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Definitions

• the present application is in the field of cellular biology and more particularly, in the field of the stem cells. It relates to the method for reprogramming induced pluripotent stem cells (iPSC) comprising a transduction of somatic differentiated cells with vector expressing p53 isoform, to the induced pluripotent stem cells obtained by the method of the invention and their use.
• iPSC induced pluripotent stem cells
• the present invention also relates to a method for detecting induced pluripotent stem cells (iPSC).
• Induced pluripotent stem cells are a type of pluripotent stem cell generated directly from adult cells.
• the iPSC technology was pioneered by Japanese Shinya Yamanaka, demonstrating in 2006 that the introduction of four specific genes (Oct4, Sox2, cMyc and Nanog) encoding transcription factors could convert adult cells into pluripotent stem cells.
• pluripotent stem cells hold great promise in the field of regenerative medicine. Its objectives are to repair tissues altered by accident, diseases or aging. This represents a new therapeutic field with tremendous medical impact because it offers the possibility to treat and cure diseases currently without adequate treatment.
• Regenerative medicine applies to most medical domains and constitutes one of the most promising developments of the biotechnology industry. Ischemic, degenerative and/or ageing- associated diseases are the major causes of mortality in the population of developed countries.
• stem cells could be a source of cells for cellular therapy for these diseases because of their proliferative and differentiating capacity.
• medical applications of embryonic stem cells are hampered by immunological and ethical concerns.
• iPSCs offer autologous cell sources for replacement cell therapy, to replace or regenerate tissues by autologous transplantation. Moreover, patient-specific iPSCs can serve as in vitro models for disease mechanism modeling and drug screening.
• the original set of reprogramming factors (also called Yamanaka factors) comprises the genes Oct4, Sox2, cMyc and Nanog. More recently, some specific combinations including three of these factors were reported to also generate iPSC. However, reprogramming of iPSC using these factors only occurs in a very small percentage of transfected cells demonstrating that the therapeutic use of iPSCs has to face many barriers among which safety, regulatory issues, financial viability, low reprogramming efficiency and an uncertain stability of derived iPSC.
• the original Yamanaka factors include c-myc, an oncogene that is known to alter DNA repair machinery and to result in DNA lesions and chromosomal aberrations. While this factor is in fact not necessary to induce iPSCs, using only the other Yamanaka factors results in decreased reprogramming efficiency.
• p53 in stem cell (SC) homeostasis and in pluripotency induction. p53 not only ensures genomic integrity after genotoxic insults but also controls SC proliferation and differentiation. Additionally, p53 provides an effective barrier to the generation of iPSCs from terminally differentiated cells. If wild-type (wt) p53 has inhibitory effects, some p53 mutants display completely opposite effects (Sarig et al, 2010).
• wt wild-type
• p53 mutants display completely opposite effects (Sarig et al, 2010).
• a recent genome wide study demonstrated that p53 regulates approximately 3600 genes in mouse ES cells (Li et al, 2012). Out of these, about 2000 genes are positively regulated while 1600 are repressed. Positively regulated genes are enriched in genes responsible for differentiation while negatively regulated genes, in maintaining ES cell status.
• p53 represses key regulators of ES phenotype like Oct 4, Nanog, Sox2, c-myc (Li et al, 2012; Lin et al, 2005). Consequently, recent reports showed that p53 is an obstacle to cellular reprogramming through p21 signalling pathway (Hong et al, 2009).
• generating iPSC by p53 depletion or expression of mutated p53 proteins carries great risk due to the fact that these cells present tumor like features and develop malignant tumors when injected in mice.
• the permanent suppression of p53 may thus lower the quality of iPSCs, in particular by accumulating DNA lesions and chromosomal aberrations during iPSC derivation and maintenance (Tapia & Scholer, 2010). So the use in cell therapy of iPSC obtained by the depletion of p53 genes or the expression of mutated p53 proteins is hazardous and unsure.
• the TP53 gene encodes at least twelve different physiological isoforms [TAp53 ( ⁇ , 6 and ⁇ ), ⁇ 40 ⁇ 53 ( ⁇ , ⁇ and ⁇ ), ⁇ 1 33 ⁇ 53 ( ⁇ , ⁇ and ⁇ ) and ⁇ 160 ⁇ 53 ( ⁇ , ⁇ and ⁇ )] (Bourdon, 2007) via several mechanisms: use of alternative promoters (the TA and ⁇ 1 33 isoforms), alternative intron splicing (intron 2: ⁇ 40 isoforms and intron 9: ⁇ , ⁇ and ⁇ isoforms) and alternative translational initiation sites ( ⁇ 40 isoforms and ⁇ 160 isoforms).
• FIG. 1 A scheme summarizing the features of isoforms a, B and ⁇ of TAp53, ⁇ 40 ⁇ 53, and ⁇ 1 33 ⁇ 53 is presented in Figure 1 .
• the TAp53a isoform is the best described and classically mentioned in the literature as p53.
• p53 isoforms can be divided in two groups: long isoforms that contain the transactivation domain (TA and ⁇ 40) and short isoforms without transactivation domain ( ⁇ 1 33 and ⁇ 160).
• the 6 and ⁇ isoforms do not contain the canonical C-terminal oligomerization domain, but an additional domain with unknown function(s) to date (Khoury and Bourdon, 201 1 ).
• p53 isoforms can regulate p53 transcriptional activity and have different outcomes on cell fate by regulating cell cycle progression, programmed cell death, replicative senescence, viral replication, cell differentiation and angiogenesis (Aoubala et al, 201 1 ; Bourdon et al, 2005; Hofstetter et al, 2012; Nutthasirikul et al, 201 3; Terrier et al, 2012).
• WO 2012/044979 discloses the use of ⁇ 1 33 ⁇ 53 ⁇ isoform for increasing reprogramming efficiency and thus iPSCs induction in the presence of at least one reprogramming factor selected from Yamanaka factors.
• this document contains no data showing that iPSC obtained with the method disclosed are genetically stable and do not have DNA damages or alterations in DNA repair machinery. Consequently, there is still a need for methods allowing efficient reprogramming of somatic cells to induced pluripotent stem cells (iPSC) that are free of genetic damages and have normal somatic cells functions and in particular an unaltered DNA repair machinery, thus guaranteeing their genetic stability and their possible clinical use.
• iPSC induced pluripotent stem cells
• ⁇ 1 33 ⁇ 536 isoform and/or ⁇ 1 33 ⁇ 53 ⁇ is involved in stem cell reprogramming.
• ⁇ 1 33 ⁇ 536 and/or ⁇ 1 33 ⁇ 53 ⁇ isoforms is (are) able to induce stem cell reprogramming of somatic differentiated cells in the absence of heterologous induction of any of the Yamanaka transcription factors (Oct4, Sox2, cMyc and Nanog) or without depleting or inducing mutations in p53 genes .
• the overexpression of these isoforms does not alter DNA in the reprogrammed iPSC.
• DNA repair checkpoint response also remains functional.
• the inventors further demonstrated that among ⁇ 133 ⁇ 53 isoforms, only isoforms 6 and Y are able to induce a reprogramming in the absence of all Yamanaka factors, the expression of ⁇ 133 ⁇ 53 ⁇ isoform not permitting to induce iPSCs in the absence of Yamanaka factors. They further demonstrated that expression of ⁇ 133 ⁇ 536 isoform does not alter the DNA repair machinery of reprogrammed iPSCs, contrary to expression of ⁇ 133 ⁇ 53 ⁇ isoform, the expression of which results in decrease of the accumulation of p21 WAF1 in the presence of DNA damage, indicating that the DNA repair function of p53 is altered in cells expressing ⁇ 133 ⁇ 53 ⁇ isoform.
• the present invention thus relates to a reprogramming method induced pluripotent stem cells (iPSC) comprising
• iPSC induced pluripotent stem cells
• ⁇ 133 ⁇ 53 ⁇ and particularly ⁇ 133 ⁇ 536 are highly expressed in the obtained iPSC.
• a high expression indicates that an elevated level of these isoforms is not associated with genetic instability or alteration of the DNA repair response, contrary to the one due to the overexpression of Yamanaka transcription factors or to the depletion or the expression of mutated p53 proteins. Consequently, using physiological p53 isoforms in the reprogramming the method of the present invention allows reducing the risk of genetic alteration because the ⁇ 133 ⁇ 536 isoforms do not alter the DNA repair response) and thus obtaining induced pluripotent stem cells with better efficiency and stability, which may be used in cell therapy.
• the present invention thus relates to induced pluripotent stem cells (iPSC) obtained by the reprogramming method of the invention.
• the iPSC obtained by the method of the invention contain the vector expressing ⁇ 133 ⁇ 53 ⁇ and/or ⁇ 133 ⁇ 53 ⁇ isoforms.
• iPSC may be used in cell therapy and /or as in vitro model to study a variety of diseases.
• the present invention thus relates to iPSC as obtained from the reprogramming method of the invention for use in cell therapy and/or as models to study a variety of diseases.
• induced pluripotent stem cells specifically express ⁇ 133 ⁇ 536 isoform, ⁇ 133 ⁇ 53 ⁇ isoform, or bothA133p536 and ⁇ 133 ⁇ 53 ⁇ isoforms also allows detecting iPSC by detecting ⁇ 133 ⁇ 536, ⁇ 133 ⁇ 53 ⁇ isoform, or bothA133p536 and ⁇ 133 ⁇ 53 ⁇ isoforms in a cell population containing iPSC.
• the present invention thus relates to a method for detecting Induced pluripotent stem cells (iPSC) comprising detecting the presence of ⁇ 133 ⁇ 536 isoform ⁇ 133 ⁇ 53 ⁇ isoform, or both ⁇ 133 ⁇ 536 and ⁇ 133 ⁇ 53 ⁇ isoforms by detecting ⁇ 133 ⁇ 536 isoform, ⁇ 133 ⁇ 53 ⁇ isoform, or both ⁇ 133 ⁇ 536 and ⁇ 133 ⁇ 53 ⁇ isoforms polypeptide and/or a fragment thereof, and/or by detecting ⁇ 133 ⁇ 536, ⁇ 133 ⁇ 53 ⁇ isoform, or both ⁇ 133 ⁇ 536 and ⁇ 133 ⁇ 53 ⁇ isoforms mRNA and/or a fragment thereof.
• iPSC Induced pluripotent stem cells
• FIG. 1 Schematic representation of human p53 isoforms.
• the human p53 gene contains a proximal (P1 ) and an internal (P2) promoter, regulating the expression of p53 and ⁇ 133 ⁇ 53 transcripts, respectively.
• the p53 transcript encodes two p53 isoforms: p53 and ⁇ 40 ⁇ 53, produced by internal initiation of translation using ATG 40.
• the p53 gene expresses three alternatively spliced carboxy-terminal isoforms ( ⁇ , 6 and ⁇ ).
• ⁇ 53 ⁇ and ⁇ 53 ⁇ protein isoforms differ from full-length p53 (FLp53) in that they lack the classical oligomerization domain.
• the ⁇ 1 33 ⁇ 53 transcript encodes both ⁇ 1 33 ⁇ 53 and ⁇ 160 ⁇ 53 isoforms, which combined with the alternatively spliced an°6nand°Y.exons generates ⁇ 1 33 ⁇ 53 ⁇ ⁇ 1 33 ⁇ 53 ⁇ , ⁇ 1 33 ⁇ 53 ⁇ and ⁇ 160 ⁇ 53 ⁇ ⁇ 160 ⁇ 53 ⁇ , ⁇ 160 ⁇ 53 ⁇ (Bourdon et al, 2005; Marcel et al, 2010). These isoforms are deleted of the N-terminal transactivation domains and part of the DNA-binding domain.
• Figure 2 Level of expression of ⁇ 1 33 ⁇ 53 ⁇ versus TBP in cells infected with 4 factors (Oct4, Sox2, Klf4, c-myc) and 6 factors (Oct4, Sox2, Klf4, c-myc, Nanog and Lyn28) after three weeks.
• ⁇ 1 33 ⁇ 53 ⁇ is highly expressed in human cells expressing the 6 Factors OSKMNL compared to 4 factors OS M
• Figure 3 ⁇ 1 33 ⁇ 536 isoform expression in fibroblasts and stem cells.
• ⁇ 1 33 ⁇ 53 ⁇ is highly expressed in human embryonic stem cells and in iPSCs.
• ⁇ 1 33 ⁇ 53 ⁇ mRNA levels were analyzed by qPCR in human embryonic stem cells before (ES) and after (ES-F) fibroblastic differentiation, in skin fibroblasts before (Sk-F) and after (iPSCs) reprogramming and after re-differentiation (iPSC-F) and from human neonatal (HFFSM3).
• ⁇ 1 33 ⁇ 53 ⁇ is clearly increased in embryonic stem cells and in iPSCs compared to fibroblasts.
• Figure 4 Images of iPSC-like cells, produced only with the expression of ⁇ 1 33 ⁇ 53 ⁇ 10, 14 and 22 days post infection as indicated
• Figure 5 Influence of ⁇ 1 33 ⁇ 536 isoform on the expression of the pluripotency factors. Expression of ⁇ 1 33 ⁇ 536 elicits an increase in Sox2, Nanog, Oct 3/4 and to a lesser extent c-myc mRNA levels between the first and the third weeks after reprogramming.
• Figure 6 Human skin fibroblasts expressing either empty vector (Empty vector) or ⁇ 1 33 ⁇ 53 ⁇ isoform ( ⁇ 1 33 ⁇ ) or ⁇ 1 33 ⁇ 536 isoform ( ⁇ 1 336).
• A mRNA expression of p53 isoforms ( ⁇ 1 33 ⁇ 53 ⁇ and ⁇ 1 33 ⁇ 53 ⁇ ) measured by qPCR with specific primers to detect all isoforms (ALL) and primers to detect all isoforms except ⁇ 1 33 (TAP53). qPCR results are expressed as target gene/ reference gene ratio (TBP, TATA binding protein).
• B mRNA expression of p21 and MDM2 measured by qPCR.
• Table 1 below provides amino acids and nucleic acid sequences of full- length p53 (denoted as "p53”), and of ⁇ 133 ⁇ 536 and ⁇ 133 ⁇ 53 ⁇ isoforms.
• iPSC Induced pluripotent stem cells
• induced pluripotent stem cells or “iPSC” refer to pluripotent stem cells obtained from a non-pluripotent cell, typically an adult somatic cell (a cell of the body, rather than gametes or an embryo), by inducing a "forced” expression of certain genes.
• iPSCs are believed to be similar to natural pluripotent stem cells, such as embryo stem cells (ESCs) in many respects.
• ESCs embryo stem cells
• iPSCs are not adult stem cells, but reprogrammed cells given pluripotent capabilities. iPSCs do not refer to cells as they are found in nature.
• somatic cells or “somatic differentiated cell” refer to differentiated adult cells.
• the somatic cells or somatic differentiated cells may be non-cancerous (also called “normal cells”) or cancerous cells.
• the somatic cells or somatic differentiated cells used in the present application are non-cancerous cells.
• reprogramming refers to a process that alters or reverses the differentiation status of a somatic cell that is either partially or terminally differentiated.
• DNA damage refers to an alteration in the chemical structure of DNA, such as a single stranded or double stranded DNA break ithat may be cause by internal or external agents.
• Damage to DNA may occur naturally as a result of metabolic alterations (leading to increased production of reactive oxygen species, reactive nitrogen species, reactive carbonyl species, lipid peroxidation products and alkylating agents) can result from metabolic (metabolism releases compounds that damage DNA including reactive oxygen species, reactive nitrogen species, reactive carbonyl species, lipid peroxidation products and alkylating agents) or hydrolytic processes (leading to cleavage of chemical bonds in DNA). DNA damage may also be caused by external factors like UV exposition or gamma irradiations.
• DNA repair or “DNA repair checkpoint response” or just “DNA repair response” or “DNA repair mechanisms” or “DNA repair machinery” refers to several processes by which a cell identifies and corrects the DNA damage. For each type of DNA damage, the cell has evolved a specific method of repairing the damage or eliminating the damaging compound.
• Various cell proteins are known to be involved in DNA repair and may thus be tested in order to determine the presence or absence of DNA repair alterations in a given cell. Examples of such proteins known to be involved in DNA repair include two p53 targets: p21 WAF-1 (inhibitors of cyclin complex/Cdks) and Mdm2 (proteins involved in p53 degradation).
• Plasmid vector refers to a replicable DNA construct.
• viral vector refers to a nucleic acid vector that includes at least one element of a virus genome and may be packaged into a viral particle.
• virus refers to a nucleic acid vector that includes at least one element of a virus genome and may be packaged into a viral particle.
• viral refers to viral particles that are formed when the nucleic acid vector is transduced into an appropriate cell or cell line according to suitable conditions allowing the generation of viral particles.
• viral vector has to be understood broadly as including nucleic acid vector (e.g. DNA viral vector) as well as viral particles generated thereof.
• infectious refers to the ability of a viral vector to infect and enter into a host cell or subject.
• regulatory elements or “regulatory sequence” refers to any element that allows, contributes or modulates the expression of nucleic acid molecule(s) in a given host cell or subject, including replication, duplication, transcription, splicing, translation, stability and/or transport of the nucleic acid(s) or its derivative (i.e. mRNA).
• mRNA Ribonucleic acid
• an aging-associated disease refers to a disease that is seen with increasing frequency with increasing senescence.
• Age-associated diseases are to be distinguished from the aging process itself because all adult humans age, but not all adult humans experience all age-associated diseases.
• degenerative diseases refers to medical problems that worsen over time. These degenerative diseases may affect the central nervous system (brain and spinal cord), bones, blood vessels or heart.
• ⁇ 133 ⁇ 536 isoform ⁇ 133 ⁇ 53 ⁇ isoform, or both ⁇ 133p536 and ⁇ 133 ⁇ 53 ⁇ isoforms have the capacity:
• the present invention thus relates to a reprogramming method for producing induced pluripotent stem cells (iPSC), comprising:
• the reprogramming method of the present invention is an in vitro method.
• the reprogramming method of the invention does not comprise a step of expression of Yamanaka transcription factors (Oct4, Sox2, cMyc and Nanog) or a depletion of p53 or an expression of p53 mutated proteins.
• the somatic differentiated cells are not and have not been previously transduced with one or more vector(s) expressing one or more of Yamanaka transcription factors (Oct4, Sox2, cMyc and Nanog), one or more p53 mutated proteins, or an inhibitor of p53.
• Somatic dedifferentiated cells for use with the reprogramming method of the present invention may be primary cells or immortalized cells.
• Such cells may be primary cells (non- immortalized cells), such as those freshly isolated from an animal, or may be derived from a cell line (immortalized cells).
• the somatic cells are mammalian cells, such as, for example, human cells or mouse cells. They may be obtained by well-known methods, from different organs, such as, but not limited to skin, lung, pancreas, liver, stomach, intestine, heart, reproductive organs, bladder, kidney, urethra and other urinary organs, or generally from any organ or tissue containing living somatic cells.
• Mammalian somatic cells useful in the present invention include, by way of example, adult stem cells, Sertoli cells, endothelial cells, granulosa epithelial cells, neurons, pancreatic islet cells, epidermal cells, epithelial cells, hepatocytes, hair follicle cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, lymphocytes (B and T lymphocytes), erythrocytes, macrophages, monocytes, mononuclear cells, fibroblasts, cardiac muscle cells, other known muscle cells, and generally any live somatic cells. These cells may be non-cancerous or cancerous cell, preferably these cells are non-cancerous cells.
• the somatic differentiated stem cells used with the reprogramming method of the invention are selected from the group consisting of human peripheral blood mononuclear cells (PBMC), CD4+ lymphocytes or fibroblasts. More preferably, the somatic differentiated cells are human fibroblasts.
• ⁇ 133 ⁇ 536 isoform In a preferred embodiment of the method for producing induced pluripotent stem cells according to the invention, it is the ⁇ 133 ⁇ 536 isoform, ⁇ 133 ⁇ 53 ⁇ isoform or both ⁇ 133 ⁇ 536 and ⁇ 133 ⁇ 53 ⁇ isoforms (preferably only the ⁇ 133 ⁇ 536 isoform) that is/are transduced in somatic differentiated cells in step b).
• expression of ⁇ 133 ⁇ 536 isoform is particularly associated to induction of pluripotent stem cells.
• Any appropriate vector expressing ⁇ 133 ⁇ 536 isoform, ⁇ 133 ⁇ 53 ⁇ isoform, or both ⁇ 133 ⁇ 536 and ⁇ 133 ⁇ 53 ⁇ isoforms may be used.
• a suitable vector comprises a nucleic acid molecule encoding ⁇ 133 ⁇ 536 isoform, ⁇ 133 ⁇ 53 ⁇ isoform, or both ⁇ 133 ⁇ 536 and ⁇ 133 ⁇ 53 ⁇ isoforms and elements necessary to allow expression thereof.
• Suitable vectors notably include plasmid vectors and viral vectors.
• Viral vectors can be replication-competent or -selective (e.g. engineered to replicate better or selectively in specific host cells), or can be genetically disabled so as to be replication-defective or replication-impaired.
• such vectors are commercially available (e.g. in Invitrogen, Stratagene, Amersham Biosciences, Promega, etc.) or available from depositary institutions such as the American Type Culture Collection (ATCC, Rockville, Md.) or have been the subject of numerous publications describing their sequence, organization and methods of producing, allowing the artisan to apply them.
• a plasmid vector expressing ⁇ 133 ⁇ 536 isoform, ⁇ 133 ⁇ 53 ⁇ isoform, or both ⁇ 133 ⁇ 536 and ⁇ 133 ⁇ 53 ⁇ isoforms is used.
• plasmid vectors include, without limitation, pREP4, pCEP4 (Invitrogen), pCI (Promega), pVAX (Invitrogen) and pGWiz (Gene Therapy System Inc).
• a plasmid vector may be complexed to lipids or polymers to form particulate structures such as liposomes, lipoplexes or nanoparticles.
• a viral vector expressing ⁇ 133 ⁇ 536 isoform, ⁇ 133 ⁇ 53 ⁇ isoform, or both ⁇ 133 ⁇ 536 and ⁇ 133 ⁇ 53 ⁇ isoforms is used (i.e a viral vector comprising a nucleic acid molecule encoding ⁇ 133 ⁇ 536 isoform, ⁇ 133 ⁇ 53 ⁇ isoform, or both ⁇ 133 ⁇ 536 and ⁇ 133 ⁇ 53 ⁇ isoforms and elements necessary to allow expression thereof).
• viral vectors are generated from a variety of different viruses (e.g. retrovirus, adenovirus, adenovirus-associated virus (AAV), poxvirus, herpes virus, measles virus, foamy virus, alphavirus, vesicular stomatis virus, etc).
• viruses e.g. retrovirus, adenovirus, adenovirus-associated virus (AAV), poxvirus, herpes virus, measles virus, foamy virus, alphavirus, vesicular stomatis virus, etc.
• retrovirus e.g. retrovirus, adenovirus, adenovirus-associated virus (AAV), poxvirus, herpes virus, measles virus, foamy virus, alphavirus, vesicular stomatis virus, etc.
• AAV adenovirus-associated virus
• poxvirus e.g. retrovirus, adenovirus-associated virus (AAV), poxvirus, herpes virus,
• a retroviral vector expressing ⁇ 133 ⁇ 536 isoform, ⁇ 133 ⁇ 53 ⁇ isoform, or both ⁇ 133 ⁇ 536 and ⁇ 133 ⁇ 53 ⁇ isoforms isoform is used (i.e a retroviral vector comprising a nucleic acid molecule encoding ⁇ 133 ⁇ 536 isoform, ⁇ 133 ⁇ 53 ⁇ isoform, or both ⁇ 133 ⁇ 536 and ⁇ 133 ⁇ 53 ⁇ isoforms and elements necessary to allow expression thereof).
• Retroviruses have the property of infecting, and in most cases integrating into, dividing cells and in this regard are particularly appropriate for use in the context of the present invention for producing induced pluripotent stem cells.
• a suitable retrovirus generally contains the LTR sequences, an encapsidation region and a nucleic acid molecule encoding ⁇ 133 ⁇ 536 0 ⁇ ⁇ 133 ⁇ 53 ⁇ isoform.
• the recombinant retrovirus can be derived from a retrovirus of any origin (murine, primate, feline, human, etc.) and in particular from the MoMuLV (Moloney murine leukemia virus), MVS (Murine sarcoma virus), Friend murine retrovirus (Fb29), Murine Embryonic Stem Cell Virus (MESV), LN virus or Murine Stem Cell Virus (MSCV). It is propagated in an encapsidation cell line which is able to supply in trans the viral polypeptides gag, pol and/or env which are required for constituting a viral particle.
• MoMuLV Moloney murine leukemia virus
• MVS Murine sarcoma virus
• Fb29 Friend murine retrovirus
• MEV Murine Embryonic Stem Cell Virus
• MSCV Murine Stem Cell Virus
• the retroviral vector according to the invention can contain modifications, in particular in the LTRs (replacement of the promoter region with a eukaryotic promoter) or the encapsidation region (replacement with a heterologous encapsidation region).
• the vector used for transducing cancer cells in step b) is a Murine Stem Cell Virus (MSCV), which is derived from the Murine Embryonic Stem Cell Virus (MESV) and the LN retroviral vectors (Grez, M., et al. 1990; Miller, A. D. et al. 1989).
• MSCV Murine Stem Cell Virus
• the transducing vector may be obtained by cloning a molecule encoding ⁇ 133 ⁇ 536 isoform, ⁇ 133 ⁇ 53 ⁇ isoform, or both ⁇ 133 ⁇ 536 and ⁇ 133 ⁇ 53 ⁇ isoforms into a pMSCV vector commercialized by Clontech, such as pMSCVhyg, pMSCVneo, or pMSCVpuro.
• viral vectors examples include adenoviral vectors, which may be derived from a variety of human or animal sources (e.g. canine, ovine, simian adenovirus, etc). Any serotype can be employed with a special preference for human adenoviruses and a specific preference for subgenus C such as Ad2, Ad5, Ad6, and subgenus B such as Ad11 , Ad34 and Ad35.
• adenovirus are available from ATCC or have been the subject of numerous publications describing their sequence, organization and methods of producing, allowing the artisan to apply them.
• an adenoviral vector When an adenoviral vector is used, it is preferably an E1 -defective adenoviral vector with an E1 deletion extending from approximately positions 459 to 3328 or from approximately positions 459 to 3510 (by reference to the sequence of Ad5 disclosed in the GenBank under the accession number M73260.1 ).
• the cloning capacity can further be improved by deleting additional portion(s) of the adenoviral genome (all or part of the non-essential E3 region (e.g. deletion from approximately positions 27867 to 30743) or of other essential E2 and/or E4 regions.
• nucleic acid molecule encoding ⁇ 133 ⁇ 536 isoform, ⁇ 133 ⁇ 53 ⁇ isoform, or both ⁇ 133 ⁇ 536 and ⁇ 133 ⁇ 53 ⁇ isoforms can then be inserted in any location of the adenoviral genome, with a specific preference for insertion in replacement of the E1 and /or E3 region. They may be positioned in sense or antisense orientation relative to the natural transcriptional direction of the region in question.
• viral vectors examples include poxvirus vectors such as fowlpox vectors (e.g. FP9), canarypox vectors (e.g. ALVAC) and vaccinia virus vectors, the latter being preferred.
• poxvirus vectors such as fowlpox vectors (e.g. FP9), canarypox vectors (e.g. ALVAC) and vaccinia virus vectors, the latter being preferred.
• Suitable vaccinia viruses include without limitation the Copenhagen strain, the Wyeth strain, NYVAC and the modified Ankara (MVA) strain.
• the general conditions for constructing and producing recombinant poxvirus are well known in the art.
• the nucleic acid molecule encoding ⁇ 133 ⁇ 536 isoform, ⁇ 133 ⁇ 53 ⁇ isoform, or both ⁇ 133 ⁇ 536 and ⁇ 133 ⁇ 53 ⁇ isoforms is preferably inserted within the poxviral genome in a non-essential locus.
• Thymidine kinase gene is particularly appropriate for insertion in Copenhagen vaccinia vectors and deletion II or III for insertion in MVA vector.
• viral vectors suitable in the context of the invention are morbillivirus which can be obtained from the paramyxoviridae family, with a specific preference for measles virus. Insertion of the nucleic acid molecule encoding ⁇ 133 ⁇ 536 isoform, ⁇ 133 ⁇ 53 ⁇ isoform, or both ⁇ 133 ⁇ 536 and ⁇ 133 ⁇ 53 ⁇ isoforms between P and M genes or between H and L genes is particularly appropriate.
• the nucleic acid molecule encoding ⁇ 133 ⁇ 536 isoform, ⁇ 133 ⁇ 53 ⁇ isoform, or both ⁇ 133 ⁇ 536 and ⁇ 133 ⁇ 53 ⁇ isoforms are in a form suitable for expression in somatic cells, which means that each of the nucleic acid molecules set forth herein is operably linked to appropriate regulatory sequences.
• Suitable promoters for constitutive expression in eukaryotic systems include viral promoters, such as SV40 promoter, the cytomegalovirus (CMV) immediate early promoter or enhancer, the adenovirus early and late promoters, the thymidine kinase (TK) promoter of herpes simplex virus (HSV)-1 and retroviral long-terminal repeats (e.g.
• PGK phosphoglycero kinase
• suitable promoters for a Murine Stem Cell Virus (MSCV) vector include those present in pMSCV vector commercialized by Clontech, such as pMSCVhyg, pMSCVneo, or pMSCVpuro.
• transduced somatic differentiated cells are cultured in a medium supporting their expansion.
• a medium may be a basal medium (comprising inorganic salts, amino acids, vitamins and glucose, such as DMEM), which may be supplemented with a reducing agent (such as ⁇ -mercaptoethanol), at least one antibiotic (such as Penicillin- Streptomycin), and/or at least one growth factor able to sustain expansion of the type of transduced somatic differentiated cells (including, but not limited to, Epidermal Growth Factor (EGF) and/or basic Fibroblast Growth Factor (bFGF)).
• EGF Epidermal Growth Factor
• bFGF basic Fibroblast Growth Factor
• Step b) is performed for a period sufficient in order to recover induced pluripotent stem cells from the transduced somatic differentiated cell culture.
• a suitable period should be optimized for each type of somatic differentiated cells, but will generally be between 3 and 30 days, in particular between 7 and 20 days, more particularly, between 9 and 24 days.
• iPSC having genetic stability i.e. without DNA damages or damages of DNA repair response
• step c induced pluripotent stem cells are isolated from the transduced somatic differentiated cells culture.
• induced pluripotent stem cells may be isolated based on selection of any feature specific to induced pluripotent stem cells compared to other somatic differentiated cells.
• iPSCs can be identified and isolated by any one of means of: '
• iPSCs are isolated based on iPSC-specific cell surface markers.
• transduced differentiated somatic cells are stained using antibodies directed to one or more iPSC-specific cell surface markers, and cells having the desired surface marker phenotype are sorted.
• Those skilled in the art know how to implement such isolation based on surface cell markers. For instance, flow cytometry cell-sorting may be used, transduced somatic cells are directly or indirectly fluorescently stained with antibodies directed to one or more iPSC-specific cell surface markers and cells by detected by flow cytometer laser as having the desired surface marker phenotype are sorted.
• magnetic separation may be used.
• antibody labelled transduced somatic cells (which correspond to iPSCs if an antibody directed to a iPSC marker is used, or to non-iPSC if an antibody specifically not expressed by iPSCs is used) are contacted with magnetic beads specifically binding to the antibody (for instance via avidin/biotin interaction, or via antibody-antigen binding) and separated from antibody non-labelled transduced somatic cells.
• magnetic beads specifically binding to the antibody (for instance via avidin/biotin interaction, or via antibody-antigen binding) and separated from antibody non-labelled transduced somatic cells.
• Several rounds of magnetic purification may be used based on markers specifically expressed and non-expressed by iPSCs.
• the most common surface markers used to distinguish iPSCs are SSEA3, SSEA4, TRA-1 -
• TRA-1 -60 and TRA-1 -81 The expression of SSEA3 and SSEA4 by reprogramming cells usually precedes the expression of TRA-1 -60 and TRA-1 -81 , which are detected only at later stages of reprogramming. It has been proposed that the antibodies specific for the TRA-1 -60 and TRA-1 -81 antigens recognize distinct and unique epitopes on the same large glycoprotein Podocalyxin (also called podocalyxin-like, PODXL)1. Other surface modifications including the presence of specific lectins have also been shown to distinguish iPSCs from non-iPSCs.
• Podocalyxin also called podocalyxin-like, PODXL
• CD30 tumor necrosis factor receptor superfamily, member 8, TNFRSF8
• CD9 leukocyte antigen, MIC3
• CD50 intercellular adhesion molecule-3, ICAM3
• CD200 MRC OX-2 antigen, MOX2
• CD90 Thy-1 cell surface antigen, THY1
• iPSC may be selected by the expression of the Yamanaka transcription factors (Oct4, Sox2, cMyc and Nanog).
• iPSCs are isolated by flow cytometry cell-sorting based on DNA dye side population (SP) phenotype.
• SP DNA dye side population
• This method is based on the passive uptake of cell-permeable DNA dyes by live cells and pumping out of such DNA dyes by a side population of stem cells via ATP-Binding Cassette (ABC) transporters allowing the observation of a side population that has a low DNA dye fluorescence at the appropriate wavelength.
• ABC pumps can be specifically inhibited by drugs such as verapamil (100 ⁇ final concentration) or reserpine (5 ⁇ final concentration), and these drugs may be used to generate control samples, in which no SP phenotype may be detected.
• Appropriate cell-permeable DNA dyes that may be used include Hoechst 33342 (the main used DNA dye for this purpose, see Golebiewska et al., 2011 ) and Vybrant® DyeCycleTM stains available in various fluorescences (violet, green, and orange; see Telford et al-2010).
• iPSC are isolated by embryoid body (EB) formation.
• Embryoid bodies (EB) are the three dimensional aggregates formed in suspension by induced pluripotent stem cells (iPSC).
• iPSC induced pluripotent stem cells
• the aggregate formation is induced by using different reagents. According to used protocol it is possible to obtain different EB formation such as self-aggregated EBs, hanging drop EBs, EBs in AggreWells ect (Lin et a/., 2014).
• iPSC are isolated by iPSC colony picking.
• a protocol for iPSC colony picking is described in the Examples given below. However, skilled artisan may implement other protocols of iPSC colony picking well known in the art.
• the reprogramming method of the present invention comprises, before the step a), a step a1 ) of isolating and culturing the somatic pluripotent cells which will be transduced with the expression vector.
• step a1 ) is performed about 2 days before step a) by culturing the somatic differentiated cells in appropriate cell medium in order to achieve 50 to 80%, particularly, 60 to 70% of confluency.
• appropriate cell medium is provided with « Invitrogen kit » : CytoTune®-iPS 2.0 Sendai Reprogramming Kit (Catalog Numbers A16517, A16518).
• the viruses are removed of transduced somatic differentiated cells by replacing the cell medium with fresh cell medium.
• iPSC Induced pluripotent stem cells
• the present invention relates to induced pluripotent stem cells (iPSC) obtained by the reprogramming method of the invention.
• the reprogramming method of the invention does not require performing (and in one embodiment does not comprise) a step of expression of Yamanaka transcription factors (Oct4, Sox2, cMyc and Nanog) or a depletion of p53 or an expression of p53 mutated proteins, and the iPSC obtained by the reprogramming method of the invention are stable and non-cancerous and have better capacity to be re-differentiated in non-cancerous somatic multipotent, unipotent or differentiated somatic cells.
• Yamanaka transcription factors Oct4, Sox2, cMyc and Nanog
• the genetic stability of the iPSC obtained by the method of the invention is preserved because of the use of one of these or both ⁇ 133p536 and ⁇ 133 ⁇ 53 ⁇ isoforms since their expression does not create DNA damage DNA, and also does not alter DNA repair mechanisms.
• the iPSC produced by the method of the invention contain the expression vector encoding ⁇ 133 ⁇ 536 and/or ⁇ 133 ⁇ 53 ⁇ isoforms.
• the iPSC produced by the method of the invention also express ⁇ 133 ⁇ 536 and/or ⁇ 133 ⁇ 53 ⁇ isoforms.
• the iPSC obtained from reprogramming method of the present invention may be differentiated to hematopoietic stem cells.
• the iPSCs as produced by the reprogramming method of the invention are used in cell therapy.
• the iPSCs produced by the reprogramming method of the invention are used as therapeutic agent in the treatment of aging- associated and /or degenerative diseases.
• aging-associated diseases are diseases selected from the group comprising or consisting of atherosclerosis, cardiovascular disease, cancer, arthritis, cataracts, osteoporosis, type 2 diabetes, hypertension, Alzheimer's disease and Parkinson disease.
• degenerative diseases are diseases selected from the group comprising or consisting of diseases affecting the central nervous system (Alzheimer's disease and Parkinson disease, Huntington diseases), bones (Duchene and Becker muscular dystrophies), blood vessels or heart.
• the iPSCs produced by the reprogramming method of the invention are used as therapeutic agent for the treatment of aging -associated and degenerative diseases selected from the group consisting of cardiovascular diseases, diabetes, cancer, arthritis, hypertension , myocardial infection, strokes, amyotrophic lateral sclerosis, Alzheimer's disease and Parkinson disease.
• the iPSCs produced by the reprogramming method of the invention are used in vitro as model for studying diseases selected from the group comprising or consisting of amyotrophic lateral sclerosis, adenosine deaminase deficiency- related severe combined immunodeficiency, Shwachman-Bodian-Diamond syndrome, Gaucher disease type III, Duchene and Becker muscular dystrophies, Parkinson's disease, Huntington's disease, type 1 diabetes mellitus, Down syndrome and spinal muscular atrophy.
• diseases selected from the group comprising or consisting of amyotrophic lateral sclerosis, adenosine deaminase deficiency- related severe combined immunodeficiency, Shwachman-Bodian-Diamond syndrome, Gaucher disease type III, Duchene and Becker muscular dystrophies, Parkinson's disease, Huntington's disease, type 1 diabetes mellitus, Down syndrome and spinal muscular atrophy.
• ⁇ 133 ⁇ 536 isoform As mentioned above, the Inventors have found that ⁇ 133 ⁇ 536 isoform, ⁇ 133 ⁇ 53 ⁇ isoform or both ⁇ 133 ⁇ 536 and ⁇ 133 ⁇ 53 ⁇ are specifically expressed in iPSC.
• iPSC Induced pluripotent stem cells
• the invention thus relates to a method for detecting Induced pluripotent stem cells (iPSC) in cell population comprising detecting the presence of ⁇ 133 ⁇ 536 isoform, ⁇ 133 ⁇ 53 ⁇ isoform or both ⁇ 133 ⁇ 536 and ⁇ 133 ⁇ 53 ⁇ by detecting a ⁇ 133 ⁇ 536, ⁇ 133 ⁇ 53 ⁇ isoform or both ⁇ 133 ⁇ 536 and ⁇ 133 ⁇ 53 ⁇ isoform polypeptide and/or a fragment thereof, and/or by detecting a ⁇ 133 ⁇ 536 isoform, ⁇ 133 ⁇ 53 ⁇ isoform or both ⁇ 133p536 and ⁇ 133 ⁇ 53 ⁇ mRNA and/or a fragment thereof.
• iPSC Induced pluripotent stem cells
• the detecting method according to the invention is based on detecting the presence of ⁇ 133 ⁇ 536 isoform.
• the detecting of presence of ⁇ 133 ⁇ 536 isoform is carried out by using a probe capable of specifically hybridizing with said ⁇ 133 ⁇ 536 isoform mRNA, complementary sequence thereof or a fragment thereof having at least 10, 15, 20, 25, 30, 35, 40, 45, 50 or more nucleotides length which is specific of said ⁇ 133p536 isoform.
• probes and fragments are described in international patent application WO201 1 /000891 (the probes and fragments preferably hybridizing with ⁇ 133p536 isoform mRNA having the sequence SEQ ID NO: 2) and may be used in the method for detecting Induced pluripotent stem cells (iPSC) of the present invention.
• iPSC Induced pluripotent stem cells
• ⁇ 133 ⁇ 53 ⁇ isoform may be also detected by using a probe capable of specifically hybridizing with said ⁇ 133 ⁇ 53 ⁇ isoform mRNA, complementary sequence thereof or a fragment thereof having at least 10, 15, 20, 25, 30, 35, 40, 45, 50 or more nucleotides length which is specific of said ⁇ 133 ⁇ 53 ⁇ isoform.
• probes and fragments are also described in international patent application WO201 1 /000891 (the probes and fragments preferably hybridizing with ⁇ 133p536 isoform mRNA having the sequence SEQ ID NO: 6) and may be used in the method for detecting Induced pluripotent stem cells (iPSC) of the present invention.
• iPSC Induced pluripotent stem cells
• the reprogramming method for iPS production by the human ⁇ 133 ⁇ 536 isoforms is adapted from the « Invitrogen kit » : CytoTune®-iPS 2.0 Sendai Reprogramming Kit (Catalog Numbers A16517, A16518).
• CytoTune®-iPS 2.0 Sendai Reprogramming Kit Catalog Numbers A16517, A16518.
• the above mentioned kit will be used according to the manufacturer instructions as follows:
• KSR KnockOutTM Serum Replacement
• Penicillin-Streptomycin (optional) 1 mL
• iPSC colonies gelatin-coated 12-well feeder plates were prepared. Before picking colonies, 1 ml hES medium containing 10 ⁇ Y27632 (ROCK (rho-associated kinase inhibitor) to each well of the plate was added and incubated it in a 37° C incubator. Then, the iPSC colonies were examined under a microscope in a hood and the colonies were marked on the bottom of the dish. One colony was cut into small pieces with a 200 ⁇ pipette and the cut pieces were transferred to the 12-well feeder plates with a 200 ⁇ pipettes. The plate containing the picked colonies was incubated in a 37° C incubator for 24-48 hours. After the colonies attach the plates, the medium was replaced with fresh hES medium and had been changed every day.
• ROCK rho-associated kinase inhibitor
• the human ⁇ 133 ⁇ 53 isoforms (6 and ⁇ ) are cloned in the pMSCV-hyg (Clontech Laboratories, sold as a part of the Cat No 634401 ) vector for viral production.
• Viral particles were produced following next procedure: On Day 1 : 1 -2x10 6 HEK 293T cells were plated into 100 mm plate dishes (growth medium: MEM supplemented with 10% FCS, sodium pyruvate, glutamine and antibiotics) On Day 2: the first transfection was performed as follow:
• JetPei transfection reagent Polyplus Cat No 101 -40
• Tube 1 and 2 were mixed.
• the mixture was incubated 30 min at room temperature. In meantime the cells were washed and plated in 5 ml of growth medium without antibiotics.
• Tubes 1 and 2 After incubation, the mixture of Tubes 1 and 2 was added in drop wise manner to the cells and leaved for 4 hours. The cells were washed with PBS and 10 ml of growth medium with antibiotics was added.
• Example 1 ⁇ 133 ⁇ 53 ⁇ is highly expressed in ES and iPSC compared to dedifferentiated cells
• ⁇ 133 ⁇ 53 ⁇ mRNA levels were analyzed by qPCR in iPSCs induced from fibroblasts with 4 factors (Oct4, Sox2, Klf4, c-myc) or 6 factors (Nanog and Lyn28 in addition of the 4 factors) after 3 weeks of reprogramming.
• A133p53G_mRNA levels were also analyzed in human embryonic stem cells before (ES) and after (ES-F) fibroblastic differentiation, in skin fibroblasts before (Sk-F) and after (iPSCs) reprogramming and after re-differentiation (iPSC-F) and from human neonatal (HFFSM3).
• mRNA expression of ⁇ 133 ⁇ 53 ⁇ is clearly increased in embryonic stem cells and in iPSCs compared to fibroblasts, indicating that elevated level of this isoform is not associated with genetic instability. This would provide a major advantage compared to mutant p53, which use in iPSC technology is hampered by genetic instability they confer.
• Human primary fibroblast cells were established from normal human foreskin and display a maximum of 72 population doublings before the onset of senescence. As shown on figure 5, ⁇ 133 ⁇ 536 splice isoform of p53 produces iPSC-like cells, which express key regulators of pluripotency (Figure 4).
• Example 3 The expression of ⁇ 133 ⁇ 53 ⁇ isoform preserves the ability of cells to repair DNA
• p53 is a limiting factor in iPSC reprogrammation from normal cells and it is also considered that the alteration of p53 expression during the reprograming somatic cells to iPSC could lead to the formation of iPSC containing DNA damage and/or chromosomal abnormalities.
• ⁇ 133 ⁇ 53 isoforms do not affect the DNA damage and DNA repair response, the inventors have tested the transactivational function of p53 in normal fibroblasts expressing ⁇ 133 ⁇ 53 ⁇ isoform or ⁇ 133 ⁇ 536 isoform in the presence of DNA breaks caused by a radiomimetic agent (bleomycin).
• ⁇ 133 ⁇ 53 ⁇ thus prevents the accumulation of p21 WAF1 in the presence of DNA damage, indicating that the transcriptional function of wild type p53 is altered in these cells.
• Histone variant H2AX and Chk2 kinase proteins two proteins involved in the repair of DNA damages upstream of p53, are activated in cells expressing ⁇ 133 ⁇ 53 ⁇ and ⁇ 1 33p53a.
• ⁇ 160 ⁇ 53 is a novel N-terminal p53 isoform encoded by ⁇ 133 ⁇ 53 transcript.

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