miR-30c MI0000254, MI0000736, MIMAT0000244, MIMAT0004550,

MIMAT0004674

hsa-miR-30c-2* MIMAT0004550

hsa-miR-30d MI0000255

hsa-miR-330-3p MI0000803

hsa-miR-340 MI0000802

hsa-miR-34a MI0000268

hsa-miR-361-5p MI0000760

hsa-miR-365 MI0000767, MI0000769

hsa-miR-367* MI0000775, MIMAT0004686

hsa-miR-372 MI0000780

hsa-miR-373 MI0000781

hsa-miR-378* MIMAT0000731, MI0000786, MI0003840, MI0014154,

MI0015825

hsa-miR-378* MI0016749, MI0016750, MI0016756, MI0016761,

cont/d MI0016808

hsa-miR-378* MIOO 16902, MIMAT0000731, MIMAT0000732

cont/d

hsa-miR-410 MI0002465

hsa-miR-424 MI0001446

hsa-miR-432 MI0003133

hsa-miR-452 MI0001733

hsa-miR-483-5p MI0002467

hsa-miR-491-3p MI0003126

hsa-miR-492 MI0003131

hsa-miR-497* MIMAT0004768 , MI0003138

hsa-miR-500 MI0003184, MIOO 15903

hsa-miR-503 MI0003188

hsa-miR-518c* MIMAT0002847, MI0003159

hsa-miR-518e* MI0003169

hsa-miR-519a* MI0003178, MI0003182, MIMAT0002869, MIMAT0005452 hsa-miR-519b-5p MI0003151

hsa-miR-519c-5p MI0003148

hsa-miR-522 MI0003177, MIMAT0005451

hsa-miR-522* MI0003177, MIMAT0005451

hsa-miR-523* MIMAT0005449, MI0003153

hsa-miR-541* MI0005539, MIMAT0004919

hsa-miR-544 MI0003515, MI0014159, MIMAT0003164

hsa-miR-548a-3p MI0003593, MI0003598, MI0003612

hsa-miR-548i MI0006421, MI0006422, MI0006423, MI0006424

hsa-miR-548o MI0006402

hsa-miR-576 MI0003583

hsa-miR-578 MI0003585

hsa-miR-588 MI0003597

hsa-miR-589* MIMAT0003256, MI0003599

hsa-miR-593* MIMAT0003261, MI0003605

hsa-miR-596 MI0003608

hsa-miR-658 MI0003682

hsa-miR-671-3p MI0003760

hsa-miR-7-1* MIMAT0004553, MI0000263

hsa-miR-7-2* MIMAT0004554, MI0000264

hsa-miR-744* MIMAT0004946, MI0005559

hsa-miR-767-5p MI0003763

hsa-miR-874 MI0005532

hsa-miR-9 MI0000466, MI0000467, MI0000468 The miRNA nomenclature in miRBase is based on the precursor microRNAs (pre -miRNA, Figure 8), therefore there usually will be a miRNA-xx and a miRNA-xx* form. miRNA and miRNA* are the two strands of the double-stranded RNA product of Dicer processing of the stem loop precursor miRNA (i.e. hsa-miR-26b and hsa- miR26b* are expressed from the same genetic locus as a single RNA). Because of the sequence complementarity they form a double stranded RNA product. The abbreviation "hsa" refers to the species of origin being Homo sapiens, and generally the SA-miRNAs that are targeted in the present invention are human SA-miRNAs.

SA-miRNAs include and are derived from the following Homo sapien pre-miRNAs as follows (which can also be targeted in the present invention): hsa-let-7b, hsa-let-7c, premiR-1180, premiR-1184, premiR-1205, premiR-122, premiR-1253, premiR-125a-5p, premiR-125b, premiR-1269, premiR-1270, premiR-1271, premiR-127-3p, premiR-128, premiR-1283, premiR- 1288, premiR-1301, premiR-130b, premiR-134, premiR-146b-5p, premiR-148a, premiR-181a, premiR-182, premiR-1826, premiR-184, premiR-191, premiR-193a-3p, premiR-193b, premiR-196a, premiR-197, premiR- 19b-2, premiR-200b, premiR-203, premiR-206, premiR-208a, premiR-208b, premiR-21, premiR-210, premiR- 218, premiR-22*, premiR-26a, premiR-26b, premiR-30a, premiR-30c, premiR-30c-2, premiR-30d, premiR-330- 3p, premiR-340, premiR-34a, premiR-361-5p, premiR-365, premiR-367, premiR-372, premiR-373, premiR-378, premiR-410, premiR-424, premiR-432, premiR-452, premiR-483-5p, premiR-491-3p, premiR-492, premiR-497, premiR-500, premiR-503, premiR-518c, premiR-518e, premiR-519a, premiR-519b-5p, premiR-519c-5p, premiR-522, premiR-523, premiR-522, premiR-541, premiR-544, premiR-548a-3p, premiR-548i, premiR-548o, premiR-576-3p, premiR-576-5p, premiR-578, premiR-588, premiR-589, premiR-593, premiR-596, premiR-658, premiR-671-3p, premiR-7-1, premiR-7-2, premiR-744, premiR-767-5p, premiR-874 and premiR-9. The sequences of these are freely available from miRBAse http://www.mirbase.org/. The sequences of the mature SA-miRNAs are also provided in SEQ ID NOs. 26 to 115.

However, the present inventors concentrated on a particular subset whose expression increases during senescence and is correlated with pi 6 levels. Accordingly, in some embodiments, example SA-miRNAs that are targeted by the anti- SA-miRNAs of the invention include hsa-miR-26b, -181a, -210 and -424, the sequences of which are provided in Table 5.

Anti-miRNAs are generally small, chemically modified single -stranded nucleic acids, such as RNA molecules, that specifically bind to and inhibit endogenous miRNA molecules and enable miRNA functional analysis by down-regulation of miRNA activity. References to anti- SA-miRNAs therefore include single stranded nucleic acid (for example DNA or RNA) molecules that target the desired SA-miRNA. Anti- SA-miRNAs can be purchased from commercial suppliers (such as Invitrogen, although other vendors are available) and can be "designed" to the desired target by techniques known to the skilled person. In particular, as anti-miRNAs are single stranded nucleic acids that specifically bind to and inhibit endogenous miRNA molecules, their sequence is generally determined by the target sequence, although they may undergo some chemical modification to improve their stability or bioavailability. As noted above, miRNA sequences are freely available via miRBase

Accordingly, the anti-SA-miRNAs may have a sequence that is complementary, or substantially complementary, to the SA-miRNA being targeted (SEQ ID NOs 16, 18, 20, 22, 24 or 26 to 115) or to a segment of the SA-miRNA being targeted. In particular, the anti-SA-miRNA molecules may comprise a sequence according to any one of SEQ ID NOs 17, 19, 21, 23, 25 or 116 to 205, which are the reverse complement sequences of SEQ ID NOs: 16, 18, 20, 22, 24 and 26 to 115, respectively (all sequences in the sequence listing are provided in the 5' to 3' direction). SEQ ID NOs 16, 18, 20, 22, 24 or 26 are pre-miRNA sequences.

The pl6 inhibitors (such as the pl6-targeting siRNAs or anti-SA-miRNAs) of the invention can be used to inhibit, prevent or reverse senescence (such as replicative senescence) in any suitable cell type, for example human cells, in particular adult human cells or cells obtained from an adult or adult tissue sample or biopsy. For example, senescence may be reversed in human mammary epithelial cells or keratinocytes. Other suitable cell types include any human cells that can be isolated and then cultured ex vivo. Further examples include: cardiac myocytes, chondrocytes, endothelial cells (large vessels), endothelial cells (microvascular), epithelial cells, fibroblasts, follicle dermal papilla cells, hepatocytes, keratinocytes, melanocytes, osteoblasts, preadipocytes, primary cells of the immune system, skeletal muscle cells, smooth muscle cells, adipocytes, neurons, glial cells, contractile cells, exocrine secretory epithelial cells, extracellular matrix cells, hormone secreting cells, keratinising epithelial cells, islet cells, lens cells, mesenchymal stem cells, pancreatic acinar cells, paneth cells of the small intestine, primary cells of haemopoietic linage, primary cells of the nervous system, sense organ and peripheral neuron supporting cells and wet stratified barrier epithelial cells. Any of these cell types can be used in methods of the invention. Generally, human cells that express pi 6 are used in the methods of the present invention. Suitably, the present invention uses non-fetal cells/tissues/organs. Some embodiments use neonatal cells, although non-neonatal cells can also be used. In some embodiments, the cells are not human embryonic stem cells. In some embodiments, the methods may be carried out on gametes (oocytes and/or spermatozoa), for example during in vitro fertilisation.

Reversal of senescence generally refers to the reversal of senescence (in particular pl6-mediated senescence) after the cells have senesced. It is particularly surprising that the present invention can achieve this aim, as all previous literature had considered pl6-mediated senescence to be irreversible once established. pl6-mediated senescence refers to senescence induced, controlled and/or maintained by pl6 expression. Classically, cells that have undergone pl6-mediated senescence have an enlarged and flattened morphology, they may accumulate vacuoles, be beta-galactosidase positive, have elevated levels of reactive oxygen species (associated with increased levels of 8-oxoguanine, detectable by immunofluorescence), increased expression of cytokines, (such as IL-6 and IL-8), and/or fail to incorporate 5-bromo-2'deoxyuridine (BrdU).

Accordingly, in a second aspect of the invention there is also provided a pi 6 inhibitor, such as a pl6-targeted siRNA or an anti-SA-miRNA, for use in the ex vivo manipulation of cells or biological tissue. For example, there is provided a pi 6 inhibitor for use in extending the lifespan of a cell, for example in cell culture. The use of a pi 6 inhibitor, such as a pl6-targeted siRNA or an anti-SA-miRNA, in the ex vivo manipulation of cells or biological tissue and in extending the lifespan of a cell, for example in cell culture {in vitro or ex vivo), is also provided. In a third aspect of the invention, there is provided a method of preventing, inhibiting or reversing senescence of a cell, such as replicative senescence, comprising contacting the cell with a pi 6 inhibitor, such as a pl6-targeted siRNA or an anti-SA-miRNA. There is also provided a method of extending the life span of a cell, comprising inhibiting the function of pi 6, for example by contacting the cell with a pi 6 inhibitor, such as a pl6-targeted siRNA or an anti-SA-miRNA. Furthermore, there is also provided a method of inhibiting pl6 expression in a cell by contacting the cell with a pl6 inhibitor, such as a pl6-targeted siRNA or an anti-SA-miRNA. The methods may be in vivo, in vitro or ex vivo methods. Generally, contacting the cell includes transfection with a pi 6 inhibitor, such as a pl6-targeted siRNA or an anti- SA-miRNA. Transfection is well known to the skilled person and can be carried out according to any suitable protocol. For example, transfection may be chemical-based transfection, such as methods utilising calcium phosphate, dendrimers (highly branched organic compounds), liposomes or cationic liposomes (also known as lipofection), or using cationic polymers (such as DEAR-dextran or polyethylenimine). Alternatively, the transfection may be non-chemical transfection, for example electroporation, sonoporation, optical transfection, impalefection, or a particle -based method of transfection, such as those involving the use of a gene gun (where the material to be transfected is coupled to a nanoparticle of an inert material (such as gold) which is then "shot" into the cell), or magnetofection (or magnet assisted transfection). Transfection may alternatively be carried out using viral methods, such as viral transduction. Other methods include nucleofection and heat shock transfection. Further methods will be apparent to the skilled person.

Short-RNA transfection can be used to introduce siRNA into a cell and has led to the development of siRNA molecules as a new class of macromolecular drugs. The present inventors have used a commercially available lipid reagent, DharmaFect (Thermo Scientific), although any suitable method or reagent could be used, as would be apparent to the skilled person.

However, a step of transfection need not be included in all methods of the present invention. For example, a pi 6 inhibitor, such as a pl6-targeted siRNA or an anti-SA-miRNA, of the invention could simply be included in a cell culture medium to delay or reverse the onset of replicative senescence. Alternatively, the pl6 inhibitor of the invention may be included in a reperfusion solution that may be used during organ, tissue or cell transplantation to keep the organ perfused before re-implantation. In such methods, the cells may uptake the siRNA or anti-SA-miRNA of the invention by molecular mechanisms such as phagocytosis. However, suitable transfection reagents to promote uptake into the cells could also be included. Accordingly, in a fourth aspect of the invention, there is provided a reperfusion (or aqueous) solution comprising a pi 6 inhibitor, such as a pl6-targeted siRNA or an anti-SA-miRNA, and one or more volume expanders. In one embodiment, the volume expander is a crystalloid or a colloid, or a combination of a crystalloid and a colloid.

A crystalloid is an aqueous solution of salts comprising at least two ions selected from the group consisting of sodium ions, chloride ions, lactate ions, potassium ions and calcium ions. In some embodiments, the crystalloid is an aqueous solution comprising at least three ions selected from the group consisting of sodium ions, chloride ions, lactate ions, potassium ions and calcium ions. In some embodiments, the crystalloid is an aqueous solution comprising at least four ions selected from the group consisting of sodium ions, chloride ions, lactate ions, potassium ions and calcium ions. In some embodiments, the crystalloid is an aqueous solution comprising sodium ions, chloride ions, lactate ions, potassium ions and calcium ions. The crystalloid may also comprise bicarbonate ions and/or glucose.

Example crystalloids include aqueous solutions of mineral salts (such a saline, Ringer's lactate or Hartmann's solution) or other water-soluble molecules. One litre of Ringer's lactate solution (also known as lactated Ringer's solution or Ringer's Lactate) can contain about 100 to 150 mEq of sodium ions (about 100 to 150 mmol/L), about 90 to 120 mEq of chloride ions (about 90 to 120 mmol/L), about 20 to 30 mEq of lactate (about 20 to 30 mmol/L), about 2 to 6 mEq of potassium ions (about 2 to 6 mmol/L) and about 1 to 5 mEq of calcium ions (about 1 to 5 mmol )L In particular, one litre of Ringer's lactate solution may contain about 130 mEq of sodium ions (130 mmol/L), about 109 mEq of chloride ions (109 mmol/L), about 28 mEq of lactate (28 mmol L), about 4 mEq of potassium ions (4 mmol/L) and about 3 mEq of calcium ions (1.5 mmol/L).

Generally, the sodium, chloride, potassium and lactate comes from NaCl (sodium chloride), NaC₃H₅0₃ (sodium lactate), CaCl₂ (calcium chloride), and KC1 (potassium chloride). However, it would be apparent to a person of skill in the art that other components could be used to reach the desired ion concentrations. The pH of Ringer's lactate can be in the range of 6 to 7, for example 6.5, although it is generally an alkalizing solution.

One litre of Hartmann's solution (also known as compound sodium lactate) can contain 131 mEq of sodium ions (131 mmol/L), 111 mEq of chloride ions (111 mmol/L), 29 mEq of lactate (29 mmol/L), 5 mEq of potassium ions (5 mmol/L.) and 4 mEq of calcium ions (2 mmol/L).

Accordingly, in one embodiment of the invention, the reperfusion solution of the invention comprises a pi 6 inhibitor and a crystalloid volume expander, wherein the crystalloid volume expander is an aqueous solution comprising at least 3 ions selected from the group consisting of sodium ions, chloride ions, lactate ions, potassium ions and calcium ions. In another embodiment of the invention, the reperfusion solution of the invention comprises a pi 6 inhibitor and a crystalloid volume expander, wherein the crystalloid volume expander is an aqueous solution comprising at least 4 ions selected from the group consisting of sodium ions, chloride ions, lactate ions, potassium ions and calcium ions. In a further embodiment of the invention, the reperfusion solution of the invention comprises a pi 6 inhibitor and a crystalloid volume expander, wherein the crystalloid volume expander is an aqueous solution comprising sodium ions, chloride ions, lactate ions, potassium ions and calcium ions.

In another aspect of the invention, there is provided a reperfusion solution comprising a pi 6 inhibitor and at least three ions (optionally at least 4 ions) selected from the group consisting of sodium ions, chloride ions, lactate ions, potassium ions and calcium ions. In another embodiment of the invention, there is provided a reperfusion solution comprising sodium ions, chloride ions, lactate ions, potassium ions, calcium ions and a pi 6 inhibitor. This aspect of the invention extends to the use of a pi 6 inhibitor in the manufacture of a solution for organ or cell reperfusion, for example for organ or cell reperfusion during transplantation. In the reperfusion solutions of the invention, the ions may be present at any suitable concentration known to the skilled person. For example, the sodium ions may be present in a concentration of about 100 mmol/L to about 150 mmol/L. The chloride ions may be present in a concentration of about 90 mmol/L to about 120 mmol/L. The lactate ions may be present in a concentration of about 20 mmol L to about 30 mmol L. The potassium ions may be present in a concentration of about 2 mmol/L to about 6 mmol/L. The calcium ions may be present in a concentration of about 1 mmol/L to 5 about mmol/L. Bicarbonate ions (if present) may be present in a concentration of about 10 mmol L to about 50 mmol/L. Glucose (if present) may be present at a concentration of about 2% to about 10% by weight, for example about 3% to about 6% by weight.

Accordingly, in another embodiment of the invention, the reperfusion solution of the invention comprises an aqueous solution comprising pi 6 inhibitor and about 100 mmol/L to about 150 mmol/L of sodium ions, about 90 mmol/L to about 120 mmol/L of chloride ions, about 20 mmol/L to about 30 mmol/L of lactate, about 2 mmol/L to about 6 mmol/L of potassium ions and about 1 mmol/L to about 5 mmol/L of calcium ions

As will be apparent to the skilled person, the above aqueous solution is an example of a suitable crystalloid volume expander comprising a pi 6 inhibitor.

The reperfusion solution may alternatively include a colloid volume expander, or it may contain a mixture of the crystalloid volume expander described above and a colloid volume expander. Examples of suitable colloids include gelatin, succinylated gelatin, albumin, dextran (for example dextran 40, dextran 70 or dextran 75), blood, or etherified starch (also known as hydroxyethyl starch, tetrastarch, hetastarch or pentastarch). The colloids are generally aqueous solutions comprising these components. For example, the colloid may comprise at least one component selected from the groups consisting of gelatin, succinylated gelatin, albumin, dextran, blood and etherified starch.

Commercially available colloids include Haemaccel® (Piramal, containing degraded gelatin polypeptides cross- linked via urea bridges), Gelofusine® (Braun, succinylated gelatin (modified fluid gelatin, average molecular weight 30 000) 40 g (4%), Na+ 154 mmol, CI- 120 mmol/litre), Gelopasma® (Fresenius Kabi, partially hydrolysed and succinylated gelatin (modified liquid gelatin) (as anhydrous gelatin) 30 g (3%), Na⁺ 150 mmol, K⁺ 5 mmol, Mg²⁺ 1.5 mmol, Cl^~ 100 mmol, lactate 30 mmol/litre), Isoplex® (Beacon, succinylated gelatin (modified fluid gelatin, average molecular weight 30 000) 40 g (4%), Na⁺ 145 mmol, K⁺ 4 mmol, Mg²⁺ 0.9 mmol, C 105 mmol, lactate 25 mmol/litre), Volplex® (Beacon, succinylated gelatin (modified fluid gelatin, average molecular weight 30 000) 40 g (4%), Na⁺ 154 mmol, Cl^~ 125 mmol/litre), Voluven® (Fresenius Kabi, 6% hydroxyethyl starch (weight average molecular weight 130 000) in 0.9% sodium chloride injection), Volulyte® (Fresenius Kabi, 6% hydroxyethyl starch (weight average molecular weight 130 000) in sodium chloride intravenous infusion 0.6%, containing Na⁺ 137 mmol, K⁺ 4 mmol, Mg²⁺ 1.5 mmol, Cl^~ 110 mmol, acetate 34 mmol/litre), Venofundin® (Braun, 6% hydroxyethyl starch (weight average molecular weight 130 000) in 0.9% sodium chloride injection), Tetraspan® (Braun, hydroxyethyl starch (weight average molecular weight 130 000) 6% or 10% in sodium chloride 0.625%, containing Na⁺ 140 mmol, K⁺ 4 mmol, Mg²⁺ 1 mmol, Ο 118 mmol, Ca²⁺ 2.5 mmol, acetate 24 mmol, malate 5 mmol/litre), HAES-steril® (Fresenius Kabi, pentastarch (weight average molecular weight 200 000) 10% in sodium chloride intravenous infusion 0.9%), Hemohes® (Braun, 6% or 10% pentastarch (weight average molecular weight 200 000), in sodium chloride intravenous infusion 0.9%), HyperHAES® (Fresenius Kabi, hydroxyethyl starch (weight average molecular weight 200 000) 6% in sodium chloride intravenous infusion 7.2%) and RescueFlow® (Vitaline, dextran 70 intravenous infusion 6% in sodium chloride intravenous infusion 7.5%).

Accordingly, in one embodiment of the invention, the reperfusion solution of the invention comprises a pi 6 inhibitor and a colloid volume expander and/or a crystalloid volume expander, wherein the colloid volume expander comprises one or more components selected from the group consisting of gelatin, succinylated gelatin, albumin, dextran, blood, and etherified starch, and wherein the crystalloid volume expander is an aqueous solution comprising at least three ions selected from the group consisting of sodium ions, chloride ions, lactate ions, potassium ions and calcium ions.

In a further embodiment of the invention there is provided a reperfusion solution comprising a pi 6 inhibitor and a colloid volume expander and/or a crystalloid volume expander, wherein the colloid volume expander comprises one or more components selected from the group consisting of gelatin, succinylated gelatin, albumin, dextran, blood, and etherified starch, and wherein the crystalloid volume expander comprises about 100 to 150 mmol/L of sodium ions, about 90 to 120 mmol/L of chloride ion, about 20 to 30 mmol/L of lactate, about 2 to 6 mmol/L of potassium ions and about 1 to 5 mmol/L of calcium ions. The reperfusion solutions of the invention may be aqueous solutions. The concentrations of each of the components can be determined by a person of skill in the art according to requirements. For example, the concentration of the compound of Formula I (or a pharmaceutically acceptable salt or ester thereof) may be present in an amount of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or 30 nM, for example in the range of 1 to 500nM or 10 to 100 nM.

The reperfusion solutions of the invention may be hypotonic, hypertonic or isotonic. In some embodiments of the invention, the volume expander is an isotonic aqueous solution. Accordingly, the invention provides an isotonic aqueous solution comprising a pi 6 inhibitor. This aspect of the invention extends to the use of a pl6 inhibitor, such as a pl6-targeted siRNA or an anti-SA- miRNA, and a volume expander in the manufacture of a reperfusion solution.

Although the present invention include reperfusion solutions, such solutions are not essential for working the invention. For example, the pi 6 inhibitor may be present in any suitable solution that allows their introduction into the target cell or tissues. For example, a particular transfection protocol may require the use of a particular solution in order for the transfection to be effective, and accordingly solutions may include transfection reagents. Transfection reagents include calcium phosphate, dendrimers (highly branched organic compounds), liposomes, cationic liposomes, or cationic polymers (such as DEAR-dextran or polyethylenimine). Suitable transfection reagents will be apparent to the skilled person. Accordingly, solutions comprising the pi 6 inhibitor, such as the pl6-targeting siRNA or anti-SA-miRNAs, of the invention may further comprise a transfection reagent.

In a fifth aspect of the invention, there is provided the use of a pi 6 inhibitor, such as a pl6-targeted small interfering RNA (siRNA) or an anti-SA-miRNA, in the prevention, inhibition or reversal of senescence. There is also provided a pl6-targeted small interfering RNA (siRNA) or an anti-SA-miRNA, for use in the prevention, inhibition or reversal of senescence. Furthermore, the present invention also provides the use of a pi 6 inhibitor such as a pl6-targeted small interfering RNA (siRNA) or an anti-SA-miRNA, in the manufacture of a solution, medicament, cell culture medium or reperfusion solution for use in the prevention, inhibition or reversal of replicative senescence. In a sixth aspect of the invention, there is provided the use of a pi 6 inhibitor, such as a pl6-targeted small interfering RNA (siRNA) or an anti-SA-miRNA, in cell culture or as a component of a cell culture medium. In a seventh aspect of the invention, there is provided a cell culture medium comprising a pl6 inhibitor, such as a pl6-targeted small interfering RNA (siRNA) or an anti-SA-miRNA. Methods of cell culture comprising culturing or expanding a population of cells in vitro in a cell culture medium of the invention or in the presence of pi 6 inhibitors are also provided.

Suitably, the cell culture medium of the present invention comprises one or more components selected from the group consisting of a source of amino acids (and/or a source of nitrogen), a source of carbon and a salt. The source of amino acids can be an amino acid. For example, the culture medium of the present invention may further comprise an amino acid selected from the group consisting of isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, histidine, glutamine, alanine, arginine, aspartate, cysteine, glutamate, glycine, proline, serine, tyrosine and asparagine. Generally, references to amino acids in this application refer to the L-isomer of the amino acid, unless specified otherwise.

In some embodiments, the source of amino acids can be one or more of the essential amino acids, wherein the essential amino acids are isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, histidine and glutamine. In some embodiments of the invention, the source of amino acids can be glutamine or a source of L-glutamine. Sources of L-glutamine include dipeptides of glutamine (such as alanyl-L-glutamine and glycyl-L-glutamine) and gluten hydrolystates. If present, the L-glutamine or source L-glutamine can be present in a concentration range of between 0.1 mM and 100 mM, for example between 0.5 and lOmM. In some embodiments, the L- glutamine or source of L-glutamine is present in an amount of at least 0.1 mM, for example at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1 mM. In some embodiments of the invention, the source of amino acids can be ammonia or ammonium ions. The source of nitrogen can be any suitable source known to the skilled person, for example glutamine or a source of L-glutamine. The salt can be any suitable salt, for example calcium chloride, ferric nitrate, magnesium sulfate, potassium chloride, sodium chloride, sodium bicarbonate and sodium phosphate. Accordingly, the culture medium general includes metal ions (such as calcium, iron, magnesium, potassium and/or sodium) as well as non-metallic ions (such as chloride, nitrate, sulfate, bicarbonate and/or phosphate). In some embodiments of the invention, the culture medium may further comprise a source of carbon. The source of carbon can be any suitable source, for example glucose or galactose.

The cell culture medium may comprise metal irons (such as zinc or copper) or a source of metal irons. The cell culture medium may comprise metal chelators (such as EDTA). In some embodiments, the cell culture medium may comprise an acid (for example arachidonic acid, linoleic acid, linolenic acid, lipoic acid, oleic acid or palmitic acid). In some embodiments, the cell culture medium may comprise one or more vitamins, for example vitamin B12.

In some embodiments, the cell culture medium may further comprise one or more components selected from the group consisting of albumin, arachidonic acid, ascorbate, biotin calcium, ceruloplasmin, citrate, copper, cysteine, cystine, one or more fatty acids, folate, glucose, glutamine, glutathione, iron, linoleic acid, linolenic acid, lipoic acid, oleic acid, palmitic acid, pyridoxal, riboflavin, selenium, stearic acid, thiamine, tocopherol, transferrin, urate, vitamin B12 and zinc. The cell culture medium of the present invention may further comprise a transfection reagent, or a transfection reagent may be added to the medium during use to facilitate the introduction of the pl6-targeted small interfering RNA (si NA) or an anti-SA-miRNA into the cell.

The cell culture medium of the present invention may be a defined culture medium, wherein a defined culture medium is one that does not include any undefined animal or organ extracts.

In some embodiments of the invention, the cell culture medium may be a selective medium and therefore could include an antibiotic, such as ampicillin, tetracycline, neomycin or gentamycin. In some embodiments of the invention, the cell culture medium may include one or more factors or hormones that induce differentiation into a particular cell type. Hormones, such as oestrogen, progesterone, placental lactogen, prolactin, and oxytocin are required for development and function of human mammary cells in vivo and can be supplemented as required either directly or via analogues into the cell culture medium. Growth hormones, such as corticosteroids, thyroid hormone, and insulin help regulate metabolic responses to nutrient uptake and may also be supplemented into the medium. Examples of growth factors include vascular endothelial growth factor (VEGF), transforming growth factor (TGF) and epidermal growth factor (EGF). In some embodiments, the cell culture medium may further comprise one or more components selected from the group consisting of glucose, sodium bicarbonate, amino acids, phenol red, pyridoxine, sodium pyruvate and oxytocin.

In some embodiments of the invention, the cell culture medium may comprise serum, for example fetal bovine serum, donor goat serum, donor horse serum, newborn calf serum or trout serum.

Suitably, the cell culture medium of the present invention may have a pH of between 7.0 and 8.0. Many cells require a pH of between 7.2 - 7.4, and close control of pH may be important for optimum culture conditions. The optimal range for fibroblasts is pH 7.4 - 7.7 and transformed cell lines a pH of around 7.0 - 7.4. The appropriate pH value will be apparent to the skilled person.

In some embodiments of the invention, the cell culture medium may comprise a buffer to maintain a suitable pH. Cell culture medium commonly use natural bicarbonate/C0₂ buffering systems. To ensure optimal pH, these should preferably be maintained in an atmosphere of 5-10% C0₂. Alternatively, buffers, such as HEPES, with a buffering capacity of 7.2-7.4 can be employed.

Generally, the skilled person could easily determine the required components of a cell culture medium according to the cells to which the medium is going to be applied. For example, when culturing human mammary epithelial cells (HMECs), the following cell culture can be used (as reported in Garbe et ah, Cancer Res., 2009, 69:7557-7568).

Table 2

Table 4 - MM4 DMEM/F12 (Gibco/Invitrogen #11039) 500ml

Insulin (lmg/mL) (SIGMA # 15500) 5ml

FCS (Gibco/Invitrogen #26140) 2.5ml

Hydrocortisone lmg/mL (SIGMA #H4001) 50μ1

Tri-iodothyronine 2 x 10^"* M (SIGMA #T2877) 25μ1

β-estradiol 2 x 10^"5 M (SIGMA #E8875) 25μ1

Epidermal Growth Factor 0.1 mg/ml (Invitrogen

25μ1

#PHG0064)

Glutamine 200mM (Invitrogen #25030) 5ml

In the present invention, the pl6 inhibitor, such as the pl6-targeted siRNA or anti-SA-miRNA, can be added to such cell culture mediums in order to realise the advantages of the present invention. In a further aspect of the invention, there is provided a kit of parts comprising a cell culture medium and an aqueous solution comprising a pi 6 inhibitor. The pl6 inhibitor can be added to the cell culture medium at the appropriate time to realise the advantages of the present invention. The kit of parts may optionally further comprise a transfection reagent. The kit of parts may also optionally further comprise instructions for use. The discovery of the present inventors has numerous applications, not least of all as tissue culture medium supplements to help extend in vitro life span of primary cells during ex vivo cell manipulation or in tissue engineering and tissue transplantation. The present invention can be used to extend the viability of biopsies and cell lines in cell culture. Accordingly, in a further aspect of the invention, there is provided the use of a pi 6 inhibitor, such as a pi 6- targeted small interfering RNA (siRNA) and/or an anti-SA-miRNA, in cell culture, ex vivo cell or tissue manipulation or in tissue engineering or tissue or organ transplantation. The tissue is biological tissue, for example a tissue sample, a biopsy or a graft. The cell can be of any suitable cell type that can be cultured in vitro. Suitably, the cell is a human cell, such as an adult human cell, or the tissue is a human tissue sample or biopsy, suitably a tissue sample or biopsy obtained from an adult or adolescent. "Adult" generally refers to any cells obtained from a patient post birth, so excludes embryonic cells but may include neonatal cells.

Methods of cell culture, ex vivo cell or tissue manipulation, tissue engineering and/or tissue and organ transplantation are also provided, wherein the method includes inhibiting the expression of pl6. This may be achieved by contacting the cell or tissue or organ with a pl6 inhibitor, such as a pl6-targeted small interfering RNA (siRNA) and/or an anti-SA-miRNA of the invention or a reperfusion solution or cell culture medium of the invention. The pl6 inhibitor may be transfected into the cell or tissue, so any of the methods of the invention may comprise such a step of transfection (chemical or otherwise) or addition of a transfection reagent. Accordingly, methods (such as methods of cell culture or methods of cell manipulation) of the present invention can include:

obtaining a cell sample or tissue sample from a patient; and

inhibiting pi 6 expression in the cell sample or tissue sample. The methods of the invention may include a step of culturing or expanding the cells obtained from a patient. If the cells senesce, the methods can include the step of reversing senescence (in particular pl6-mediated senescence) by inhibiting pl6 expression in the cells or tissue sample.

Inhibition of pi 6 may be achieved by contacting or transfecting the cell sample or tissue (biopsy) with a pi 6 inhibitor, such as a pl6-targeted small interfering RNA (siRNA) and/or an anti-SA-miRNA, of the invention.

The methods of the present invention may be entirely ex vivo methods, so are carried out on cells or tissues that have already been removed or obtained from a patient, or they can include the steps of the removal from a donor patient. The donor patient may be a living donor patient or may be cadaverous (deceased). Following obtaining a tissue sample or biopsy, and prior to contacting the tissue sample or biology with a pi 6 inhibitor, the tissue sample or biopsy may undergo enzymatic and/or mechanical separation. Methods of the present invention may include a step of culturing the cells or tissue, for example in a cell culture medium of the present invention, or a step of reperfusing a tissue or organ in a reperfusion solution of the invention. In some embodiments of the invention, the step of inhibiting pi 6 expression (for example by contacting or transfecting the cell or tissue with the pi 6 inhibitor such as a pl6-targeted small interfering RNA (siRNA) and/or an anti-SA-miRNA) can comprise a step of culturing the cell or tissue in a cell culture medium of the invention or it may comprise a step of contacting the cell or tissue with a reperfusion solution of the invention. In some embodiments, the step of inhibiting pi 6 expression can comprise contacting the cell or tissue with a pl6 inhibitor (such as the pl6-targeted small interfering RNA (siRNA) and/or an anti-SA-miRNA) or it can comprise transfecting the cell or tissue, for example by any suitable means known to the skilled person (for example by adding a transfection reagent, or by other means). In methods of the invention, pl6-mediated senescence can be reversed using the pi 6 inhibitors. In addition, the methods of the invention may be carried out on cell or tissue samples, or on biopsies or organs, or alternatively may be carried out on cell lines.

Accordingly, in one embodiment of the invention there is provided a method of cell culture comprising:

obtaining a cell sample, tissue sample or biopsy from a patient;

optionally subjecting the cell sample, tissue sample or biopsy to enzymatic and/or mechanical separation; and

reversing, inhibiting or preventing pl6-mediated senescence by culturing the cell sample, tissue sample or biopsy in the presence of a pl6 inhibitor, such as a pl6-targeted siRNA or an anti-SA-miRNA (or a combination thereof). Alternatively the method of cell culture may be carried out on cell lines or a cell sample, tissue sample or biopsy that was previously obtained from a patient. Transfection reagents may be added to facilitate uptake of the pl6 inhibitor into the cells to reverse, prevent or inhibit senescence. Optionally, cells can be administered to a patient (usually the same patient as the donor patient). Cells can also be cryogenically stored until required.

In another embodiment of the invention there is provided a method of cell, tissue or organ transplantation, comprising: obtaining a cell sample, tissue sample or organ from a patient;

optionally subjecting a cell sample or tissue sample to enzymatic and/or mechanical separation; and reversing, inhibiting or preventing pl6-mediated senescence by culturing the cell sample or tissue sample in the presence of a pi 6 inhibitor or by reperfusing the organ in a reperfusion solution of the invention. The cells, tissue or organ can be administered to a patient (the same patient as the donor patient or a difference patient, that is, autologous or allogeneic transplantation). Cells, tissue samples or organs may be cryogenically stored until required. The method of tissue or organ transplantation may be carried out using esophagus, stomach, liver, gallbladder, pancreas, adrenal, glands, bladder, gallbladder, large intestine, small intestine, kidneys, liver, pancreas, colon, stomach, thymus, spleen, brain, spinal cord, nerves, adipose tissue, heart, lungs, eyes, corneal, skin or islet tissue or organs. Methods may also be carried out on oocytes and/or spermatozoa, mesenchymal stem cells, adipocytes, central nervous system neurons and glial cells, contractile cells, exocrine secretory epithelial cells, extracellular matrix cells, hormone secreting cells, keratinising epithelial cells, islet cells, kidney cells, lens cells, mesenchymal stem cells, pancreatic acinar cell, paneth cell of small intestine, primary cells of haemopoietic lineage, primary cells of the nervous system, sense organ and peripheral neuron supporting cells or wet stratified barrier epithelial cells.

Since the 1960s, it has been known that explanted human cells display replicative senescence in culture (Hayflick, L., Cell. Res. , 1965, 37:614-36). The activation of pl6 in this context is a highly documented, universal phenomenon (for example fibroblasts (Alcorta et al, Proc. Natl. Acad. Sci. USA, 1996, 93(24): 13742- 7), keratinocytes (Lee et al., Arch. Oral Biol , 2000, 45:809-818; Loughran et al., Oncogene, 1996, 13:561-568), mammary epithelial cells (Brenner et al., Oncogene, 1998, 17: 199-205), prostate epithelial cells (Jarrard et al., Cancer Res. , 1999, 59:2957-2964), and T-lymphocytes (Erickson et al, Oncogene, 1998, 17:595-602)).

The accumulation of pi 6 is also observed in many tissues as a function of advancing age. As a consequence, the ability of cells to replicate in vitro diminishes with donor age and this directly correlates with pl6 status (for example human corneal endothelial cells (Enomoto et al, Invest. Ophthalmol. Vis. Sci., 2006, 47:4330-4340)). Furthermore, cellular senescence by chronological ageing or repeated sub-culture has been shown to induce loss of stem cell fractions, (e.g. in keratinocytes (Nakamura & Nishioka, Br. J. Dermatol , 2003, 149:560-565). Given that this fraction plays a critical role in the expansion of ex vivo cultures, preserving it through repression of pl6 would also be beneficial.

The ex vivo manipulation of cells would benefit from the repression of pl6 (by pl6 si NA(s) and/or anti-SA- miRNA(s), referred to herein as the "inhibition of pi 6" or "inhibiting pi 6 expression" and/or an inhibition or reversal of senescence. Taken together, the present invention is of particular benefit for patients of advanced age. For example, the uses and methods of the present invention may be carried out on samples obtained from human patients that are at least 30, 40, 50, 60 or 70 years of age, or indeed the methods may be carried out on such patients themselves.

Inhibitors of pl6 expression include the pl6-targeted siRNA and the anti-SA-miRNAs of the present invention. Accordingly, any aspect of the invention involving the use of a pl6-targeted siRNA or an anti-SA-miRNA can similarly be carried out using an inhibitor of pi 6 expression. Inhibitors of pi 6 expression can also include molecules that inhibit pi 6 expression indirectly by acting on other genes that modulate pi 6 expression, for example any of the genes noted above that modulate the expression of pl6.

Below are some examples of the types of ex vivo cell manipulations that would benefit from the inhibition of pl6. In each instance, the inhibition of pl6 is used to improve cell yields, and/or reduce protocol time from isolation to re -implantation. A reduced protocol time has the advantage of reducing the risk of mutations arising as a consequence of ex vivo manipulations, reducing costs and improving patient care. The re-introduction of cells with a reduced pl6+/senescent population is also potentially desirable. Skin tissue engineering e.g. after burns

Both allogeneic (i.e. where donor and recipient are different) and autologous (i.e. where donor and recipient are the same) cultured skin cells can be used to augment skin repair, such as in the treatment of severe burns, blisters and ulcers (such as diabetic ulcers, recalcitrant ulcers (common in individuals with diabetes), pressure ulcers and neuropathic ulcers.

Biopsies are subjected to enzymatic/mechanical segregation as appropriate and kerotinocytes isolated. These cells are then cultured in vitro in the presence/absence of lethally irradiated 3T3 fibroblasts and expanded with the aim of generating enough cells to apply to the wound bed, either as a confluent sheet of cells or in single-cell suspension. More recently, protocols have been devised that replace the irradiated mouse -derived 3T3 fibroblasts with non-irradiated autologous fibroblasts (Jubin et al., Cytotechnology, 2011. 63:655-662). During the isolation and expansion process the inhibition of pi 6 in the keratinocytes and/or autologous fibroblasts will be beneficial.

Accordingly, in a further aspect of the invention there is provided a method of skin tissue engineering comprising removal of skin cells (for example keratinocytes and/or autologous fibroblasts) from a patient and inhibiting pi 6 expression. Inhibition may be achieved by contacting the cells with a pi 6 inhibitor, such as a pl6-targeted siRNA or an anti-SA-miRNA, of the invention, or a solution comprising a pl6 inhibitor, such as a pl6-targeted siRNA or anti-SA-miRNA, of the invention, or a cell culture medium of the invention, or a reperfusion solution of the invention. The contacted cells can then be administered to a recipient patient in need thereof after senescence has been suitably reversed and/or inhibited. The donor and recipient patients may be the same or different patients.

There is also provided the use of a pi 6 inhibitor, such as a pl6-targeted siRNA or an anti-SA-miRNA, in tissue engineering, ex vivo cell manipulation or tissue transplantation. Furthermore, there is provided the use of a pi 6 inhibitor, such as a pl6-targeted siRNA or an anti-SA-miRNA, of the invention in the treatment of burns injury, as well as methods of treatment of burns injury comprising administering a pi 6 inhibitor, such as a pl6-targeted siRNA or an anti-SA-miRNA, of the invention to a patient in need thereof.

At any stage in this method or in other methods of the invention, the cells can be cryogenically preserved using standard protocols, which a skilled individual will be familiar with. This will enable the long-term storage of the cells. The cells can be stored as a preventative step, to be thawed and expanded at a later date should they be required. Alternatively, cells surplus to requirements can be stored following expansion until such time as they are needed. This protocol can be applied to any of the ex vivo or in vitro uses described herein.

Bone marrow transplantation (BMT)

During bone marrow transplantation, pi 6 has been found to be induced, as a function of the transplant protocol (Janzen et al., Nature, 2006, 443:421-426). Thus, BMT is a pro-ageing protocol. This can be ameliorated by pl6 inhibition (for example using pl6 siRNA(s) and/or anti-SA-miRNA(s)) in vitro during transplant. Indeed, bone marrow cells obtained from mice lacking pi 6 (amongst others) have shown improved ability to reconstitute the blood long term (Akala et al, Nature, 2008, 453:228-232).

Accordingly, in one aspect of the present invention, there is provided a method of stem cell transplantation (such as hematopoietic stem cell transplantation in bone marrow transplantation) comprising inhibiting pi 6 expression. Inhibition may be achieved by contacting donor cells extracted from a donor patient with a pi 6 inhibitor, such as a pl6-targeted siRNA or anti-SA-miRNA, of the invention. For example, in one aspect of the invention there is provided a method of hematopoietic stem cell transplantation (for example bone marrow transplantation) comprising removal of donor cells from a donor patient and subsequently contacting the donor cells with a pi 6 inhibitor, such as a pl6-targeted siRNA or anti-SA-miRNA, of the invention, or a solution comprising a pl6 inhibitor, such as a pl6-targeted siRNA or anti-SA-miRNA, of the invention, or a cell culture medium of the invention, or a reperfusion solution of the invention. The contacted cells can then be administered to a recipient patient in need thereof. As with other ex vivo manipulation methods of the invention, the cells will suitably be cultured/expanded prior to transplantation. The cells may also be cryogenically stored, as described above.

In another aspect of the invention, there is provided a pl6 inhibitor, such as a pl6-targeted siRNA or anti-SA- miRNA, of the invention for use in hematopoietic stem cell transplantation (for example bone marrow transplantation).

The protocols for the isolation of bone marrow and bone marrow transplantation are fully established and published, and can be carried out by a skilled individual. Renal allograft

Renal allograft survival is related directly to cell senescence (Naesens, Discov. Med. , 2011, 11(56):65-75). In the transplantation scenario many cellular events - participating as immunological and non-immunological factors - could contribute to accelerate this biological process, responsible for the ultimate fate of the graft. In combination with immunosuppressive treatment, the repression of pi 6 and/or SA-miRNAs could improve patient outcome.

Accordingly, in one aspect of the present invention, there is provided a method of renal transplantation comprising inhibiting pi 6 expression. Inhibition may be achieved by contacting donor cells, tissue or organ extracted (removed) from a donor patient with a pl6 inhibitor, such as a pl6-targeted siRNA or anti-SA-miRNA, of the invention. For example, in one aspect of the invention there is provided a method of renal transplantation comprising removal of donor cells, tissue or organ from a donor patient and subsequently contacting the donor cells with a pl6 inhibitor, such as a pl6-targeted si NA or anti-SA-miRNA, of the invention, or a solution comprising a pl6 inhibitor, such as a pl6-targeted siRNA or anti-SA-miRNA, of the invention, or a cell culture medium of the invention, or a reperfusion solution of the invention. The contacted cells can then be administered to a recipient patient in need thereof.

In another aspect of the invention, there is provided a pl6 inhibitor, such as a pl6-targeted siRNA or anti-SA- miRNA, of the invention for use in renal transplantation.

The protocols for renal transplantation are fully established and published, and can be carried out by a skilled individual.

Human corneal epithelium

The expansion of human corneal epithelium is used in a variety of contexts (corneal burns, cateracts, etc). Corneal cells are pl6 limited in culture (Enomoto et ah, Invest. Ophthalmol. Vis. Sci. , 2006, 47:4330-4340), and whilst cells from older donors can proliferate, they respond more slowly and to a lesser extent than cells from young donors (Senoo & Joyce, Invest. Ophthalmol. Vis. Sci. , 2000, 41 :660-667).

Accordingly, in one aspect of the present invention, there is provided a method of corneal transplant, comprising inhibiting pi 6 expression. Inhibition may be achieved by contacting a donor cornea removed from a patient with a pl6 inhibitor, such as a pl6-targeted siRNA or anti-SA-miRNA, of the invention, or a solution comprising a pl6 inhibitor, such as a pl6-targeted siRNA or anti-SA-miRNA, of the invention, or a cell culture medium of the invention, or a reperfusion solution of the invention. The cornea can then be transplanted to a donor patient.

The protocols for the isolation and transplantation of human corneal epithelial are fully established and published, and can be carried out by a skilled individual.

Cells derived from human embryonic stem cells or induced pluripotent stem cells

The emerging field of regenerative medicine promises to solve the problem of organ transplant rejection by regrowing organs in the lab, using the patients' own cells (stem cells, or healthy cells extracted from the donor site.) The usefulness of such cells can be extended using the compositions and methods of the present invention by inhibiting, preventing or reversing the onset of replicative senescence. In some embodiments, the stem cell in a non-human embryonic stem cell. The protocols for the isolation of human embryonic stem cells (and other stem cells) and the generation of a broad spectrum of induced pluripotent stem cells from a range of starting cell types are published, and can be carried out by a skilled individual.

As outlined above, with the exception of pluripotent embryonic stem cells, primary cells in vitro and in vivo gradually lose the ability to divide and become senescent. It is now known that epithelial cells derived from human embryonic stem cells undergo pl6-mediated senescence (Dabelsteen et ah, Stem Cell, 2009, 27: 1388- 1399). Therefore, the inhibition of pl6 may prove critical in the development of stem cell therapies. Similarly, the repression of pi 6 aids in the generations of induced pluripotent stem cells (Banito and Gil, EMBO Rep. , 2010, 11, 353-359; Banito et al, Genes Dev, 2009, 23, 2134-2139; Li et al, Nature, 2009, 460, 1136-1139). Accordingly, in one aspect of the invention there is provided a pi 6 inhibitor for use in rejuvenative medicine, such as stem cell rejuvenation therapy, and corresponding methods. There is also provided the use of a pi 6 inhibitor in the manufacture of a medicament for rejuvenative medicine or stem cell rejuvenation therapy. The pl6 inhibitor may be a pl6-targeted si NA or an anti-SA-miRNA of the invention. Methods of stem cell therapy can be carried out according to the methods of the present invention disclosed herein relating to cell culture and ex vivo cell manipulation.

In a further aspect of the invention, there is provided a method of treating an age-related disease or condition comprising inhibiting pi 6 expression, for example by administering a pi 6 inhibitor. Treatment may involve the in vivo inhibition of pi 6 expression using any technique described herein. The treatment may involve in vitro or ex vivo methods steps. For example, inhibition may be achieved by administering a pl6-targeted siRNA or an anti-SA-miRNA to a patient in need thereof. There is also provided an inhibitor of pi 6 expression, such as a pl6-targeted siRNA or an anti-SA-miRNA, for use in the in vivo treatment of an age-related disease or condition. There is further provided the use of an inhibitor of pi 6 expression, such as a pl6-targeted siRNA or an anti-SA-miRNA, in the manufacture of a medicament for the treatment of an age-related disease or condition The age-related disease or condition may be selected from the group consisting of Alzheimer's disease, coronary heart disease, Parkinson's disease, myocardial infarction, ischemic stroke, abdominal aortic aneurism, arterial stiffness, frailty, perdiodontal disease, type 2 diabetes, neurodegeneration and ageing, pi 6 inhibitors, such as the pl6-targeted siRNA or an anti-SA-miRNA, can also be used to treat degenerative traits, such as synaptic loss, white matter atrophy, cognitive impairment, skin thinning, vision impairment, cataracts and muscle wastage, and so methods for treating such conditions are also provided. The pi 6 inhibitors can also be used to diagnose such age-related diseases or condition, pi 6 inhibitors can also be used to promote wound healing, for example of traumatic or stalled wounds.

In further aspects of the invention, there is provided a method of treating infection, cancer, alopecia (such as alopecia areata, alopecia areata monolocularis, alopecia areata multilocularis, baldness or male pattern baldness) neurodegeneration, gum disease, bone degeneration, or degeneration of the cardiovascular, digestive, integumentary, lymphatic or musculoskeletal system comprising inhibiting pi 6 expression by any means disclosed in the present invention, pi 6 inhibitors, such as the pl6-targeted small interfering RNA (siRNA) and/or the anti-SA-miRNA of invention, for use in such methods, and for use in the manufacture of medicaments for such diseases, are also provided.

In methods and uses of the invention relating to the treatment of certain conditions, inhibitors of pi 6 expression (such as a pl6-targeted siRNA or an anti-SA-miRNA) may be in the form of a pharmaceutical composition. There is therefore also provided a pharmaceutical composition comprising an inhibitor of pi 6, such as a pi 6- targeted siRNA or an anti-SA-miRNA, and a pharmaceutically acceptable carrier, diluent or excipient. The pharmaceutical composition may be in any suitable form, depending upon the desired method of administering it to a patient, pi 6 inhibitors for use in medicine are also provided. The pharmaceutical composition may be adapted for administration by any appropriate route, for example by topical administration. Such compositions may be prepared by any method known in the art of pharmacy, for example by admixing the active ingredient with the carrier(s) or excipient(s) under sterile conditions. Pharmaceutical compositions adapted for transdermal administration may be presented as discrete patches intended to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. For example, the active ingredient may be delivered from the patch by iontophoresis as generally described in Pharmaceutical Research, 3 (6), page 318 (1986). Pharmaceutical compositions adapted for topical administration may be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols or oils. In some embodiments, the pharmaceutical composition is applied as a topical ointment or cream. When formulated in an ointment, the active ingredient may be employed with either a paraffinic or a water-miscible ointment base. Alternatively, the active ingredient may be formulated in a cream with an oil-in- water cream base or a water-in-oil base. Pharmaceutical compositions adapted for topical administration to the eye include eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent. Pharmaceutical compositions adapted for topical administration in the mouth include lozenges, pastilles and mouth washes.

Pharmaceutical compositions adapted for parenteral administration include aqueous and non-aqueous sterile injection solution which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation substantially isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. Excipients which may be used for injectable solutions include water, alcohols, polyols, glycerine and vegetable oils, for example. The compositions may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze -dried (lyophilized) condition requiring only the addition of the sterile liquid carried, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.

The pharmaceutical compositions may contain preserving agents, solubilising agents, stabilising agents, wetting agents, emulsifiers, sweeteners, colourants, odourants, salts (substances of the present invention may themselves be provided in the form of a pharmaceutically acceptable salt), buffers, coating agents or antioxidants. They may also contain therapeutically active agents in addition to the substance of the present invention.

Dosages of the pharmaceutical compositions can vary between wide limits, depending upon the disease or disorder to be treated, the age and condition of the individual to be treated, etc. and a physician will ultimately determine appropriate dosages to be used.

In order to increase the stability of the siRNA or anti-SA-miRNA, they may be chemically modified. For example, they may be incorporated into vectors, liposomes, vesicles, beads (for example resin or agar beads) or microparticles, or any other encapsulation technology, to assist in their delivery. Incorporation of medically active substances into microparticles is described in WO2006/97725. The vector, liposome, vesicle, bead or microparticle may be targeted to the appropriate tissue by means know to the skilled person.

Other delivery methods will be apparent to the skilled person. For example, lentivirus-expressed siRNA vectors against Alzheimer disease are described in Peng & Masliah, Methods in Molecular Biology, 2010, 614:215-224. RNA interference as a tool for Alzheimer's disease therapy is described in Orlacchio et al, Mini-Reviews in Medicinal Chemistry, 2007, 7(11): 1166-76. RNAi-based therapeutic strategies for metabolic disease are described in Czech et al, Nature Reviews Endocrinology, 2011, 7:473-484. Local administration of siRNAs to the eye is described in Reich et al, Molecular Vision, 2003, 9:210-216. Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs is described in Soutschek et al , Nature, 2004, 432: 173- 178. siRNA delivery for treatment of muscular dystrophy is described in Hagstrom et al. , Molecular Therapy, 2004, 10:386-398.

As described in Dykxhoorn et al, Gene Therapy, 2006, 13:541-552, there are two general ways for delivering siRNA molecules to patients: delivery using double stranded siRNA molecules or delivery using gene therapy to express precursor RNAs from viral vectors. Accordingly, embodiments of the present invention include such strategies, for example double stranded pl6-targeted siRNA molecules for use in methods and compositions of the invention. Further discussion on formulation and deliver of siRNA molecules can be found in, for example, Burnett et al., Biotechnol J., 2011, 6(9): 1130-46, Peer et al, Gene Ther., 2011, 18(2): 1127-33, Manjunath & Dykxhoorn, Discov. Med , 2010, 9(48):418-30 and Zheng et al, Proc Natl Acad Sci USA, 2012, 109(30)11975- 80.

Once delivered, siRNA molecules may target mRNA (such as mRNA encoding pi 6 protein) for degradation or may prevent their translation, thereby silencing gene expression. Precursor RNA may be processed by action of the enzyme Dicer, which cleaves long double-stranded RNA (dsRNA) molecules into short double stranded fragments suitable for use in siRNAs and miRNAs. Accordingly, when carrying out the present invention, a skilled person may use a suitable precursor that is processed in situ (in vivo) by action of the enzyme Dicer into a suitable and effective siRNA molecule. In some embodiments, siRNAs may target genomic DNA and prevent or interfere with transcription of a gene.

The half-life of siRNA molecules can be extended by, for example, incorporating the siRNA into particles (to avoid renal filtration) and by chemically modifying the sugar backbone. For example the siRNA may be 2'-0- methyl or 2'-fluoro substituted, or it may be modified at the 2' position to 2'deoxyribose. siRNA molecules may be conjugated to lipids, such as cholesterol, to increase their half-life, or they may be incorporated into microparticles or liposomes, and other techniques will be apparent to the skilled person.

The siRNA or other pl6 inhibitors of the invention may be administered to a patient with separate, simultaneous or subsequent delivery of suitable transfection reagents, for example during local delivery to tissues such as the lung, vagina, a subcutaneous tumor, muscle, eye or the nervous system. Systemic delivery may be achieved by, for example, rapid high-pressure intravenous injection ("hydrodynamic therapy") or receptor-mediated systemic delivery. An in-depth review is provided in Dykxhoorn et al. Anti-mi NA molecules may also be modified to increase their stability and efficacy, for example as discussed in Lennox & Behlke, Gene Therapy, 2011, 18: 1111-1120, and may be formulated according to methods known for the formulation and delivery of siRNAs, as discussed above. Anti-miRNA molecules may comprise antisense oligonucleotides. The oligonucleotides may be modified, for example 2'-0-methyl RNA, which have a higher binding affinity than DNA when targeting RNA, and they are more resistant to nuclease degradation. The oligonucleotides may incorporate one or more P-S bonds, or other chemical modifications may be present as deemed suitable by the skilled person. In a still further aspect of the invention, there is provided an anti-ageing or anti- wrinkle composition, such as a cream, comprising an inhibitor of pl6 expression, for example a pl6-targeted siRNA or an anti-SA-miRNA of the invention. The anti-ageing or anti-wrinkle composition may be in the form of an emulsion, for example a water-in-oil or an oil-in-water emulsion. The composition may include a number of other components, for example one or more components selected from the group consisting of emollients, antioxidants, surfactants, fats, proteins, coenzymes (such as coenzyme Q10), vitamins (such as vitamin D), UV filters (i.e. sunscreens), lubricants and/or water. The pl6 inhibitors may be incorporated into vesicles, liposomes or microparticles to allow delivery of the pi 6 inhibitors to the skin. The composition may also be used to promote wound healing, and hence the present invention also provides the use of pi 6 inhibitors and compositions comprising the same in the promotion of wound healing (such as traumatic wounds and stalled wounds), as well as methods of promoting wound healing comprising administering a composition comprising an inhibitor of pi 6 expression to a patient in need thereof. Topical delivery of siRNA is described in Zheng et ah, Proc Natl Acad Sci USA, 2012, 109(30): 11975-80. The compositions and creams of the invention may be used in ex vivo methods of the present invention. Composition, solutions, cell culture media and other embodiments of the invention incorporating pl6 inhibitors may include more than one type of pl6 inhibitor. For example, they may include one or more pl6-targeted siRNAs as well as one or more anti-SA-miRNAs. Similarly, methods of preventing, inhibiting or reversing senescence may include use the use of more than one type of pi 6 inhibitor, for example the administration of both an pl6-targeted siRNA and one or more anti-SA-miRNAs.

There is provided a method of reversing senescence by inhibiting the expression of pi 6 in the cell. In a preferred embodiment, this is achieved by transfecting the cell with a pl6-targeted siRNA or a miRNA antagonist that targets a senescence-associated miRNA (suitably an SA-miRNA listed in Table 5). The cell to be contacted can be any cell capable of being cultured in vitro, for example HMEC cells. As a result of the inhibition of pl6, the cells can re-enter the cell cycle. pl6-mediated replicative senescence can be prevented by transfecting cells with the pl6-targeted siRNA or the anti-SA-miRNA of the invention before the senescent state is entered. In this way, the effective lifespan of the cells in vitro can be extended and the possible adverse consequences of entering senescence (for example when carrying out bone marrow transplants) avoided.

Features of the first aspect of the invention apply to the second and subsequent aspects of the invention, mutatis mutandis. The present invention will now be further described by reference to specific Examples. However, these examples are presented for illustrative purposes only and are not to be construed as limiting on the scope of the invention. In the Examples, references are made to a number of Figures, in which:

FIGURE 1 shows replicative senescence in HMECs: A - HMECs in culture undergo a gradual decay in proliferative potential, and reach replicative senescence at passage 11. B - Replicative senescence in HMECs is associated in an upregulation of pi 6 expression FIGURE 2 shows reversal of senescence in RS-HMECs. In particular:

A - Transmitted light images of RS-HMECs 9 days post transfection which demonstrate reversal of senescence accompanied by proliferation following transfection with pi 6 siRNA (right panel), relative to the negative control siRNA siGLO (left panel).

B - Quantitation of the reversal of senescence, demonstrating increased cell number, increased % BrdU positive cells and reduced % 8-oxoguanine negative cells at 2, 5, 7 and 9 days post transfection with pi 6 siRNA or anti- SA-miRNAs 26b, 181a, 210 or 424 relative to the siGLO siRNA negative control.

C - Quantitation of γΗ2ΑΧ foci per nucleus (0-1, 2-4, 5-9 or >10) shows no change in the number of foci per nucleus following transfection with siGLO siRNA, pl6 siRNA or anti- SA-miRNAs 26b, 181a, 210 or 424 at 2, 5, 7 or 9 days post transfection. Mitomycin C (mitoC, Day 2) serves as a positive control for the induction of double strand DNA breaks.

FIGURE 3 shows: A. Telomerase expression is not detectable (NT) in RS-HMECs transfected with siGLO. Transfection of RS-HMECs with pi 6 siRNA results in a re-activation of telomerase expression. This data is represented as a percentage of the expression level detected in HMECs stable transduced with a retroviral construct over expression hTERT (Telomerase), referred to as HMEC.hTERT. B. Serial dilution of HMEC.hTERT RNA used for the normalisation of the data shown in Figure 3 A.

FIGURE 4 shows: A. SA-miRNA Target prediction analysis highlights known regulators of the INK/ARF locus, including members of the Polycomb group of proteins (Polycomb), pl6 inhibitors/repressors (pl6 Inh), pl4 inhibitors/repressors (pl4 Inh) and modifying enzymes. B. miRNA target prediction for a control group of miRNAs highlighting the enrichment of predicted targets within the SA-miRNA group.

FIGURE 5 shows: A. qPCR for Polycomb members EZH2, EED, Suzl2, CBX7 and BMIl showing a down regulation of each member during replicative senescence (HMEC-P6 versus HMEC-P11). B. qPCR for Polycomb members CBZ7, EED, EZH2 and SUZ12 following transfection with siRNAs targeting siGLO, CBX7, pl6, or with SA-miRNAs 26,181,210 or 424 or anti-SA-miRNA-26,181,210 or 424. This confirms that the transient introduction of any one of the SA-miRNAs to HMECs generates a down regulation of each of the Polycomb members examined. Similarly, transfection with any one of the anti- SA-miRNAs generates an upregulation of the corresponding Polycomb genes. Of interest regulation of pi 6 also generates an upregulation of Polycomb gene expression, suggesting the existence of a feedback loop. FIGURE 6 shows: A. ChIP analysis reveals a reduction in the abundance of the Polycomb repressive mark H3K27Me3 at the genetic locus of SA-miRNA-181a, -181a-2, -210 and -424 during replicative senescence (HMEC-P6 versus RS-HMECs). We predict that the same Polycomb-mediated regulation occurs at other SA- miRNA genetic loci.

FIGURE 7 shows a cartoon representation of the model of SA-miRNA control of replicative senescence. Model of the pl6-coupled miRNA pathways that mediate replicative senescence. PcG members CBX7, EED, EZH2 and Suzl2 function to retrain replicative senescence by epigenetically repressing the expression of senescence- associated miRNAs (SA-miRNAs) SA-miRNA-26b, 181a, 210 and 424 and pi 6. CBX7 functions to positively promote the expression of other PcG members. The onset of replicative senescence disrupts this equilibrium and SA-miRNA expression is stimulated. SA-miRNAs directly target PcG mRNAs for degradation. This, together with SA-miRNA cross-talk, ensures continued expression of the SA-miRNA signature, and as a direct consequence, PcG-mediated repression of pi 6 is relieved and the senescence programme enforced, pi 6 , in turn, ensures self-reinforcement through a positive feedback loop with SA-miRNAs and a negative feedback loop with PcG members.

FIGURE 8 The miRNA nomenclature in miRBase is based on the precursor microRNAs (pre-miRNA, Figure 4), therefore there will be a miRNA-xx and a miRNA-xx* form. miRNA and miRNA* are the two strands of the double-stranded RNA product of dicer processing of the stem loop precursor miRNA (i.e. hsa-miR-26b and hsa- miR26b* are expressed from the same genetic locus as a single RNA). Because of the sequence complementarity they form a double stranded RNA product.

EXAMPLES Cells and Reagents

Normal finite life-span HMECs were obtained from reduction mammoplasty tissue of a 21 -year-old individual, specimen 184, and were cultured as previously described (Garbe et al, 2009) and were used for reconstruction, screening, and follow-up miRNA studies. HMEC Cell culture and antibody staining

Primary HMECs were cultured as previously described (Garbe et al., 2009). Cells at passage 6 (P6) were classified as a proliferative culture. Cells at passage 11 (PI 1) were termed replicative senescent HMECs (RS- HMECs) and were defined by the following criteria, each determined by immunofluorescence: 1. >90% of cells having the characteristic enlarged flattened shape of senescent cells. Cell area was quantified following staining with the fluorescent dye Cell mask (Invitrogen);

2. >95% of cells failing to incorporate BrdU after a 24 hour labelling procedure with lOuM BrdU (mouse anti-BrdU-FITC, Abeam);

3. >90% of cells positive for the senescence marker pi 6 (mouse anti-pl6 antibody, provided through collaboration with James Koh, Duke University Medical Center, USA); and 4. >90% of cells positive for the oxidative damage marker 8-oxoguanine (mouse anti-8-oxoguanine antibody, Millipore).

In each case cells were imaged using the IN Cell 1000 microscope (GE) and subjected to image analysis and quantitation using the Developer Analysis software (GE).

High-Content miRNA Screening

The screen was performed using the human genome miRNA library from Qiagen (miRBase Version 11.0, 837 miRNAs), together with control siRNAs targeting Cyclophilin B (siGLO, Dharmacon), CBX7 (Ambion AM 16704- 137077), and pl6 (Qiagen #SI02664403). HMECs at P6 were reverse transfected in triplicate with 60 nM siRNA/miRNA in 384-well format using HiperFect (Qiagen). Plates were incubated for 46 hours, medium changed, and fixed/stained 72 hours later with Moapl6 JC2, GtaMo AlexaFluor488 (Invitrogen), DAPI, and Cell Mask (Invitrogen). High-content images were acquired with the IN Cell 1000 automated microscope (GE), and analysis was performed using the Developer Analysis software (GE). miRNAs that induced pl6-associated senescence were defined according to the criteria described in (Bishop et ah, Mol. Cell., 2010, 40:533-547).

The present inventors sought senescence-associated (SA-miRNAs) that regulate pi 6 in HMECs by combining high-content screening with miRNA expression profiling. The high-content screening was performed in HMEC- P6 as described previously (Bishop et al, 2010) using a library of 837 miRNAs (Qiagen, miRBase Version 11.0). The miRNA expression profiling compared the relative expression levels of miRNAs from HMEC-P6 and RS-HMECs (Exiqon Array miRBase Version 13). This revealed a common list of SA-miRNAs, whose expression increased during senescence and correlated with increased pi 6 levels. A subset of these (miR-26b, 181a, 210 and 424) were explored further either individually or in combination. We predict that anti-miRNAs targeting the remaining SA-miRNAs may also have the potential to reverse senescence. miRNA inhibitors (anti-miRNAs) are small, chemically modified single-stranded RNA molecules designed to specifically bind to and inhibit endogenous miRNA molecules and enable miRNA functional analysis by down- regulation of miRNA activity. Anti-SA-miRNAs were purchased from Invitrogen, although other vendors are available. miRNA sequences are freely available via miRBase (http://www.mirbase.org/), as shown in Table 5:

Table 5: SA-miRNA sequences (pre-miRNA sequences prior to Dicer processing)

hsa-miR-424 CGAGGGGAUACAGCAGCAAUUCAUGUUUUGAAGUGUUCUAAAUGGUUCAAAAC GUGAGGCGCUGCUAUACCCCCUCGUGGGGAAGGUAGAAGGUGGGG (SEQ ID NO: 24; reverse complement sequence in SEQ ID NO: 25) miRNA Expression Profiling

HMECs were continually cultured from passage 4 through to RS-HMECs. miRNAs were isolated from biologically independent triplicates as HMEC-P6 and HMEC-P10 and subjected to miRNA microarray expression profiling (Exiqon Array miRBase Version 13.0). Following technical quality assessment and data normalisation, the Hy3/Hy5 ratios (log2 transformed) were calculated and two-way hierarchical clustering performed.

Identification of senescence-associated miRNAs

The hits from the high-content miRNA screen were cross referenced with those whose expression increased during replicative senescence to reveal the subset of senescence-associated miRNAs. A subset of these, miR- 26b, 181a, 210 and 424, were subjected to further investigation.

Immunofluorescence

Primary antibodies used were mouse anti-pl6 (Μοαρΐό) JC2 (Bishop et al., Mol. Cell., 2010, 40:533-547); MoaBrdU-FITC (Abeam ab74545); ΜοαγΗ2ΑΧ (Upstate 05-636); and Moa8-oxoguanine (Millipore MAB3560). Secondary antibodies were the appropriate AlexaFlur-488 or AlexaFlur-546 antibody (Invitrogen). DAPI and CellMask Deep Red (Invitrogen) were also included. Images were collected with the IN Cell 1000 microscope (GE) and Developer Software (GE) used for image analysis.

HMEC Transfection for reversal of senescence

RS-HMECs were seeded at 5000cells/cm² and incubated for 12-24 hours. Forward transfection was performed using 30-60nM siRNA (siGLO or pl6 siRNA, as above) or 60-90nM anti-miRNA complexed to the DharmaFect transfection reagent (Thermo Scientific). Cells were medium changed every 48 hours and fixed for immunofluorescence studies at appropriate intervals post transfection (typically 2,5,7 or 9 days).

The results shown in Figure 1 demonstrate the accumulation of pi 6 in cells in replicated cells. The results shown in Figure 2 demonstrate the highly surprising ability of pl6-targetting siRNA molecules and anti-SA- miRNAs to reverse replicative senescence. miRNA Target prediction analysis

miRNA target prediction analysis was performed for the SA-miRNAs as described in (Betel et al., Genome Biol., 2010, 11, R90) using the cut off of a miRSV score of <-0.4. The control group of miRNAs were defined as miRNAs that were expressed HMECs, showed no significant difference in expression levels (HMEC-P6 vs HMEC-P10), and were not family members of miRNAs defined as SA-miRNAs. miRNA Reporter Luciferase Assays

The 3' UTR of CBX7, EED, EZH2 and SUZ12 were cloned into the pGL3 Luciferase reporter construct (Promega). HeLa or HT1080 cells were transfected with SA-miRNAs or controls and then cotransfected with 3' UTR-pGL3 together with the Renilla Luficerase control construct (internal control for luciferase activity). After 48 hours, the cells were lysed and luciferase assays were conducted using the dual luciferase assay system (Promega, Madison, WI). Each experiment was performed in triplicate. Quantitative RT-PCR (qPCR)

qPCR reactions were performed with SYBR Green Master Mix (ABI) or Quantifast. For siRNA/SA-miRNA experiments, RNA (including miRNAs) was extracted from 1 x 105 cells 48 hr posttransfection. Alternatively, the same extraction procedure was followed to quantitate the levels of endogenous mRNA/miRNA levels. GAPDH levels were quantified for each cDNA sample in separate qPCR reactions and were used as an endogenous control. Target gene -expression levels were quantified using target specific probes. Values were normalized to the internal GAPDH control and expressed relative to siGLO transfected control levels (100%). All qPCR reactions were run in duplicate for two independent samples.

Chromatin immunoprecipitation (ChIP)

ChIP experiments were performed as previously described (Bishop et al, 2010). Briefly, exponentially growing cells (HMEC-P6) or fully senescence cells (RS-HMECs) were used for the ChIP protocol using ChIP grade antibodies to H3K27Me3 (Millipore/Upstate). qPCR was then performed using primers specific to the region of the genetic loci of SA-miRNAs.

Claims

1. A pi 6 inhibitor for use in the prevention, inhibition or reversal of senescence.

2. The pi 6 inhibitor for use as claimed in claim 1, wherein the prevention, inhibition or reversal of senescence occurs during the ex vivo manipulation of cells or biological tissue, optionally wherein the ex vivo manipulation of cells comprises culturing the cells in a cell culture medium.

3. The pl6-inhibitor for use as claimed in claim 1 or 2, wherein the pl6-inhibitor is a pl6-targeted small interfering RNA (siRNA) or an anti senescence-associated miRNA (anti-SA-miRNA).

4. The pl6-targeted siRNA for use as claimed in claim 3, wherein the siRNA targets a 10 to 50 nucleotide segment of the pi 6 gene or corresponding mRNA.

5. The anti-SA-miRNA for use as claimed in claim 3, wherein the SA-miRNA targeted by the anti-SA- miRNA is selected from the group consisting of hsa-miR-26b, hsa-miR-181a-l, hsa-miR-181-2, hsa-miR-210 and hsa-miR-424.

6. The anti-SA-miRNA for use as claimed in claim 3 or 5, wherein the anti-SA-miRNA is an antisense oligonucleotide.

7. An in vitro or ex vivo method of preventing, inhibiting or reversing senescence of a cell, comprising contacting the cell with a pl6-inhibitor, or comprising contacting a tissue comprising the cell with a pi 6 inhibitor, optionally wherein the pi 6 inhibitor is a pl6-targeted siRNA or anti-SA-miRNA.

8. An in vitro or ex vivo method of cell culture or cell manipulation, comprising contacting the cell with a pl6-inhibitor, or comprising contacting a tissue comprising the cell with a pl6-inhibitor, optionally wherein the pl6 inhibitor is a pl6-targeted siRNA or anti-SA-miRNA.

9. The method of claim 7 or 8, wherein the method further comprises obtaining the cell or tissue from a patient.

10. The method of any one of claims 7 to 9, further comprising contacting the cell or tissue with a transfection reagent.

11. The method of any one of claims 7 to 10, wherein the step of contacting the cell or tissue comprises transfecting the cell or tissue with the pl6 inhibitor, the pl6-targeted siRNA or the anti-SA-miRNA.

12. The method of any one of claims 7 to 11, wherein the method further comprises the step of culturing or expanding the cell or tissue in a cell culture medium.

13. The method of any one claims 7 to 12, further comprising the step of subjecting the cell or tissue to enzymatic and/or mechanical segregation.

14. The method of any one of claims 7 to 13, further comprising the step of administering the cell or tissue to a patient in need thereof.

15. The methods of any one of claims 7 to 14, wherein the method is a method of skin tissue engineering, stem cell transplantation, hematopoietic stem cell transplantation, corneal transplant, stem cell therapy or renal allograft transplantation.

16. Use of a pl6 inhibitor in the prevention, inhibition or reversal of senescence.

17. The use of a pl6 inhibitor as claimed in claim 16, wherein the prevention, inhibition or reversal of senescence occurs during ex vivo or in vitro cell manipulation or cell culture.

18. The use of a pl6 inhibitor as claimed in claim 16 or claim 17, wherein the pl6 inhibitor is a pie- targeted siRNA or an anti-SA-miRNA.

19. Use of a pl6-targeted siRNA or an anti-SA-miRNA in cell culture or as a component of a cell culture medium.

20. Use of a pl6-targeted siRNA or an anti-SA-miRNA in cell, tissue or organ transplantation.

21. A cell culture medium comprising a pl6-targeted siRNA or an anti-SA-miRNA.

22. The cell culture medium of claim 21 further comprising one or more components selected from the group consisting of a source of amino acids and a salt.

23. The cell culture medium of claim 21 or 21 further comprising one or more components selected from the group consisting of glucose, sodium bicarbonate, amino acids, phenol red, pyridoxine, sodium pyruvate and oxytocin.

24. The cell culture medium of any one of claims 21 to 23, wherein the cell culture medium further comprises serum.

25. The cell culture medium of any one of claims 21 to 23, wherein the cell culture medium is chemically defined.

26. The cell culture medium of any one of claims 21 to 25, wherein the cell culture medium further comprises a source of nitrogen, optionally wherein the source of nitrogen is glutamine or a source of glutamine.

27. The cell culture medium of any one of claims 21 to 26, wherein the source of amino acids is glutamine or a source of L-glutamine.

28. The cell culture medium of any one of claims 21 to 27, wherein the cell culture medium further comprises a source of carbon, optionally wherein the source of carbon is glucose or galactose.

29. The cell culture medium of any one of claims 21 to 28, wherein salt is selected from the group consisting of calcium chloride, ferric nitrate, magnesium sulfate, potassium chloride, sodium chloride, sodium bicarbonate and sodium phosphate.

30. A reperfusion solution comprising a pl6 inhibitor and one or more volume expanders, optionally wherein the pi 6 inhibitor is a pl6-targeted siRNA or an anti-SA-miRNA

31. The reperfusion solution of claim 30, wherein the volume expander is a crystalloid or a colloid, or a combination of a crystalloid and a colloid.

32. The reperfusion solution of claim 30 or 31 , further comprising:

about 100 mmol/L to about 150 mmol/L of sodium ions,

about 90 mmol/L to about 120 mmol/L of chloride ion,

about 20 mmol/L to about 30 mmol/L of lactate,

about 2 mmol/L to about 6 mmol/L of potassium ions; and

about 1 mmol/L to about 5 mmol/L of calcium ions.

33. A kit of parts comprising a cell culture medium and a pl6 inhibitor, optionally wherein the pl6 inhibitor is a pl6-targeted siRNA or an anti-SA-miRNA.

34. The kit of parts of claim 33 further comprising a transfection reagent.

35. The kit of parts of claim 33 or claim 34, further comprising instructions for use.

36. A method of treating an age-related disease or condition, comprising administering a pl6 inhibitor to a patient in need thereof, optionally where in the pi 6 inhibitor is a pl6-targeted siRNA or an anti-SA-miRNA

37. A pl6-inhibitor for use in the treatment of an age-related disease or condition, optionally wherein the pl6 inhibitor is a pl6-targeted siRNA or an anti-SA-miRNA

38. The method of claim 36, or the pl6-inhibitor, the pl6-targeted siRNA or the anti-SA-miRNA for use as claimed in claim 37, wherein the age-related disease or condition is Alzheimer's disease, coronary heart disease, Parkinson's disease, myocardial infarction, ischemic stroke, abdominal aortic aneurism, arterial stiffness, frailty, perdiodontal disease, type 2 diabetes, ageing, synaptic loss, white matter atrophy, cognitive impairment, skin thinning, vision impairment, cataracts or muscle wastage.

39. A method of treating infection, cancer, alopecia, neurodegeneration, gum disease, bone degeneration, or degeneration of the cardiovascular, digestive, integumentary, lymphatic or musculoskeletal system comprising administering a pi 6 inhibitor to a patient in need thereof, optionally where in the pi 6 inhibitor is a pl6-targeted siRNA or an anti-SA-miRNA.

40. A pl6-inhibitor for use in the treatment infection, cancer, alopecia, neurodegeneration, gum disease, bone degeneration, or degeneration of the cardiovascular, digestive, integumentary, lymphatic or musculoskeletal system, optionally wherein the pl6 inhibitor is a pl6-targeted siRNA or an anti-SA-miRNA

41. An anti-ageing or anti-wrinkle composition comprising a pi 6 inhibitor, optionally wherein the pi 6 inhibitor is a pl6-targeted siRNA or an anti-SA-miRNA.

42. The anti-ageing or anti-wrinkle composition of claim 40, wherein the composition further comprising a pharmaceutically acceptable carrier.

43. The anti-ageing or anti-wrinkle composition of claim 40 or claim 41, wherein the composition is an emulsion.

44. A pharmaceutical composition comprising a pl6 inhibitor and a pharmaceutically acceptable carrier, optionally wherein the pl6 inhibitor is a pl6-targeted siRNA)or an anti-SA-miRNA.

45. A pi 6 inhibitor for use in rejuvenative medicine or for use in stem cell rejuvenation therapy, optionally wherein the pi 6 inhibitor is a pl6-targeted siRNA or an anti-SA-miRNA.

46. A method of cell, tissue or organ transplantation, comprising:

obtaining a cell sample, tissue sample or organ from a patient;

optionally subjecting a cell sample or tissue sample to enzymatic and/or mechanical separation; and culturing the cell sample or tissue sample in the presence of a pi 6 inhibitor or reperfusing the organ in a reperfusion solution as defined in any one of claims 30 to 32.

47. The pl6-targeted siRNA for use as claimed in any one of claims claim 3, 4, 37, 38, 40 or 45, the method of any one of claims 7 to 15, 36, 38, 39 or 46, the use of pl6-targeted siRNA of any one of claims 17 to 20, the cell culture medium of any one of claims 21 to 29, the reperfusion solution of any one of claims 30 to 32, the kit of parts of any one of claims 33 to 35, the anti-ageing or anti-wrinkle composition of any one of claims 41 to 43, or the pharmaceutical composition of claim 44, wherein the pl6-targeted siRNA is a double stranded RNA molecule that hybridizes with an mRNA encoding pi 6 protein, optionally wherein the siRNA hybridizes with the nucleic acid sequence TACCGTAAATGTCCATTTATA or a corresponding mRNA sequence thereof.

48. The anti-SA-miRNA for use as claimed in any one of claims claim 3, 5, 6, 37, 38, 40 or 45, the method of any one of claims 7 to 15, 36, 38, 39 or 46, the use of pl6-targeted siRNA of any one of claims 17 to 20, the cell culture medium of any one of claims 21 to 29, the reperfusion solution of any one of claims 30 to 32, the kit of parts of any one of claims 33 to 35, the anti-ageing or anti-wrinkle composition of any one of claims 41 to 43, or the pharmaceutical composition of claim 44, wherein the anti-SA-miRNA is a nucleic acid that hybridizes with an miRNA selected from the group consisting of hsa-miR-26b, hsa-miR- 181 a- 1 , hsa-miR-181-2, hsa-miR- 210 and hsa-miR-424.