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Definitions

• the present invention relates to a SoxB1 factor variant comprising a) the HMG (high-mobility group) domain of any one of the amino acid sequences of SEQ ID NOs: 1 to 3, wherein the amino acid alanine at position 61 of the HMG-domain is substituted with an amino acid selected from valine, leucine, isoleucine, phenylalanine, methionine, tryptophan or proline, preferably valine; or b) an amino acid sequence sharing at least 82% sequence identity with the HMG domain as defined in a), provided that the substitution as defined in a) is retained.
• iPSCs induced pluripotent stem cells
• Shinya Yamanaka brought transcription factor-based cell fate conversion into the spotlight, and rightly so
• Pluripotent cells are unique in their ability to give rise to all the tissues of an animal.
• the induction of pluripotency amounts to the ultimate cell rejuvenation, once thought to be impossible (Waddington, 1957).
• iPSC technology has already made enormous contributions to human developmental biology studies, allowed new strategies for drug discovery, and even provided a source for cell replacement therapy.
• Oct4, Sox2, Klf4, and cMyc are pioneer transcription factors capable of opening silent chromatin, which allows cells to maintain the plastic pluripotent state, or initiate differentiation later (King and Klose, 2017).
• Oct4 stands out as the master regulator of the pluripotency network. Oct4 is the only factor for which knock out in ESCs leads to an inevitable collapse of pluripotency; forced expression of Oct4 can even compensate for the loss of Sox2 (Masui et al., 2007; Nishimoto et al., 2005; Niwa et al., 2002). Oct4 is the only reprogramming factor that cannot be replaced by other members of its family (Nakagawa et al., 2008).
• Oct4 plays divergent roles in establishing pluripotency during mouse and human development: Oct4 knockout mouse blastocysts still develop a Nanog + inner cell mass, while human OCT4-null blastocysts fail to do so (Fogarty et al., 2017; Wu et al., 2013).
• SKM induction is sufficient to induce pluripotency In mouse somatic cells (An et al., 2019; Velychko et al., 2019a), however, SKM reprogramming has not been demonstrated for human or other species.
• Oct4 cooperates with Sox2 to co-regulate the majority of its targets in pluripotent cells (Chen et al., 2008).
• Oct4/Sox2 cooperativity is mediated by DNA allostery and by protein-protein interaction between their DNA-binding domains (Merino et al., 2015).
• Mcerino et al., 2015 DNA-binding domains
• Oct4/Sox2 cooperativity was shown to be essential forthe induction and maintenance of pluripotency (Tapia et al., 2015).
• Sox17 cooperates with Oct4 on compressed, non-canonical SoxOct motifs and cannot induce pluripotency but rather controls primitive endoderm and germline specification (Aksoy et al., 2013; Merino et al., 2014; Ng et al., 2012, Irie et al, 2015). Jauch et al.
• the present invention addresses this need and provides alternative Sox reprogramming factors and cocktails and methods for highly efficient and improved cell reprogramming. Accordingly, the present invention relates in a first aspect to a SoxB1 factor variant comprising a) the HMG domain of any one of the amino acid sequences of SEQ ID NOs: 1 to 3, wherein the amino acid alanine at position 61 of the HMG-domain is substituted with an amino acid selected from valine, leucine, isoleucine, phenylalanine, methionine, tryptophan or proline, preferably valine; or b) an amino acid sequence sharing at least 82% sequence identity with the HMG domain as defined in a), provided that the substitution as defined in a) is retained.
• a SoxB1 factor variant comprising a) the HMG domain of any one of the amino acid sequences of SEQ ID NOs: 1 to 3, wherein the amino acid alanine at position 61 of the HMG-domain is substituted with an amino acid selected from valine
• Sox factor refers to a member of a known family of transcription factors (TFs), which are involved in maintaining pluripotency. In general, they exert their functions by binding to regulatory elements containing a Sox DNA motif.
• the members of the Soxfamily of TFs are classified into different groups (SoxA-SoxJ) based on their level of amino acid sequence identity within their HMG-domains. All naturally occurring Sox TFs are composed of an N-terminal domain (NTD), a DNA-binding high-mobility (HMG)-domain and a C-terminal domain (CTD), containing either a transactivation or a transrepression function.
• NTD N-terminal domain
• HMG DNA-binding high-mobility
• CTD C-terminal domain
• Sox members belonging to the same group have a high degree of amino acid sequence identity (-70% to 95%) with respect to both, their HMG domain and the regions outside their HMG domains.
• Sox proteins from different groups have only partial (246%) amino acid sequence identity with respect to their HMG domains (Aksoy et al., 2013). Because Sox TFs generally have similar DNA binding specificities, their ability to trigger specific biological processes is thought to be mediated by their selective interaction with specific cofactors, such as Oct4.
• SoxB1 factors refers to the B1 subclass of Sox (TFs), including, among others, the factors Sox1 , Sox2, and Sox3, which share more than 90% sequence identity in respect to their HMG domains.
• Sox17 EK mutant i.e., Sox17 E57K
• the inventors found that replacing Sox2 with the Sox17 EK mutant (i.e., Sox17 E57K) in the reprogramming cocktail rescues otherwise detrimental Oct4 mutants as well as allows reprogramming with POU factors other than Oct4. Subsequently, the inventors generated a library of chimeric Sox2-Sox17 TFs to find the structural elements of Sox17 responsible for this peculiar phenotype.
• Sox17 refers to a member of the F subclass of Sox TFs (“SoxF factors”), which is a subclass of the Sox TFs, distinct from the SoxB1 factors.
• Sox A61v allows reprogramming with Oct4 orthologs, such as Bm2, Bm4, Oct6, Oct2 and otherwise unfunctional Oct4 mutants.
• Oct4 orthologs such as Bm2, Bm4, Oct6, Oct2 and otherwise unfunctional Oct4 mutants.
• the tetrapioid complementation assay - the most stringent test for pluripotency that measures the ability of IPSCs to generate the entire animal - showed that the Sox A61v mutant dramatically enhances the developmental potential of OSKM IPSCs (demonstrated in the examples herein below).
• amino acid sequences of SEQ ID NOs: 1 to 3 correspond to the HMG domains of the human wild-type SoxB1 factors Sox1 , 2 and 3, respectively.
• the SoxB1 factor variant of the first aspect of the present invention comprises a) the HMG domain of any one of Sox1 , 2 or 3 (i.e. SEQ ID NO: 1 , 2 or 3), wherein the amino acid alanine at position 61 is substituted with an amino acid selected from valine, leucine, isoleucine, phenylalanine, methionine, tryptophan or proline, preferably valine; or b) an amino acid sequence sharing at least 82% sequence identity with the HMG domain as defined in a), provided that the substitution at position 61 of their respective HMG domain is retained.
• the amino acid sequence sharing at least 82% sequence identity display with increasing preference, at least 83% sequence identity, at least 84% sequence identity, at least 85% sequence identity, at least 86% sequence identity, at least 87% sequence identity, at least 88% sequence identity, at least 89% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity and at least 99% sequence identity with the HMG domain as defined in item a) of the first aspect of the present invention.
• percent (%) sequence identity describes the number of matches (“hits”) of identical amino acids/nucleotides of two or more aligned amino acid or nucleic acid sequences as compared to the number of amino acid residues or nucleotides making up the overall length of the template nucleic acid or amino acid sequences.
• hits the number of matches of identical amino acids/nucleotides of two or more aligned amino acid or nucleic acid sequences as compared to the number of amino acid residues or nucleotides making up the overall length of the template nucleic acid or amino acid sequences.
• percentage of amino acid residues or nucleotides that are the same e.g.
• sequence identity describes the sequence match between two (poly)peptides or nucleic acids.
• the (poly)peptide or nucleic acid sequences to be compared are aligned and compared fortheir identical aligned sequence, wherein an identical alignment means the occupation of the same position in the sequences to be compared by the same nucleobase or amino acid residue. Accordingly, the "percent identity” is a function of the number of matching positions divided by the number of positions compared and multiplied by 100%. For example, if 7 out of 10 sequence positions are identical, then the identity is 70%.
• Nucleotide and amino acid sequence analysis and alignment in connection with the present invention are preferably carried out using bioinformatics tools for pair wise alignment such as EMBOSS Needle (https://www.ebi.ac.uk/Tools/psa/emboss_needle/; see also Madeira F, et al. The EMBL-EBI search and sequence analysis tools APIs in 2019. Nucleic Acids Research. 2019 Jul;47(W1):W636-W641. DOI: 10.1093/nar/gkz268).
• the “identity” or “percent (%) identity” between two amino acid sequences can, e.g., be determined by using the Needleman-Wunsch algorithm (Needleman, S.B. and Wunsch, CD.
• nucleic acids for example, two molecules having the same sequence but different linkage components such as thiophosphate instead of phosphate are identical by this definition.
• Another tool for assessing biological sequence alignments determining regions of similarity of biological sequences with the same number of residues is the NCBI BLAST algorithm (https://blast.ncbi.nlm.nih.gov/Blast.cgi; Stephen F. Altschul, Thomas L. Madden, Alejandro A. Schaffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), Nucleic Acids Res. 25:3389-3402).
• amino acid sequences of wild-type SoxB1 factors, as well as the corresponding protein encoding nucleotide sequences are known for a number of species, including human and mouse Sox1 , Sox2 and Sox3, and are available, e.g., from the NCBI Database: https://www.ncbi.nlm.nih.gov.
• amino acid sequences of the wild-type full-length human Sox1 , Sox2 and Sox3 proteins are available from the NCBI database under the following accession numbers: Sox1 : NP_005977, such as NP_005977.2; Sox2: NP_003097, such as NP_003097.1 ; Sox3: NP_005625, such as NP_005625.2 and are defined herein by SEQ ID NOs: 4 to 6, respectively. It is understood that all sequences under the respective NCBI reference sequence numbers are included.
• homologs of the human Sox1 , Sox2 and Sox3 HMG domain from other species are specifically also included, e.g., such as the HMG domains of Sox1 , Sox2 or Sox3 from mouse, cynomolgus macaque, rat, horse, pig, bovine or other animal species.
• the SoxB1 factor variant in accordance with the first aspect of the present invention may further comprise one or more additional amino acids or amino acid sequences flanking the C-terminal and/or the N-terminal end of the HMG domain.
• additional amino acids or amino acid sequences may, e.g., correspond to a naturally or non-naturally occurring N-terminal domain and/or a C-terminal domain flanking the HMG domain, respectively.
• the N-terminal or C-terminal domain naturally flanking the HMG domain of a Sox factor may for example be replaced by the N-terminal or C-terminal domain of another Sox factor.
• Sox'! 7 C-terminal (transactivator) domain is larger and more potent than the C-terminal domain of Sox2 and that replacing the Sox2 CTD with the Sox'! 7 CTD enhances the reprogramming ability of Sox2 (Aksoy et al., 2013).
• the SoxB1 factor variant further comprises the amino acid sequence of SEQ ID NO: 9, wherein preferably the amino acid sequence of SEQ ID NO: 9 is linked to the C-terminal end of the HMG domain as defined in accordance with the first aspect of the present invention.
• the term “linked” means one (poly)peptide is attached, preferably, to another by a peptide bond between one amino acid of the (poly)peptide to one amino acid of the other (poly)peptide.
• the term “linked” as used herein in particular means “operably linked”, which refers to the juxtaposition of at least two (poly)peptide(s) to each other in a way that both (poly)peptides function normally and allow the possibility that at least one of the (poly)peptides can mediate a function that is affected by the other (poly)peptide.
• the SoxB1 factor variant may also represent a full-length variant of wild-type Sox1 , Sox2 or Sox3 (corresponding to SEQ ID NOs: 4 to 6) comprising a C- and N-terminal domain, wherein the amino acid alanine at position 61 within their respective HMG domains as defined by SEQ ID NO: 1 , 2 or 3 is substituted with an amino acid selected from valine, leucine, isoleucine, phenylalanine, methionine, tryptophan or proline, preferably valine.
• amino acid alanine at position 61 within their respective HMG domains as defined by SEQ ID NOs: 1 , 2 or 3 is substituted with an amino acid selected from valine, leucine, and isoleucine, most preferably valine.
• the SoxB1 factor variant comprises or consists of any one of the amino acid sequences of SEQ ID NOs: 10 to 12; or an amino acid sequence sharing at least 82%, sequence identity with any one of SEQ ID NOs: 10 to 12, provided that the amino acid valine corresponding to position 109 of SEQ ID NO: 10, position 99 of SEQ ID NO: 11 or position 197 of SEQ ID NO: 12 is retained.
• SEQ ID NOs: 10 to 12 correspond to the full-length amino acid sequences of human wild-type Sox1 , Sox2 and Sox3, respectively, wherein the amino acid alanine at position 61 within their respective HMG domains as defined by SEQ ID NO: 1 to 3, respectively, is substituted with valine.
• the amino acid sequence according to the above-mentioned preferred embodiment of the first aspect of the invention sharing at least 82% sequence identity displays, with increasing preference, at least 83% sequence identity, at least 84% sequence identity, at least 85% sequence identity, at least 86% sequence identity, at least 87% sequence identity, at least 88% sequence identity, at least 89% sequence identity, , at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, and at least 99.5% sequence identity to SEQ ID NOs: 10 to 12 and has most preferably 100% sequence identity to SEQ ID NOs: 10 to 12.
• the SoxB1 factor variant comprises or consists of the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14.
• the SoxB1 factor variant shares, with increasing preference, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, and at least 99.5% sequence identity, and is most preferably 100% identical to SEQ ID NOs: 13 or 14, provided that the substitution as defined in a) is retained.
• the present invention relates to a Sox17 factor variant comprising: a) the HMG domain of the amino acid sequence of SEQ ID NO: 15, wherein the amino acids at positions 24 to 28 of SEQ ID NO: 15 are substituted with the amino acid sequence of SEQ ID NO: 16; or b) an amino acid sequence sharing at least 82%, sequence identity with the HMG domain as defined in a), provided that the substitution as defined in a) is retained.
• SEQ ID NO: 15 corresponds to the amino acid sequence of the human wild-type HMG domain of Sox17.
• SEQ ID NO: 16 corresponds to the amino acid sequence at positions 24 to 28 of the human wild-type HMG domain of Sox2.
• the amino acid sequence according to the above-mentioned preferred embodiment of item b) of the second aspect of the invention sharing at least 82% sequence identity displays, with increasing preference, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% and at least 99% sequence identity with the HMG domain as defined in a), provided that the substitution as defined in a) is retained.
• homologs of the human Sox17 HMG domain from other species are specifically also included, such as the HMG domains of Sox17 from mouse or cynomolgus macaque, rat, horse, pig, bovine, or other animal species.
• first aspect of the invention as far as being amenable for combination with the second aspect of the invention, apply mutatis mutandis to the second aspect of the invention.
• the definitions and preferred embodiments of the first and second aspect as far as being amenable for combination with the third aspect apply mutatis mutandis to the third aspect of the invention, etc.
• SoxB1 factor variants of the present invention not only allow the generation of IPSCs with significantly improved developmental potential, but also allow reprogramming with otherwise detrimental Oct4 mutants and allows reprogramming with POU factors such as Brn4, Oct2, Oct6 and Brn2, which are normally incapable of iPSC generation.
• the present invention relates to a fusion protein comprising or consisting of a) a Sox factor and a POU factor; or b) a Sox factor and a POU domain; or c) an HMG domain and a POU factor; or d) an HMG domain and a POU domain; wherein the Sox factor is selected from: i. a SoxB1 factor or the SoxB1 factor variant of the first aspect of the present invention; or ii. Sox 17 or a Sox17 factor variant of of the second aspect of the present invention; and wherein the HMG domain is selected from: iii.
• the HMG domain in accordance with item a) or item b) of the first aspect of the present invention; and wherein the POU factor is selected from iv. Oct4 or an Oct4 variant; or v. Oct2 or an Oct2 variant; or vi. Oct6 or an Oct6 variant; or vii. Brn2 or a Brn2 variant; or viii. Brn4 or a Brn4 variant; or (ix) other natural or synthetic POU factors; and wherein the POU domain is selected from x.
• SEQ ID NO: 17 corresponds to the amino acid sequence of the wild-type human Oct4 POU domain.
• SEQ ID NO: 18 corresponds to the amino acid sequence of the wild-type mouse Oct4 POU domain.
• sequence identity at least 89% sequence identity, at least 90% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, and at least 99.5% sequence identity with SEQ ID NOs: 17 or 18 and has most preferably 100% sequence identity with SEQ ID NOs: 17 or 18, respectively.
• POU domain of SEQ ID NO: 17 or 18 means an amino acid sequence as defined by the amino acid sequences set forth in SEQ ID NO: 17 and 18, respectively.
• fusion protein refers to a synthetic, semi-synthetic or recombinant single poly(peptide) molecule that comprises all or a portion of two or more different poly(peptides).
• the fusion can be an N-terminal fusion, a C-terminal fusion or an internal fusion.
• the fusion protein furthermore can include a linker connecting two or more different poly(peptides). The skilled person is aware of linkers which may suitably be used for connecting two or more different polypeptides.
• POU factors refers to transcription factors (TFs) containing a bipartite DNA binding domain referred to as a POU domain, flanked by N- and C-terminal transactivator domains (NTD and CTD).
• the bipartite POU domain consists of a POU-specific domain (POU S ) and a POU- homeodomain (POU H D) joined by a flexible non-conserved linker domain, which can be of variable length.
• POU S POU-specific domain
• POU H D POU- homeodomain
• the structure of the POU domain allows the binding of DNA and also participation in proteinprotein interactions.
• the term “other natural or synthetic POU factors” as used herein generally specifically encompasses to (poly)peptides comprising a POU domain as described herein above.
• the (poly)peptides may include both naturally occurring products and the products of recombinant DNA or other synthetic techniques.
• POU factors have different binding profiles and different preferences for hetero- versus homodimerisation (Jerabek et al., 2017; Malik et al., 2019; Mistri et al., 2015).
• Examples of POU factors include POU class 2 (including Octi and Oct2), POU class 3 (including Oct6, Brn1 , Brn2, Brn4, etc.) and POU class 5 (including Oct4) factors.
• POU factors POU family or “Oct family” refers to the family of octamer ("Oct”) transcription factors which play a crucial role in, amongst others, maintaining pluripotency.
• POU5F1 POU domain, class 5, transcription factor 1
• Oct4 is one representative of POU family. Further examples include Octi , Oct2, Oct6, Brn2 and Brn4.
• Exemplary Oct4 proteins are the proteins encoded by the murine Oct4 gene (all sequences deposited under the NCBI Reference Sequence NM_013633, such as NM_013633.3) and the human Oct4 gene (all sequences deposited under the NCBI Reference Sequence NM_002701 , such as NM_002701.6).
• Oct4 refers to any of the naturally-occurring forms of the Octamer 4 transcription factor, or variants thereof that maintain Oct4 transcription factor activity of at least, (for each value) with increasing preference, 10%, 30% , 50%, 80%, 90% or 100% activity compared to wild type Oct4 as measured by methods known in the art, such as measuring the efficiency of IPSC generation in reprogramming experiments.
• variants have at least 90% amino acid sequence identity across their whole sequence compared to the naturally occurring Oct4 polypeptide.
• the Oct4 protein is the protein as identified by the Genbank reference ADW77327.1 , corresponding to SEQ ID NO: 19, or the protein as identified by SEQ ID NO: 20.
• Oct4 variants potentially not maintaining Oct4 transcription factor activity, but comprising or consisting of the amino acid sequence of any one of the SEQ ID NOs: 21 to 24 are specifically also included.
• Bm2 refers to any of the naturally-occurring forms of the Brn2 transcription factor, or variants thereof that maintain Brn2 transcription factor activity of at least, (for each value) with increasing preference, 50%, 80%, 90% or 100% activity compared to wild type Brn2 as measured by methods known in the art, such as measuring the efficiency of iPSC generation in reprogramming experiments.
• variants have at least 90% amino acid sequence identity across their whole sequence compared to the naturally occurring Brn4 polypeptide.
• the Brn4 protein is the protein as identified by all sequences deposited under the NCBI Reference Sequence NP_000298, such as NP_000298.3 or, corresponding to SEQ ID NOs: 31 or 32, respectively.
• the variant with respect to a referred (poly)peptide refers to an amino acid sequence variant of a corresponding wild-type amino acid sequence of said (poly)peptide.
• the variant maintains or essentially maintains the function of the wild-type form. Essentially means with increasing preference of at least 50%, at least 80 % at least 90% and at least 95%.
• the variant shares, with increasing preference, at least 83% sequence identity, at least 84% sequence identity, at least 85% sequence identity, at least 86% sequence identity, at least 87% sequence identity, at least 88% sequence identity, at least 89% sequence identity, at least 90% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, and at least 99.5% sequence identity with the corresponding wild-type amino acid sequence.
• the Sox factor may be a wild-type Sox factor or Sox factor variant as defined above, preferably by the Sox factor variant as defined in SEQ ID NO: 13 or 14.
• the present invention relates to a complex, or a composition, comprising or consisting of: a) a Sox factor and a POU factor; or b) a Sox factor and a POU domain; or c) an HMG domain and a POU factor; or d) an HMG-domain and a POU domain, wherein the Sox factor, the POU factor, the HMG domain and the POU domain are selected from the Sox factor, the POU factor, the HMG domain, and the POU domain as defined in accordance with the third aspect of the invention.
• the term "complex” refers to an association of two molecules that interact with each other through bonds and/or forces (e.g., van der Waals, hydrophobic, hydrophilic forces) that are not peptide bonds. Individual members of a complex may be linked by non- covalent interactions.
• the “complex” according to the fourth aspect of the invention may also include a further non-covalent interaction of at least one of the molecules as defined in items a) to d) of the fourth aspect of the invention with another macromolecule, such as a nucleic acid. It will be understood that a complex can be multimeric. Each interacting molecule of a complex is herein referred to as an "individual member" or "member” of the complex.
• nucleic acid molecule in accordance with the present invention includes DNA, such as cDNA or double- or single-stranded genomic DNA and RNA, including natural and modified DNA and RNA.
• DNA deoxyribonucleic acid
• DNA means any chain or sequence of the chemical building blocks selected from adenine (A), guanine (G), cytosine (C) and thymine (T), called nucleotide bases, that are linked together typically by a phosphodiester bond on a deoxyribose sugar backbone.
• DNA can have one strand of nucleotide bases, or two complementary strands which may form a double helix structure.
• RNA ribonucleic acid
• A adenine
• G guanine
• C cytosine
• U uracil
• nucleotide bases that are linked together typically by a phosphodiester bond on a ribose sugar backbone.
• RNA typically has one strand of nucleotide bases, such as mRNA. Included are also single- and double-stranded hybrids molecules, i.e., DNA-DNA, DNA- RNA and RNA-RNA.
• the nucleic acid molecule may also be modified by many means known in the art.
• Non-limiting examples of such modifications include methylation, "caps", substitution of one or more of the naturally occurring nucleotides with an analogue, and internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorod ithioates, etc.).
• uncharged linkages e.g., methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates, etc.
• charged linkages e.g., phosphorothioates, phosphorod ithioates, etc.
• Nucleic acid molecules in the following also referred as polynucleotides, may contain one or more additional covalently linked moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), intercalators (e.g., acridine, psoralen, etc.), chelators (e.g., metals, radioactive metals, iron, oxidative metals, etc.), and alkylators.
• proteins e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.
• intercalators e.g., acridine, psoralen, etc.
• chelators e.g., metals, radioactive metals, iron, oxidative metals, etc.
• alkylators e.g., metals, radioactive metals, iron, oxidative metals, etc.
• nucleic acid mimicking molecules known in the art such as synthetic or semi-synthetic derivatives of DNA or RNA and mixed polymers.
• nucleic acid mimicking molecules or nucleic acid derivatives according to the invention include phosphorothioate nucleic acid, phosphoramidate nucleic acid, 2’-O-methoxyethyl ribonucleic acid, morpholino nucleic acid, hexitol nucleic acid (HNA), peptide nucleic acid (PNA) and locked nucleic acid (LNA) (see Braasch and Corey, Chem Biol 2001 , 8: 1).
• LNA is an RNA derivative in which the ribose ring is constrained by a methylene linkage between the 2’-oxygen and the 4’-carbon.
• nucleic acids containing modified bases for example thio-uracil, thio-guanine and fluoro-uracil.
• a nucleic acid molecule typically carries genetic information, including the information used by cellular machinery to make proteins and/or polypeptides.
• the nucleic acid molecule of the invention may additionally comprise promoters, enhancers, response elements, signal sequences, polyadenylation sequences, introns, 5'- and 3'- non-coding regions, and the like.
• the nucleic acid molecule is an RNA, preferably a modified RNA, more preferably a modified mRNA (modRNA).
• modified RNA or “modified mRNA” refers to an RNA or mRNA, respectively which comprises at least one modified nucleoside.
• the nucleoside uridine may be modified to pseudo uridine or N1-mehyl-pseudouridine and/or the nucleoside cytosine may be modified to 5- methylcytosine, which alter the secondary structure of the RNA and can reduce recognition by the innate immune system while still allowing effective translation.
• the reprogramming factor is selected from the group including POU factor, Klf and Myc family members.
• c-Myc c-Myc
• C-Myc C-Myc
• c-Myc c-Myc transcription factor
• c-Myc c-Myc transcription factor
• variants thereof that maintain c-Myc transcription factor activity (e.g., with at least, (for each value) with increasing preference, 50%, 80%, 90% or 100% of transcription factor activity) compared to wild type c- Myc as measured by methods known in the art, such as measuring the efficiency of iPSC generation in reprogramming experiments.
• L-Myc L-Myc
• L-Myc L-Myc transcription factor
• variants thereof that maintain L-Myc transcription factor activity (e.g., with at least (for each value) 50%, 80%, 90% or 100% activity compared to wild type L-Myc) as measured by methods known in the art, such as measuring the efficiency of iPSC generation in reprogramming experiments.
• Non-limiting examples of methods for protecting a modRNA from host cell ribonucleotides include packing the modRNA into a liposome for delivery and up-take into the host cell.
• the present invention relates to (a) a cell or (b) a cell derived from said cell, wherein said cell (a) comprises or said cell (b) has been modified by the presence in the cell of:
• cell refers to and includes a single cell, a plurality of cells or a population of cells where context permits, unless otherwise specified.
• a cell (b) derived from the cell (a) wherein said cell (b) has been modified as indicated above refers to a cell that comprises or previously comprised the SoxB1 factor variant of the first aspect, the Sox'!
• the fusion protein of the third aspect and/or the complex or composition of the fourth aspect of the invention the nucleic acid molecule or the combination of nucleic acid molecules of the fifth aspect of the invention or the vector or combination of vectors of the sixth aspect of the invention, wherein the cell has been modified in its characteristics according to the expression pattern of the nucleic acid molecule or the combination of nucleic acid molecules of the fifth aspect of the invention or the vector or combination of vectors of the sixth aspect of the invention.
• the "cell” or “cell derived from the cell” (also referred to as cell derivative) as described herein is preferably an isolated cell.
• the derivative of the cell includes progeny that may or may not display the phenotype of the original cell.
• certain features such as an enhanced developmental potential, a higher expression level of at least one, preferably at least two, more preferably at least three, more preferably at least four, more preferably at least five, and most preferably at least six na’ive pluripotency-specific marker(s) selected from Klf17, Klf4, Sox2, Susd2, Argfx, and Dnmt3l, a higher expression level of at least one primitive endoderm-specific marker(s) selected from Gata6 and Sox17, an activated POU5f1 distal enhancer, a reactivated X chromosome in female lines, a reduced DNA methylation, an enhanced capacity to differentiate, preferably into a germline cell, and/or an enhanced capacity to contribute to development of an embryo(s) and/or animal(s), of the phenotype of the original cell carrying a SoxB1 factor variant of the first aspect, or the Sox17 factor variant of the second aspect, as described in this specification may be lost in the pro
• the present invention relates to a method for enhancing the cooperativity between a Sox factor and a POU factor, the method comprising increasing the average number and/or strength of interactions between the HMG domain of the Sox factor and the POU domain of the POU factor.
• Sox factor and POU factor generally refers to the interaction between Sox factors and POU factors as described herein above on their genomic targets that leads to a higher stability of the Sox/Oct heterodimer on DNA compared to either of the monomers on the same DNA. Accordingly, POU factors cooperate with Sox factors to co-regulate their genomic targets.
• Oct/Sox cooperativity is generally mediated by DNA allostery and by protein-protein interactions between their respective DNA-binding domains: the POU and the HMG-domain.
• increasing the level of the SoxB1 factor variant or the Sox'! 7 factor variant as defined in item a); the fusion protein or the complex or composition as defined in item b) and/or the SoxB1 factor variant as defined in item c); and optionally, an inhibitor of p53 function; and/or one or more additional reprogramming factor(s) in said non-pluripotent cells is achieved by (co-)expressing in said non-pluripotent cell(s) the factor variant, POU factor, fusion protein, complex, and/or Klf family member as defined in items a) to c) and optionally the inhibitor of p53 function, and/or one or more additional reprogramming factor(s).
• miRNA molecules are single-stranded RNA molecules. Endogenously present miRNA molecules regulate gene expression by binding to a complementary mRNA transcript and triggering of the degradation of said mRNA transcript through a process similar to RNA interference. Accordingly, exogenous miRNA may be employed as an inhibitor of p53 function after introduction into the respective cells.
• antisense nucleic acid molecule refers to a nucleic acid which is complementary to a target nucleic acid.
• An antisense molecule in accordance with the invention is capable of interacting with the target nucleic acid, more specifically it is capable of hybridizing with the target nucleic acid. Due to the formation of the hybrid, transcription of the target gene(s) and/or translation of the target mRNA is reduced or blocked. Standard methods relating to antisense technology have been described (see, e.g., Melani et al., Cancer Res. (1991) 51 :2897-2901 ).
• Antisense molecules, siRNAs and shRNAs of the present invention are preferably chemically synthesized using a conventional nucleic acid synthesizer.
• Suppliers of nucleic acid sequence synthesis reagents include Proligo (Hamburg, Germany), Dharmacon Research (Lafayette, CO, USA), Pierce Chemical (part of Perbio Science, Rockford, IL, USA), Glen Research (Sterling, VA, USA), ChemGenes (Ashland, MA, USA), and Cruachem (Glasgow, UK).
• aptamers in the art have been selected which bind nucleic acid, proteins, small organic compounds, and even entire organisms.
• a database of aptamers is maintained at http://aptamer.icmb.utexas.edu/. More specifically, aptamers can be classified as DNA or RNA aptamers or peptide aptamers. Whereas the former consists of (usually short) strands of oligonucleotides, the latter consist of a short variable peptide domain, attached at both ends to a protein scaffold.
• Non-modified aptamers are cleared rapidly from the bloodstream, with a half-life of minutes to hours, mainly due to nuclease degradation and clearance from the body by the kidneys, a result of the aptamer's inherently low molecular weight.
• the rapid clearance of aptamers can be an advantage in applications such as in vivo diagnostic imaging.
• Several modifications, such as 2'-fluorine-substituted pyrimidines, polyethylene glycol (PEG) linkage, etc. are available with which the half-life of aptamers easily can be increased to the day or even week time scale.
• a recent development, also useful in accordance with the present invention, is the combination of an aptamer, recognizing a small compound, with a hammerhead ribozyme.
• the conformational change induced in the aptamer upon binding the target molecule can regulate the catalytic function of the ribozyme.
• Examples of chemical inhibitors of p53 include, but are not limited to, p53 inhibitors typified by pifithrin (PFT)-a and - , which are disclosed in WO 00/44364, PFT-p disclosed in Storm et al., 2006, analogues thereof and salts thereof (for example, acid addition salts such as hydrochlorides and hydrobromides, and the like) and the like.
• PFT pifithrin
• analogues thereof and salts thereof for example, acid addition salts such as hydrochlorides and hydrobromides, and the like
• PFT-a and analogues thereof [2-(2-lmino-4, 5,6,7- tetrahydrobenzothiazol-3-yl)-1-p-tolylethanone, HBr (product name: Pifithrin-a) and 1-(4-Nitrophenyl)-2- (4,5,6,7-tetrahydro-2-imino-3(2H)-benzothiazolyl)ethanone, HBr (product name: Pifithrin-a, p-Nitro)], PFT-0 and analogues thereof [2-(4-Methylphenyl)imidazo[2,1-b]-5, 6,7, 8-tetrahydrobenzothiazole, HBr (product name: Pifithrin-a, Cyclic) and 2-(4-Nitrophenyl)imidazo[2,1-b]-5, 6, 7, 8-tetrahydrobenzothiazole (product name: Pifith),
• antibody as used in accordance with the present invention comprises, for example, polyclonal or monoclonal antibodies. Furthermore, also derivatives or fragments thereof, which still retain the binding specificity to the target are comprised in the term "antibody".
• the isotype of the antibody is not particularly limited, and is preferably IgG, IgM or IgA, particularly preferably IgG.
• polyclonal antibodies can be obtained from the blood of an animal following immunisation with an antigen in mixture with additives and adjuvants and monoclonal antibodies can be produced by any technique which provides antibodies produced by continuous cell line cultures. Examples for such techniques are described, e.g.
• recombinant antibodies may be obtained from monoclonal antibodies or can be prepared de novo using various display methods such as phage, ribosomal, mRNA, or cell display.
• a suitable system for the expression of the recombinant (humanised) antibodies may be selected from, for example, bacteria, yeast, insects, mammalian cell lines or transgenic animals or plants (see, e.g., US patent 6,080,560; Holliger P, Hudson PJ. 2005, Nat Biotechnol., vol. 23(9), 11265).
• Surface plasmon resonance as employed in the BIAcore system can be used to increase the efficiency of phage antibodies.
• antibody mimetics refers to compounds which, like antibodies, can specifically bind antigens but which are not structurally related to antibodies. Antibody mimetics are usually artificial peptides or proteins with a molar mass of about 3 to 20 kDa.
• an antibody mimetic may be selected from the group consisting of affibodies, adnectins, anticalins, DARPins, avimers, nanofitins, affilins, Kunitz domain peptides, Fynomers®, trispecific binding molecules and prododies. These polypeptides are well known in the art and are described in further detail herein below.
• anticalin refers to an engineered protein derived from a lipocalin (Beste G, Schmidt FS, Stibora T, Skerra A. (1999) Proc Natl Acad Sci U S A. 96(5): 1898-903; Gebauer and Skerra (2009) Curr Opinion in Chemical Biology 13:245-255).
• Anticalins possess an eight-stranded p-barrel which forms a highly conserved core unit among the lipocalins and naturally forms binding sites for ligands by means offour structurally variable loops at the open end.
• Anticalins although not homologous to the IgG superfamily, show features that so far have been considered typical for the binding sites of antibodies: (i) high structural plasticity as a consequence of sequence variation and (ii) elevated conformational flexibility, allowing induced fit to targets with a differing shape.
• DARPin refers to a designed ankyrin repeat domain (166 residues), which provides a rigid interface arising from typically three repeated -turns. DARPins usually carry three repeats corresponding to an artificial consensus sequence, wherein six positions per repeat are randomised. Consequently, DARPins lack structural flexibility (Gebauer and Skerra, 2009).
• a “Kunitz domain peptide” is derived from the Kunitz domain of a Kunitz-type protease inhibitor such as bovine pancreatic trypsin inhibitor (BPTI), amyloid precursor protein (APP) or tissue factor pathway inhibitor (TFPI).
• BPTI bovine pancreatic trypsin inhibitor
• APP amyloid precursor protein
• TFPI tissue factor pathway inhibitor
• Kunitz domains have a molecular weight of approximately 6kDA and domains with the required target specificity can be selected by display techniques such as phage display (Weidle et al., (2013), Cancer Genomics Proteomics; 10(4): 155-68).
• Fynomer® refers to a non-immunoglobulin-derived binding polypeptide derived from the human Fyn SH3 domain.
• Fyn SH3-derived polypeptides are well-known in the art and have been described e.g. in Grabulovski et al. (2007) JBC, 282, p. 3196-3204, WO 2008/022759, Bertschinger et al (2007) Protein Eng Des Sei 20(2):57-68, Gebauer and Skerra (2009) Curr Opinion in Chemical Biology 13:245-255, or Schlatter et al. (2012), MAbs 4:4, 1-12).
• trispecific binding molecule refers to a polypeptide molecule that possesses three binding domains and is thus capable of binding, preferably specifically binding to three different epitopes.
• the trispecific binding molecule is preferably a TriTac.
• a TriTac is a T-cell engager for solid tumors which comprised of three binding domains being designed to have an extended serum half-life and be about one-third the size of a monoclonal antibody.
• probody refers to a protease-activatable antibody prodrug.
• a probody consists of an authentic IgG heavy chain and a modified light chain.
• a masking peptide is fused to the light chain through a peptide linker that is cleavable by tumor-specific proteases. The masking peptide prevents the probody binding to healthy tissues, thereby minimizing toxic side effects.
• An anti-p53 antagonist antibody can be produced using p53 or a partial peptide thereof as an antigen, by a method of antibody or anti-serum production known per se.
• the method is performed as an in vitro or ex vivo method.
• the method is performed as an in vivo method.
• the present invention relates to a reprogramming method for rejuvenating aged cell(s), tissue(s), organ(s) or organism(s), said method comprising increasing in said aged cell(s), tissue(s), organ(s) or organism(s) the level of: a) the SoxB1 factor variant of the first aspect of the invention or the Sox17 factor variant the second aspect of the invention, and a POU factor; and/or b) the fusion protein of the third aspect of the invention or the complex or composition of the fourth aspect of the invention ; and/or c) the SoxB1 factor variant sharing the amino acid sequence comprising or consisting of SEQ ID NOs: 13 or 14, and a Klf family member; and optionally:
• the method comprises increasing the level of the SoxB1 factor variant or the Sox17 factor variant and a POU factor as defined in item a); and/or the fusion protein or the complex or composition as defined in item b); and/or the SoxB1 factor variant and a Klf family member as defined in item c); and optionally:
• the “rejuvenated cell(s)” are preferably rejuvenated senescent cells.
• telomere attrition refers to a cell that exhibit cell cycle arrest, elicited by replicative exhaustion due to telomere attrition or in response to stresses such as DNA damage, chemotherapeutic drugs, or aberrant expression of oncogenes. This arrest is implemented primarily through activation of p53 and the up-regulation of the cyclin-dependent kinase (CDK) inhibitors pl6 INK4A and p21 CIP1 (Collado et al. 2007, Cell, 130: 223-233).
• CDK cyclin-dependent kinase
• a “senescent cell” may be characterized by at least one or more or all of the following characteristics: activation of the p53/p21 CIP1 and pRb/pl6 INK4A tumor suppressor pathways (hereafter referred as senescence effectors), cells arrested irreversibly in Gl, shortening of telomere size, expression of senescent-associated p-galactosidase activity (SA P-Gal), specific chromatin modification as senescence-associated heterochromatic foci (SAHF), specific secretome, reduced/altered overall mitochondrial activity. Irreversible cell arrest in Gl may be assessed by FACS.
• Shortening of telomere size may be characterized by evaluating the mean terminal restriction fragment (TRF) length for example by Southern blot analysis.
• TRF mean terminal restriction fragment
• SA p- Gal senescent- associated p-galactosidase activity
• SAHF senescence-associated heterochromatic foci
• An aged cell is a proliferative cell that exhibits one or more of the following characteristics: upregulation of tumor suppressors, decreased ability to proliferate, changes to DNA methylation pattern, accumulation of other epigenetic changes, misregulated gene expression, decreased or lost ability to perform its function.
• the aging markers can be observed using techniques known in the art such as epigenetic clocks (Horvath, 2013).
• said aged or senescent cells display at least one or more (or all) of the following characteristics of the aging phenotype: upregulation of tumor suppressors, cells arrested irreversibly in Gl, expression of senescent-associated 0-galactosidase activity (SA 0-Gal), expression of senescence-associated heterochromatic foci (SAHF), and altered overall mitochondrial activity, changes to DNA methylation patterns, accumulation of other epigenetic changes, misregulated gene expression, decreased or lost ability to perform its function.
• upregulation of tumor suppressors cells arrested irreversibly in Gl
• SA 0-Gal senescent-associated 0-galactosidase activity
• SAHF senescence-associated heterochromatic foci
• altered overall mitochondrial activity changes to DNA methylation patterns, accumulation of other epigenetic changes, misregulated gene expression, decreased or lost ability to perform its function.
• Aged tissue is tissue that exhibits one or multiple of the following characteristics: accumulation of the senescent or aged cells within the tissue, changes to DNA methylation patterns, accumulation of other epigenetic changes, misregulated gene expression, depletion or loss of stem cells within the tissue, decrease of loss regenerative capacity, decreased or lost ability to perform its function.
• An aged organ is an organ that exhibits one or multiple of the following characteristics: accumulation of the senescent or aged cells within the organ, changes to DNA methylation patterns, accumulation of other epigenetic changes, misregulated gene expression, depletion or loss of stem cells within the organ, decrease of loss regenerative capacity, decreased or lost ability to perform its function.
• An aged organism Is an animal organism that exhibits one or multiple of the following characteristics: accumulation of the senescent or aged cells within the organism, changes to DNA methylation patterns in some or all of its cells and tissues, accumulation of other epigenetic changes in some or all of its cells and tissues, misregulated gene expression in some or all of its cells and tissues, depletion or loss of stem cells within one or multiple of its tissues or organs, loss of regenerative capacity of some or multiple of its tissues and organs, occurrence or high propensity of occurrence of age-related disease or diseases.
• the method is an in vivo method.
• Lin28 refers to a protein that is encoded by the LIN28A gene in humans. It is a marker of undifferentiated human embryonic stem cells and encodes a cytoplasmic mRNA-binding protein that binds to and enhances the translation of the IGF- 2 (Insulin-like growth factor 2) mRNA. Lin28 has also been shown to bind to the let-7 pre-miRNA and block production of the mature let-7 microRNA in mouse embryonic stem cells. Yu et al. demonstrated that it is a factor in iPSCs generation, although it is not mandatory.
• Lin28 is the protein encoded by murine gene (all sequences deposited under the NCBI Reference Sequence NM_145833, such as NM_145833.1 ) and human LIN28A gene (all sequences deposited under the NCBI Reference Sequence NM 024674, such as NM_ NM 024674.6).
• the term "Lin28” as referred to herein includes any of the naturally occurring forms of the Lin28 transcription factor, or variants thereof that maintain Lin28 transcription factor activity (e.g., within at least 50%, 80%, 90% or 100% activity) compared to wild type Lin28 as measured by methods known in the art, such as measuring the efficiency of iPSC generation in reprogramming experiments.
• variants have at least 90% amino acid sequence identity across their whole sequence compared to the naturally occurring Lin28 polypeptide.
• the Lin28 protein is the protein as identified by all sequences deposited under the NCBI references NP 078950, such as NP 078950.1 or NP 665832, such as NP 665832.1 , corresponding to SEQ ID NOs: 36 and 37, respectively.
• variants have at least 90% amino acid sequence identity across their whole sequence compared to the naturally occurring Nanog polypeptide.
• the Nanog protein is the protein as identified by all sequences deposited under the NCBI references NP_079141 , such as NP 079141.2 or NP 082292, such as NP 082292.1 , corresponding to SEQ ID NOs: 38 and 39, respectively.
• GLIS1 refers to a protein that belongs to the GLIS protein family (GLI similar family) of zinc finger-type transcription factors. Forced expression of GLIS1 promotes the formation of iPSCs (Maekawa et al., 2001).
• Exemplary GLIS1 proteins are the proteins encoded by the GLIS1 gene in humans (all sequences deposited under the NCBI Reference Sequence NM 147193, such as NM 147193.4).
• GLIS1 as referred to herein includes any of the naturally occurring forms of the GLIS1 transcription factor (including the isoforms), or variants thereof that maintain GLIS1 transcription factor activity (e.g., within at least 50%, 80%, 90% or 100%) activity compared to wild type GLIS1 as measured by methods known in the art, such as measuring the efficiency of iPSC generation in reprogramming experiments.
• the method comprises (co-)expressing in the non-pluripotent cell(s) or increasing in said aged cell(s), tissue(s), organ(s), or organism(s) the expression of: (i) the SoxB1 factor variant of the first aspect of the present invention or the Sox17 factor variant the second aspect of the invention, and Oct4; and/or (ii) the SoxB1 factor variant of the first aspect of the present invention or the Sox17 factor variant of the second aspect of the invention and Oct4 and Klf4 or GLIS1 ; and/or (iii) the SoxB1 factor variant of the first aspect of the present invention or the Sox17 factor variant the second aspect of the invention and Oct4, Klf4 and GLIS1 ; and/or (iv) the SoxB1 factor variant of the first aspect of the present invention or the Sox17 factor variant the Sox17 factor variant the Sox17 factor variant the
• proteins/factors in two different mammalian species are encoded by sequences deposited by distinct NCBI Reference sequence number.
• the proteins/factors for the purpose of the disclosure as described herein are chosen to be from the same species, preferably human.
• any further reprogramming factor selected from the group of Klf and/or Myc family and/or other reprogramming factors are preferably selected from human.
• Culturing the cells includes specific conditions, such as naive media, e.g. Rset (STEMCELL Technologies), PXGL (see Bredenkamp, Nicholas et al. 2019b), or other formulations (containing one or more small molecule inhibitors of the following molecules/ pathways: HDACs, WNT, MEK, FGF, FGFR, GSK3, ROCK, and PKC, p38, JNK, BMP, ERK, TGFB), optionally feeder layer, and/or hypoxic conditions (5% O2).
• naive media e.g. Rset (STEMCELL Technologies), PXGL (see Bredenkamp, Nicholas et al. 2019b), or other formulations (containing one or more small molecule inhibitors of the following molecules/ pathways: HDACs, WNT, MEK, FGF, FGFR, GSK3, ROCK, and PKC, p38, JNK, BMP, ERK, TGFB), optionally feeder layer, and/or hypoxic conditions (5% O2).
• Culturing the cell(s) in accordance with the eleventh aspect of the present invention may include contacting the cell(s) with the SoxB1 factor variant and the Klf family member as defined in items a) and b) of the eleventh aspect of the invention so that the respective SoxB1 factor variant and Klf family member are taken up by the cell, as well as transfecting or transducing the cell with nucleic acids encoding the SoxB1 factor variant and the Klf family member and co-expressing the SoxB1 factor variant and the Klf family member.
• the term “pluripotent cell”, refers to any pluripotent cell of lower differential potential than a high-grade na ve pluripotent cell.
• the pluripotent cell is a low-grade pluripotent cell.
• the method comprises (co-)expressing in said pluripotent cell(s): a) a SoxB1 factor or the SoxB1 factor variant of the first aspect of the invention, and a Klf family member; and/or b) the SoxB1 factor variant sharing the amino acid sequence comprising or consisting of SEQ ID NOs: 13 or 14; and optionally:
• the cells are selected from human, non-human primate, mouse, porcine, bovine, horse, dog, cat or elephant cells.
• the present invention relates to a kit of parts comprising or consisting of a) the SoxB1 factor variant of the first aspect of the invention; and/or b) the Sox17 factor variant the second aspect of the invention; and/or c) the fusion protein of the third aspect of the invention; and/or d) the complex or composition of the fourth aspect of the invention; and/or e) the nucleic acid molecule or the combination of nucleic acid molecules the fifth aspect of the invention; and/or f) the vector or the combination of vectors of the sixth aspect of the invention; and/or the cell of the seventh aspect of the invention; and optionally instructions for use of the kit.
• the increasing the level in aged cell(s), tissue(s), organ(s) or organism(s) of the factor variants and POU factor as defined in item a), and/or the fusion protein or complex or composition as defined in item b) and/or the SoxB1 factor variant and a Klf family member as defined in item c) and optionally, one or more additional reprogramming factor(s) is achieved by (co-)expressing the factor variants, the POU factor, the fusion protein, the complex and/or the Klf family member as defined in items a) to c) and optionally, one or more additional reprogramming factor(s).
• Culturing the cell in accordance with the thirteenth aspect of the present invention may include contacting the aged cell(s), tissue(s), organ(s) or organism(s) with the SoxB1 factor variant or the Sox17 factor variant, and a POU factor as defined in items a) and/or the fusion protein or complex or composition as defined in item b) and/or the SoxB1 factor variant and a Klf family member of the thirteenth aspect of the invention so that the respective SoxB1 factor variant, the Sox'!
• the POU factor, the fusion protein, the complex or composition and/or the Klf family member are taken up by the aged cell(s), tissue(s), organ(s) or organism(s), as well as transfecting or transducing the aged cell(s), tissue(s), organ(s) or organism(s) with nucleic acids encoding the respective SoxB1 factor variant, the Sox'! 7 factor variant, the POU factor, the fusion protein, the complex and/or the Klf family member and (co-)expressing the respective SoxB1 factor variant, the Sox17 factor variant, the POU factor, the fusion protein, the complex and/or the Klf family member.
• the method further comprises optionally increasing the level of one or more additional reprogramming factor(s) and/or inhibitors, wherein the inhibitor is selected from the group of TGFbeta inhbitors, Src inhibitors and other kinase inhibitors, which may be used in combination with epigenetic modifiers.
• the inhibitor is selected from the group of TGFbeta inhbitors, Src inhibitors and other kinase inhibitors, which may be used in combination with epigenetic modifiers.
• TGFbeta inhbitors Src inhibitors and other kinase inhibitors
• the method comprises co-expressing in the pluripotent cell: the SoxB1 factor variant comprising or consisting of SEQ ID NOs: 13 or 14.
• the rejuvenated cells are rejuvenated senescent cells.
• the Klf family member is Klf4.
• the present invention relates to a method for producing high-grade naTve pluripotent cell(s), the method comprising increasing in low-grade pluripotent cell(s) the level of: a) a SoxB1 factor or the SoxB1 factor variant of the first aspect of the invention or the Sox17 factor variant of the second aspect of the invention, and a Klf family member; and/or b) the fusion protein of the third aspect of the invention or the complex or composition of the fourth aspect of the invention and a Klf family member; and optionally one or more additional reprogramming factor(s); thereby producing high- grade naive pluripotent cell(s).
• Increasing the level of the factors, fusion protein or complex or the composition as recited in items a) and/or b) in accordance with the fourteenth aspect of the invention may be performed by contacting the low-grade pluripotent cell(s) with the factors, fusion proteins and/or complexes or compositions as defined in items a) and/or b) of the fourteenth aspect of the invention so that the respective factor, fusion protein, and/or complex or composition is taken up by the cell(s), as well as transfecting or transducing the cell(s) with nucleic acids encoding the factor, fusion protein and/or complex and (co-)expressing the factor, fusion protein and/or complex.
• the increasing the level of the referred factors or proteins is achieved by (co-)expression of the referred factors or proteins.
• the method comprises (co-)expressing in the low-grade pluripotent cell: (i) the SoxB1 factor variant sharing the amino acid sequence comprising or consisting of SEQ ID NOs: 13 or 14, and Klf4; or (ii) the SoxB1 factor variant sharing the amino acid sequence comprising or consisting of SEQ ID NOs: 13 or 14, Klf4 and c-Myc; or (iii) or the SoxB1 factor variant comprising or consisting of the amino acid sequence SEQ ID NOs: 13 or 14, Klf4 and Oct4.
• the method according to the fourteenth aspect further comprises prior to step a) providing the iPSC(s) by conducting the method according to the ninth aspect of the invention.
• the vector is preferably an episomal vector.
• the (co-)expression is a constitutive (co-)expression.
• the conditions suitable for producing naive pluripotent cells include a) optionally plating the cell or a plurality of cells as single cells on inactivated feeder layer or in feeder-free conditions and b) optionally supplementing the media with small molecules, that support naive reset, i.e., primed-to-naive conversion (upgrade), or using cell culture media that supports naive pluripotent cell cultures.
• Klf 17 is a gene and protein name or a transcription factor from the Klf family, which regulates pre-implantation development in human and other primates.
• An example for human is KLF17 gene is encoded by all sequences deposited under the NCBI Reference Sequence NM_173484, such as NM_173484.4 and has been described in Boroviak et al., 2017 (Development (Cambridge, England) vol. 144,2: 175-186. doi:10.1242/dev.145177) and in examples below.
• Dnmt3l is a gene and protein name for DNA (Cytosine-5-)- Methyltransferase 3-Like protein responsible for CpG methylation - an epigenetic modification important for embryonic development.
• An example for human is DNMT3Lgene is encoded by all sequences deposited under the NCBI Reference Sequence NM 013369, such as NM 013369.4, and NM 175867, such as NM_175867.3 and has been described in Bi et al. 2022 and in examples below.
• the combination of at least two refers to the combination of all features in items (i) and (ii). In another preferred embodiment, the combination of at least two refers to the combination of the features in items (i) and (ill). In another preferred embodiment, the combination of at least two refers to the combination of the features in items (i) and (vii). In another preferred embodiment, the combination of at least two refers to the combination of the features in items (ii) and (vii). In another preferred embodiment, the combination of at least two refers to the combination of the features in items (Hi) and (vii). In another more preferred embodiment, the combination of at least three refers to the combination of the features in items (i), (ii) and (iii).
• the combination of at least three refers to the combination of the features in items (i), (ii) and (vii). In another more preferred embodiment, the combination of at least three refers to the combination of the features in items (i), (iii) and (vii).
• the combination of at least five refers to the combination of the features in items (i), (ii), (iii), (iv). and (v). In another more preferred embodiment, the combination of at least five refers to the combination of the features in items (i), (ii), (iii), (iv). and (vii).
• the combination of at least six refers to the combination of the features in items (i), (ii), (iii), (iv). (v) and (vi). In another more preferred embodiment, the combination of at least six refers to the combination of the features in items (i), (ii), (iii), (iv). (v) and (vii).
• the combination of at least seven refers to the combination of the features in items (i), (ii), (iii), (iv). (v), (vi) and (vii). In another more preferred embodiment, the combination of eight refers to the combination of the features in items (i), (ii), (iii), (iv). (v), (vi), (vii) and (viii).
• corresponding in accordance with the above preferred embodiment of the fifteenth aspect of the invention means a cell from a comparable similar origin, i.e. , animal, preferably a cell identical to the parent cell before reprogramming/conversion.
• the high-grade naive pluripotent cell is a reprogrammed/converted human low-grade pluripotent cell
• the corresponding non-naive pluripotent cell is a human low-grade pluripotent cell selected from primed pluripotent cells, iPSCs, and embryonic stem cells.
• the expression level of the naive pluripotency-specific markers) and the primitive endoderm-specific marker(s) as defined in item (ii), and (iii) of the above preferred embodiment of the fifteenth aspects of the invention is a gene expression and/or protein expression, preferably the expression of the markers is a detectable expression of said marker proteins.
• the expression levels according to the present invention can be quantified by any suitable means and methods available from the art. In general, relative and absolute quantification means and methods can be used. In absolute quantification no known standards or controls are needed. The expression level can be directly quantified. As well-known in the art, absolute quantification may rely in certain further embodiments on a predetermined standard curve. In relative quantification the expression level is quantified relative to a reference such as known control expression levels. Also, in the absence of controls, one can relatively quantify the expression level when comparing e.g., fluorescence intensities.
• a higher expression level or the term “increased expression level” refers to quantification of expression levels, e.g., levels of pluripotency-specific markers in comparison to a reference.
• the increase compared to the reference is at least, with increasing preference, 5 times more, at least 10 times more, at least, 20 times more, at least 30 times more, at least 40 times more, at least
• P0U5f1 distal enhancer is also referred to as “Oct4 distal enhancer” and specifically refers to a regulatory region in the POU5F1 gene for regulating transcription of Oct4.
• Oct4 distal enhancer specifically refers to a regulatory region in the POU5F1 gene for regulating transcription of Oct4.
• the reactivated X chromosome in female lines is also described as “Xa chromosome” herein above, wherein the active status of both X chromosomes is referred to as “XaXa”.
• Reactivation of X chromosomes occurs on inactivated X chromosomes as an epigenetic process.
• inactivation of one of the X chromosomes in female lines occur to compensate for potential dosage differences of X-linked genes between female XX and male XY cells.
• mouse embryonic stem cells from the inner cell mass of the blastocyst contain two active X chromosomes, one of which will be inactivated upon exit from naive state, during in vivo or vitro priming or differentiation.
• the X chromosomes are already inactivated, because they represent primed pluripotent state.
• human iPSCs and ESCs the inactivation state can be reversed.
• Molecular dynamic simulations reveal SL configuration of Sox/Oct4 a-b, A model of Sox2/Oct4 versus Sox2 AV /Oct4 heterodimer (a) and Sox2/Oct6 versus Sox2 AV /Oct6 heterodimer (b) in POUs (S) configuration on HoxB1 DNA motif. Only DNA-binding domains are shown. Oct4/Oct6 in yellow, Sox2/Sox2 AV in blue, c-d, Computer molecular dynamic simulations (MDS) of Sox/Oct heterodimers on HoxB1 motif.
• MDS Computer molecular dynamic simulations
• the plots show the coordination numbers (the number of contacts) between the residue 61 in Sox2 (blue) or Sox2 A61v (red) either with the entire DNA binding domain of Oct4 (dark) or Oct6 (light) molecule (c), or with residue 211 in Oct (d). Calculations were made for 4.8 ps of MDS of each ternary Sox/Oct/DNA complex. 4 independent 1.2 ps long simulations were performed using two different starting structural models (2 simulations per model). To ensure stochasticity, each simulation was started with a different distribution of atomic velocities. The distance threshold for a contact between two atoms was 4.5 A.
• White arrow heads indicate nonspecific bands (ns) and black arrow heads indicate free DNA or DNA bound by Oct4, Sox2, or the heterodimer, f, Representative kinetic off-rate EMSAs of whole-cell lysates from HEK293 overexpressing Oct4, Sox2 (blue), Sox2 A61v (light red) or Sox2 43 ⁇ 7 ’ 61 ’ 6586 c17 (Sox2-17, S*, dark red) on Oct4 distal enhancer (Oct4DE), Nanog promoter, or Fgf4 enhancer DNA elements labeled with Cy5.
• White arrow heads indicated nonspecific bands (ns) and black arrow heads indicate free DNA or DNA bound by Oct4 (O/DNA), Sox (S/DNA), or Oct4/Sox heterodimer (O/S/DNA).
• h A schematic of the enabling effect of highly cooperative Sox2 AV mutant on reprogramming with tissue specific POU factors or incapable Oct4 mutants.
• b Heat maps and read pileup plots of Sox2 and Oct4 ChlP-seq for 2 dpi tetO-OKS MEF reprogramming samples comparing Sox2 AV versus wild-type Sox2.
• c Boxplots of quantile normalized ChlP-seq peaks for Oct4 and Sox2 for 2 dpi OKS and KS reprogramming samples.
• the midline indicates the median, boxes indicate the upper and lower quartiles and the whiskers indicate 1 .5 times interquartile range, d, Fraction of binding sites containing SoxOct, MORE, both or none of the motifs between 2 dpi OKS reprogramming samples, where O is either Oct4 or Oct6, and S is either Sox2 orSox2 AV .
• e Genome browser track of Oct4 and Sox2 ChlP-seq peaks for selected pluripotency-specific loci
• f Venn diagram showing a number of ESC-specific enhancers (Shen et al., 2012) bound by Sox2 versus Sox2 AV on 2 dpi tetO-OKS reprogramming samples
• g Percentage of tetrapioid (4N)-aggregated embryos derived from the indicated tet-inducible or integration-free episomal iPSCs that gave rise to full-term pups, pups that initiated breathing, pups that survived foster-nursing for at least 48h, and those survived to adulthood (at least 3 months).
• Bars represent the mean between all tested lines for each cocktail. Error bars represent SEM. The statistical significance was determined by Mann-Whitney test, h, Adult tetO-OS AV KM all-IPSC mice (9 months), i, PCR-genotyping of the progeny of tetO-OS AV KM all- iPSC mice.
• Sox2-17 enhances reprogramming in five species
• a Schematic representation of time course tet-inducible lentiviral reprogramming experiment
• b Time course reprogramming of Oct4-GFP MEFs induced with OSKM or SKM carrying either Sox2 (S) or Sox2- 17 (S*) for the given number of days; the iPSC colonies were counted on 9 dpi.
• TRA1-60 Two-factor (OS) reprogramming of human fibroblasts with monocistronic retroviral vectors.
• j Representative whole-well scan of (i).
• k TRA1-60 staining of human fetal (CRL-2097) fibroblasts reprogrammed with self-replicating RNA (VEE) vectors carrying either OKSiG or OKS*iG reprogramming casettes.
• VEE self-replicating RNA
• RNA-seq global gene expression
• TPM S1 p
• RRBS global methylome
• HEK293T cells were cultured in low-glucose DMEM (Sigma) supplemented with 10% FBS (Capricorn Scientific), 1% Glutamax, 1% penicillin-streptomycin, 1% nonessential amino acids (all from Sigma).
• Mid-log phase Sf9 cells were used to amplify the virus.
• Suspension High FiveTM cells were infected with P3 virus for two days at 27°C and 120 rpm shaking.
• crude lysates were purified on a HiTrap TALON column (GE Healthcare), cleaved on the column with 3C protease followed by size exclusion chromatography (HiLoad Superdex 200, GE Healthcare).
• the final product was collected in 25 mM HEPES pH 7.8, 150 mM NaCI, 1 mM TCEP, and 5% glycerol with ⁇ 95% purity confirmed by SDS-PAGE.
• DNA probes were generated by annealing complementary 5’ labeled Cy5 oligos (Metabion International AG) followed by purification from 10% polyacrylamide gels.
• WCL 2-4 ug of total protein
• purified proteins were incubated in binding buffer (25 mM HEPES-KOH pH 8, 50 mM NaCI, 0.5 mM EDTA, 0.07% Triton X-100, 4 mg/mL BSA, 7 mM DTT, and 10% glycerol) and 70 nM Cy5- dsDNA at 37°C for 1 h.
• Equations for decay were determined using nonlinear regression in Prism 7 for Mac (version 7.0a) and only used when goodness of fit, evaluated by R 2 values, was 0.95 or greater.
• the coordination number between two atom selections describes the number contacts between the selections using a continuous switching function with a distance threshold for contact formation as implemented in the COLVAR module of NAMD.
• RNA-seq and RRBS were performed for human iPSC at passage 10-12, human ESCs at passage 35-36 grown in StemFlex media on matrigel, human fibroblasts at passage 11-15 were grown on gelatin-coated dishes in fibroblast media.
• RNA-seq, ChlP-seq, RRBS were peformed as described before (Keshet and Benvenisty, 2021 ; Malik et al., 2019; Velychko et aL, 2019a).
• Oct4 is the only TF of the POU (Pit1 , Oct1/Oct2, UNC-86) family that can induce pluripotency in both mouse and human cells (Takahashi and Yamanaka, 2006; Takahashi et al., 2007; Yu et al., 2007), while other members of the family, such as the ubiquitously expressed Octi (Pou2f1), neural Oct6 (Pou3f1 ), and Brn4 (Pou3f3 or Oct9) cannot (Jerabek et al., 2017; Nakagawa et al., 2008; Velychko et al., 2019b).
• POU factors have different binding profiles and different preferences for hetero- versus homodimerisation (Jerabek et al., 2017; Malik et aL, 2019; Mistri et al., 2015).
• search to find what makes Oct4 unique among POU factors they discovered that the Sox17 E57K mutant (Jauch et al., 2011), but not wild type Sox2, can efficiently generate iPSCs in combination with Brn4 (Fig. 1 a).
• POU factors consist of a DNA-binding domain (POU domain), flanked by N- and C-terminal transactivator domain (NTD and CTD).
• the POU domain is bipartite, consisting of a POU-specific domain (POUs) and a POU-homeodomain (POUHD) joined by a flexible non-conserved linker.
• Oct4 s but not OctTs linker contains an alpha-helix near its N-terminus (Esch et aL, 2013; Klemm et aL, 1994).
• Replacement of the Oct4 linker with those from other POU factors, or even the point mutant L80— >A in the linker helix is detrimental for induction and maintenance of pluripotency or supporting normal development (Chen et al., 2020a; Esch et aL, 2013; Han et al., 2022a).
• Sox17 EK could also rescue the reprogramming ability of Oct4 L80A (Fig. 7a).
• FIG. 1b-c A library of chimeric TFs was constructed, where the non-conserved residues from Sox17 were swapped into Sox2 to find the structural elements responsible for the rescuing phenomenon (Fig. 1b-c).
• Monocistronic retroviral supernatants were used for reprogramming of mouse embryonic fibroblasts (MEFs) carrying endogenous Oct4-GFP reporter (OG2); the volumes of viral supernatants were adjusted based on qPCR titration (Fig. 7b-c).
• Sox17-CTD enhanced reprogramming with wild-type Oct4, but could not rescue Oct4 L80A (Sox2c17 chimera, Fig. 1 d-e) unless combined with the 61-62 region (Sox2 61- 62 c17 chimera, Fig. 1d-e).
• Other Sox17 elements e.g., 24-28 significantly decreased the reprogramming efficiency of Sox2 (Fig. 1 d).
• a second screen determined that a single A61 V swap in Sox2 rescues both Oct4 L80A and Brn4, while L62Q was inconsequential (Fig. 1 f-g).
• Sox2 A61v also allowed reprogramming with Oct2 (Pou2f2), Oct6, and Brn2 (Pou3f2 or Oct7) (Fig. 1 h).
• Octi (Pou2f1 ) was the only tested POU factor that did not yield iPSC colonies when co-expressed with Sox2 A61v and Klf4 in MEFs (Fig. 1 h).
• An A61L mutation was also tested, as leucine has even higher hydrophobicity than valine.
• Sox2 A61L performed better than wild type Sox2, it was significantly worse than Sox2 A81v (from now on Sox2 AV ), especially in rescuing Brn4 (Fig. 1 f-g).
• Sox2 43 ' 4761 ' 65 ' 86 c17 Sox2-17 or S* from now on
• Fig. 1 b-c exceeded the reprogramming ability of Sox2, Sox2c17, or Sox17 EK (Fig. 1f-g).
• A61 is located in the third helix of the high-mobility group (HMG) domain of Sox2 that faces towards POUs domain when the heterodimer is bound to a canonical SoxOct motif (Fig. 2a-b).
• HMG high-mobility group
• valine has two additional methyl groups, making it more hydrophobic.
• MDS Molecular dynamic simulations
• Sox2/Oct4 heterodimer SL configuration involves both the POUs and Linker of Oct4, as opposed to S configuration, which involves only the POUs.
• Yet another configuration described for Sox/Oct heterodimer on the rarer Fgf4 motif was also modeled, where Sox and Oct sites are spaced by a three base-pair gap. Sox2 and Oct4 cooperate more distantly on the Fgf4 motif, forming a distant S (DS) configuration that involves Sox2’s T78 and T80, but not A61 (Fig. 2f).
• DS distant S
• Episomal (epi-) vectors deliver milder overexpression, giving rise to better quality integration-free iPSCs, even in the presence of exogenous Oct4 (Velychko et al., 2019a). Yet only 4.2% of transferred epi-OSKM all-iPSC embryos survived until adulthood, compared to 22.2% of OS AV KM all-iPSC embryos. Thus, enhancing Sox2/Oct4 cooperativity significantly improves the developmental potential of mouse OSKM iPSCs.
• RNA-OKS*iG vector Human self-replicating RNA (Yoshioka et aL, 2013) OKS*iG vector encoding for OCT4, KLF4, SOX2-17, and GLIS1 also gave ⁇ 50 times more TRAI- 60+ colonies comparing to wild-type SOX2 (Fig. 5k). Moreover, using the RNA-OKS*iG vector, integration-free iPSCs from a Parkinson’s patient’s dermal fibroblasts were successfully generated and characterized, which could not yield IPSCs using original vector with wild-type SOX2 (Rosety et al., Sci Adv 2022, in press).
• SOX2-17-P2A-KLF4 S*K
• OCT4/shTP53 OCT4/shTP53
• L-MYC/LIN28 OS*KML
• the episomal vectors were tested in generating IPSCs from cynomolgus macaque (Macaca fascicularis), an important non-human primate model (Han et al., 2022b; Wunderlich et al., 2012, 2014), for which the ESCs have been derived (Chen et aL, 2015b; Fu et aL, 2020) but no transgene-independent iPSCs have been reported.
• Integration-free ciPSCs displayed morphology similar to that of hiPSCs, except they were more prone to differentiation in the middle of colonies and could only be maintained on a feeder layer (Fig. 10r). They expressed pluripotency markers NANOG and OCT4 (Fig. 10s) and could differentiate into three germ layers in teratoma assays (Fig. 10t). It was also attempted to reprogram bovine and porcine fibroblasts using XAV939-containing bFGF-based media recently developed by the Smith lab to derive and maintain cattle ESCs (Kinoshita et al., 2021).
• Loss of imprinting is a common potentially cancerous (Holm et al., 2005; Jelinic and Shaw, 2007) irreversible (Hiura et al., 2013) epigenetic aberration afflicting iPSC technology for both mouse and human (Bar et al., 2017; Carey et al., 2011 ; Keshet and Benvenisty, 2021 ; Takikawa et al., 2013).
• mice The ESC derivative of mouse pre-implantation inner cell mass and their IPSC counterparts grown in LIF containing media are called “naive” (Nichols and Smith, 2009).
• Mouse naive lines exhibit the highest developmental potential of all cultured cells - they can contribute to chimeric animals, some, but not all naive lines are even capable of generating all-PSC mice in 4N complementation assays. This, however, is not true for “primed” PSCs of other species including human, which are more similar to mouse epiblast stem cells (mEpiSCs) (Tesar et aL, 2007).
• mEpiSCs mouse epiblast stem cells
• Oct4DE is active in naive but not primed pluripotent cells (Choi et aL, 2016; Gafni et aL, 2013; Yeom et aL, 1996), and the highly-cooperative Sox factors dramatically increase stability of Sox2/Oct4 heterodimer on Oct4DE element (Fig. 3f).
• Sox/Oct cooperativity could be at the core of naive pluripotency.
• Previously published time-course ATAC-seq dataset of naive-to-primed transition samples generated for mouse ESCs upon exposure to bFGF media (Yang etaL, 2019) were analyzed.
• d1 samples also had a significant depletion of Sox/Oct footprints (Fig. 6b), which became the most depleted of all TF footprints between d1 and d2 of transition (Fig. 6c).
• Whole-cell lysate EMSAs was performed on the SoxOct element of Nanog promoter to measure the levels of heterodimerization of naturally expressed Sox2 and Oct4 in E14 mESC line grown in KSR-LIF media, GFP" E3 mEpiSC line harboring Oct4-GFP reporter (Gof18) grown in bFGF-containing hESC media, primed-to-naTve converted sorted Oct4-GFP + mEpiSCs grown in KSR-LIF or the same media containing 2i (Mek and GSK-3 inhibitors, PD0325901 and CHIR99021 , respectively (Ying et al., 2008)), as well as human iPSCs grown in conventional bFGF media (Fig.
• mCherry was cloned into the human episomal reprogramming vectors to generate pCXLE-mCherry- T2A-SOX-P2A-KLF4 carrying SOX2, SOX2 AV , or SOX2-17.
• the episomal Cherry-SK vectors were delivered into mEpiSCs using lipofection, the cells were sorted for mCherry + /Oct4-GFP" on day 2 (Fig. 11a) and plated in KSR-LIF media on inactivated feeders.
• Reprogramming factor stoichiometry influences the epigenetic state and biological properties of induced pluripotent stem cells.
• JASPAR 2022 The 9th release of the open-access database of transcription factor binding profiles. Nucleic Acids Res. 50, D165-D173.
• mice Male and female mice derived from the same embryonic stem cell clone by tetrapioid embryo complementation. Nat. Biotechnol. 20, 455-459.
• Genome editing reveals a role for OCT4 in human embryogenesis. Nature 550, 67-73.
• Klf4 reverts developmentally programmed restriction of ground state pluripotency. Development 136, 1063-1069.
• Epiblast stem cell subpopulations represent mouse embryos of distinct pregastrulation stages. Cell 143, 617-627.
• KLF17 promotes human naive pluripotency but is not required for its establishment. Development. 2021 Nov 15;148(22):dev199378. doi: 10.1242/dev.199378. Epub 2021 Nov 15. PMID: 34661235; PMCID: PMC8645209.
• KSR-based medium improves the generation of high-quality mouse IPS cells.
• Sox2 is the faithful marker for pluripotency in pig: Evidence from embryonic studies. Dev. Dyn. 244, 619-627.
• Genomic imprinting is variably lost during reprogramming of mouse IPS cells. Stem Cell Res. 11, 861-873.

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