Engineered Sox / Oct heterodimerization to induce high-grade developmental resetting

Abstract

The present invention relates to a variant of a SoxB1 factor comprising: (a) an HMG (high mobility group) domain of any 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 that shares at least 82% sequence identity with the HMG domain defined in (a), assuming that the substitution defined in (a) is retained.

Description

[Technical Field]

[0001] The present invention relates to a variant of a SoxB1 factor comprising: a) an HMG (high-mobility group) domain of any 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 defined in a), assuming that the substitution defined in a) is retained.

[0002] Several documents are cited herein. The disclosures of these documents, while not considered relevant to the patentability of this invention, are hereby incorporated by reference in their entirety. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference. [Background technology]

[0003] The ability of some transcription factors, such as MyoD, to alter cell fate has been known since 1987 (Davis et al., 1987). It wasn't until 2006, with the discovery of induced pluripotent stem cells (iPSCs) by Shinya Yamanaka, that transcription factor-based cell fate conversion came into focus, and rightly so (Takahashi and Yamanaka, 2006). Pluripotent cells are unique in their ability to give rise to all tissues of an animal. Induction of pluripotency, culminating in ultimate cellular rejuvenation, was once thought to be impossible (Waddington, 1957). iPSC technology has already contributed significantly to human developmental biology research, enabled new strategies for drug discovery, and provided a source of cell replacement therapy. However, the most radical efforts are likely still ahead: recent work by Belmonte's lab and others has demonstrated the ability of reprogramming factors to reverse aging at the whole-organism level in mice (Browder et al., 2022; Chen et al., 2021; Chondronasiou et al., 2022; Lu et al., 2020; Ocampo et al., 2016; Sarkar et al., 2020), and the Reik lab has shown that time-limited induction of reprogramming factors can rejuvenate cultured human cells (Gill et al., 2022).

[0004] All components of the Yamanaka cocktail—Oct4, Sox2, Klf4, and cMyc (OSKM)—regulate the pluripotency of the inner cell mass of the blastocyst (Nakatake et al., 2006; Wu and Schoeler, 2014). This stage of mammalian development is transient; shortly after emergence, pluripotent cells in the inner cell mass are committed to unidirectional differentiation. Pluripotent cells can be artificially propagated from the inner cell mass in the form of embryonic stem cells (ESCs) (Thomson et al., 1998). Oct4, Sox2, and Klf4 (OSK) are pioneer transcription factors that can open silent chromatin, allowing cells to maintain a plastic pluripotent state or to later initiate differentiation (King and Klose, 2017). iPSC technology exploits the pioneering ability of OSK to drive cell identity in opposite directions (Soufi et al., 2012). Oct4 stands out as a key regulator of the pluripotency network. It is the only factor whose knockout in ESCs leads to the inevitable disruption 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). Concurrently, overexpression of Oct4 during reprogramming has been shown to result in abnormal epigenetic changes in mouse iPSCs and to worsen the developmental potential of OSKM relative to SKM iPSCs (Velychko et al., 2019a). Oct4 plays diverse roles in establishing pluripotency during mouse and human development: Oct4 knockout mouse blastocysts still retain Nanog expression. +Human OCT4-null blastocysts develop an inner cell mass, whereas human OCT4-null blastocysts fail to do so (Fogarty et al., 2017; Wu et al., 2013). Correspondingly, SKM induction is sufficient to induce pluripotency in mouse somatic cells (An et al., 2019; Velychko et al., 2019a), although SKM reprogramming has not been demonstrated in humans or other species.

[0005] Oct4 cooperates with Sox2 to coregulate most of its targets in pluripotent cells (Chen et al., 2008). Oct4 / Sox2 cooperativity is mediated by DNA allostery and protein-protein interactions between their DNA-binding domains (Merino et al., 2015). At the initiation of iPSC reprogramming, when the native Oct4 and Sox2 sites are inaccessible, each factor often binds independently (Soufi et al., 2012), yet sites bound by both factors remain more open (Chronis et al., 2017; Malik et al., 2019). Indeed, Oct4 / Sox2 cooperativity, particularly on the Hoxb1-like canonical SoxOct motif, has been shown to be essential for the induction and maintenance of pluripotency (Tapia et al., 2015). On the other hand, Sox17, in cooperation with Oct4 on the compacted non-canonical SoxOct motif, fails to induce pluripotency but rather regulates primitive endoderm and germ cell characteristics (Aksoy et al., 2013; Merino et al., 2014; Ng et al., 2012, Irie et al., 2015). Jauch et al. reported that a single residue modification in Sox17, E57→K, shifts its binding preference to the canonical SoxOct motif and converts Sox17 into a pluripotent derivative (Jauch et al., 2011). The Sox17 C-terminal transactivator domain (CTD) is more potent than the Sox2 CTD. Replacing the Sox2 CTD with that from Sox17 has been shown to enhance the reprogramming ability of Sox2 (Aksoy et al., 2013).

[0006] iPSC technology remains unsatisfactory, especially for non-mouse cells, and reprogramming often results in epigenetic abnormalities that result in inconsistent iPSC quality. It is well established that OSKM reprogramming, commonly used to derive iPSCs, often results in loss of imprinting (LOI), which can be cancerous. As mentioned above, several alternative cocktails have been shown to improve the fidelity of the reprogramming process in mice, but they have failed to reprogram human cells, which appear to have stronger epigenetic barriers. Perhaps the apparent barriers are simply evidence of shortcomings in the wild-type factor-based reprogramming mechanisms we currently utilize. Thus, there remains an urgent need in the art to provide means and methods for improving the efficacy and quality of iPSC reprogramming that overcome the shortcomings of previous techniques and cocktails. The present invention addresses this need and provides alternative Sox reprogramming factors, cocktails, and methods for highly effective and improved cell reprogramming. Summary of the Invention

[0007] Thus, in a first aspect, the present invention relates to a variant of a SoxB1 factor comprising: (a) an HMG domain of any 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 defined in (a), assuming that the substitution defined in (a) is retained.

[0008] Generally, "Sox factors" refer to members of a known family of transcription factors (TFs) involved in maintaining pluripotency. They generally exert their function by binding to regulatory factors containing Sox DNA motifs. Members of the Sox family of TFs are classified into distinct groups (SoxA-SoxJ) based on their level of amino acid sequence identity within their HMG domains. All naturally occurring Sox TFs consist of an N-terminal domain (NTD), a DNA-binding high-mobility domain (HMG), and a C-terminal domain (CTD), which contain either transactivation or transrepression functions. Sox members within the same group share a high degree of amino acid sequence identity (approximately 70%-95%) for both their HMG domains and regions outside their HMG domains. In contrast, Sox proteins from different groups share only partial (≥46%) amino acid sequence identity within their HMG domains (Aksoy et al., 2013). Because Sox TFs generally have similar DNA-binding specificities, their ability to induce specific biological processes is thought to be mediated by their selective interaction with specific cofactors, such as Oct4.

[0009] The term "SoxB1 factors" as used herein refers to the B1 subclass of Sox (TFs), which includes factors Sox1, Sox2 and Sox3, which share greater than 90% sequence identity, especially over their HMG domains.

[0010] As shown herein, we use Sox2 and Sox17 in the reprogramming cocktail. EK We found that replacing the mutant Sox17 with a Sox17 E57K mutant rescued the otherwise deleterious Oct4 mutant and enabled reprogramming with POU factors other than Oct4. We then created a library of chimeric Sox2-Sox17 TFs to identify the structural components of Sox17 responsible for this unique phenotype.

[0011] The term "Sox17," as used herein, refers to a member of the F subclass of Sox TFs ("SoxF factors"), a subclass of Sox TFs distinct from SoxB1 factors.

[0012] Surprisingly, we found through library screening that a single residue modification at the Oct / Sox interface in the SoxB1 factor Sox2, namely the replacement of alanine at position 61 in the HMG domain of Sox2 with the more hydrophobic residue valine, significantly increased the stability of the Oct / Sox complex on the canonical SoxOct motif that controls naive pluripotency. Enhanced heterodimerization was observed on both naked and nucleosomal DNA using EMSA and in situ in reprogrammed cells using CHIP-seq. Furthermore, Sox A61V allows reprogramming with Oct4 orthologues, such as Brn2, Brn4, Oct6, Oct2, and otherwise non-functional Oct4 mutants. Furthermore, the tetraploid complementation assay—the most rigorous test for pluripotency, which measures the ability of iPSCs to generate whole animals—enables Sox A61V The mutants were shown to dramatically enhance the developmental potential of OSKM iPSCs (as shown in the Examples herein below).

[0013] In this context, it should be noted that the amino acid sequences of SEQ ID NOs: 1 to 3 correspond to the HMG domains of human wild-type SoxB1 factors Sox1, 2 and 3, respectively.

[0014] Thus, a SoxB1 factor variant according to a first aspect of the present invention comprises: a) the HMG domain of any of Sox1, 2 or 3 (i.e., SEQ ID NO: 1, 2 or 3), wherein the amino acid alanine at position 61 is replaced 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 defined in a), assuming that the substitution at position 61 of their respective HMG domains is retained.

[0015] In a preferred embodiment of item b) of the first aspect of the invention, the amino acid sequence sharing at least 82% sequence identity exhibits, 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 defined in item a) of the first aspect of the invention.

[0016] According to the present invention, the term "percent (%) sequence identity" describes the number of identical amino acid / nucleotide matches ("hits") of two or more aligned amino acid or nucleic acid sequences compared to the number of amino acid residues or nucleotides that make up the entire length of a template nucleic acid or amino acid sequence. In other terms, using an alignment for two or more sequences or subsequences, the percentage of amino acid residues or nucleotides that are the same (e.g., 70%, 75%, 80%, 85%, 90%, or 95% identity) can be determined when (sub)sequences are compared and aligned for maximum correspondence, or when aligned manually and visually inspected, over a window of comparison, or over a designated region as measured using sequence comparison algorithms known in the art. This definition also applies to the complementary strand of any aligned sequence.

[0017] The term "sequence identity" as used herein 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 for their identical aligned sequences, and identical alignment means that the same nucleotide base or amino acid residue occupies the same position in the compared sequences. Thus, "percent identity" is a function of the number of matching positions divided by the number of compared positions multiplied by 100%. For example, if 7 positions out of 10 sequence positions are identical, the identity is 70%.

[0018] Nucleotide and amino acid sequence analysis and alignment in the context of the present invention is preferably carried out using bioinformatics tools for pairwise 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). "Identity" or "percent (%) identity" between two amino acid sequences can be determined, for example, using the Needleman-Wunsch algorithm incorporated into EMBOSS Needle (Needleman, SB and Wunsch, CD. A general method applicable to the search for similarities in the amino acid sequence of two proteins. J Mol Biol. 1970;48(3):443-53. DOI: 10.1016 / 0022-2836(70)90057-4) using a BLOSUM62 matrix, a "gap open penalty" of 10, a "gap extension penalty" of 0.5, a pseudo "end gap penalty", an "end gap open penalty" of 10, and an "end gap extension penalty" of 0.5. Percent (%) identity is typically determined over the entire length of the query sequence over which the analysis is performed. Two molecules with the same primary amino acid or nucleic acid sequence are identical regardless of any chemical and / or biological modifications, such as glycosylation patterns. For example, in the case of nucleic acids, two molecules with the same sequence but different linkage components, eg, a thiophosphate instead of a phosphate, are identical by this definition.Another tool for evaluating biological sequence alignment, which determines the region of similarity between 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. Schaeffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), Nucleic Acids Res. 25:3389-3402).BLAST can be used for nucleotide sequences (nucleotide BLAST) and amino acid sequences (protein BLAST).Those skilled in the art will understand other suitable programs for aligning nucleic acid sequences. The preferred method for nucleotide and amino acid sequence analysis and alignment according to the present invention is best performed with EMBOSS Needle, with parameters of BLOSUM62 matrix, "Gap Open Penalty" of 10, "Gap Extension Penalty" of 0.5, pseudo "End Gap Penalty", "End Gap Open Penalty" of 10 and "End Gap Extension Penalty" of 0.5.

[0019] The amino acid sequences of wild-type SoxB1 factors and the corresponding protein-encoding nucleotide sequences are known for several species, such as human and mouse Sox1, Sox2 and Sox3, and are available, for example, from the NCBI database: https: / / www.ncbi.nlm.nih.gov.

[0020] For example, the amino acid sequences of wild-type full-length human Sox1, Sox2, and Sox3 proteins are available from the NCBI database under the following accession numbers: Sox1: NP_005977, e.g., NP_005977.2; Sox2: NP_003097, e.g., NP_003097.1; Sox3: NP_005625, e.g., NP_005625.2, and are defined herein by SEQ ID NOs: 4-6, respectively. All sequences under each NCBI reference SEQ ID NO are understood to be included. The HMG domains of human Sox1 to Sox3, defined by SEQ ID NOs: 1-3, respectively, are included in SEQ ID NOs: 4-6, respectively, e.g., the HMG domain of SEQ ID NO: 1 is included in SEQ ID NO: 4, etc.

[0021] Note that in this context, homologues of the human Sox1, Sox2 and Sox3 HMG domains from other sources, such as the Sox1, Sox2 or Sox3 HMG domains from mouse, cynomolgus monkey, rat, horse, pig, cow or other animal species, having a sequence identity level of at least 82% to SEQ ID NO: 1, 2 or 3 are also specifically included.

[0022] As shown in the Examples herein below, the inventors have further found that replacing the residue at position A61 of the HMG domain of Sox2 with an amino acid selected from valine, leucine and isoleucine, preferably valine; when combined with other beneficial elements of the Sox17 HMG domain defined herein by SEQ ID NO: 15, further enhances reprogramming efficiency.

[0023] Thus, in a preferred embodiment of item a) of the first aspect of the present invention, a SoxB1 factor variant is also contemplated herein, further comprising: a) the amino acids at positions 43 to 47 in the HMG domain of any of SEQ ID NOs: 1 to 3 are replaced with the amino acid sequence of SEQ ID NO: 7; and / or b) the amino acids at positions 65 to 86 in the HMG domain of any of SEQ ID NOs: 1 to 3 are replaced with the amino acid sequence of SEQ ID NO: 8; and / or c) the SoxB1 factor variant comprises an amino acid sequence that shares at least 82% sequence identity with the SoxB1 factor variant of a) and / or b), assuming that the substitutions defined in a) and / or b) are retained.

[0024] According to item c) of the above preferred embodiment, an amino acid sequence sharing at least 82% sequence identity exhibits, 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 SoxB1 factor variant of a) and / or b), provided that the substitutions defined in a) and / or b) are retained.

[0025] In a particularly preferred embodiment of the first aspect of the invention, the SoxB1 factor variant is a Sox2 factor variant.

[0026] Thus, according to the above-mentioned particularly preferred embodiment, the Sox2 factor variant comprises: a) an HMG domain of the amino acid sequence of SEQ ID NO: 2, 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 defined in a), assuming that the substitution defined in a) is retained.

[0027] The present inventors have further found that when the substitutions for the HMG domain described herein above are combined with additional beneficial factors, reprogramming efficiency can be further enhanced.

[0028] Thus, a variant SoxB1 factor according to the first aspect of the present invention may further comprise one or more additional amino acids or amino acid sequences adjacent to the C-terminus and / or N-terminus of the HMG domain. Such additional amino acids or amino acid sequences may, for example, correspond to naturally occurring or non-naturally occurring N-terminal and / or C-terminal domains adjacent to the HMG domain, respectively. The N- or C-terminal domain naturally adjacent to the HMG domain of a Sox factor may, for example, be replaced by the N- or C-terminal domain of another Sox factor.

[0029] Specifically, the Sox17 C-terminal (transactivator) domain was found to be larger and more potent than that of Sox2, and replacement of the Sox2 CTD with the Sox17 CTD enhanced the reprogramming ability of Sox2 ( Aksoy et al., 2013 ).

[0030] Therefore, in another preferred embodiment of the first aspect of the present invention, the SoxB1 factor variant further comprises the amino acid sequence of SEQ ID NO:9, preferably the amino acid sequence of SEQ ID NO:9 is linked to the C-terminus of the HMG domain defined according to the first aspect of the present invention.

[0031] As used herein, the term "linked" means that one (poly)peptide is attached to another, preferably by a peptide bond between one amino acid of the (poly)peptide and one amino acid of the other (poly)peptide. The term "linked" as used herein particularly means "operably linked," which refers to the juxtaposition of at least two (poly)peptides to each other in a manner that allows both (poly)peptides to function normally, such that at least one of the (poly)peptides may mediate the function performed by the other (poly)peptide.

[0032] In another preferred embodiment, 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), including the C- and N-terminal domains, in which the amino acid alanine at position 61 in the HMG domain of each of them as defined in SEQ ID NOs: 1, 2 or 3 is substituted with an amino acid selected from valine, leucine, isoleucine, phenylalanine, methionine, tryptophan or proline, preferably valine.

[0033] In another, or even more preferred, embodiment, the amino acid alanine at position 61 within the HMG domain defined in SEQ ID NO: 1, 2 or 3, respectively, is substituted with an amino acid selected from valine, leucine and isoleucine, most preferably valine.

[0034] Note that in this context, references to position 61 with respect to substitutions according to the present invention always relate to the amino acid number starting from the respective HMG domain as defined herein.

[0035] Thus, in a further preferred embodiment of the first aspect of the present invention, the SoxB1 factor variant comprises or consists of any 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 of SEQ ID NOs: 10 to 12, assuming 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.

[0036] SEQ ID NOs: 10 to 12 correspond to the full-length amino acid sequences of human wild-type Sox1, Sox2, and Sox3, respectively, in which the amino acid alanine at position 61 within their respective HMG domains as defined in SEQ ID NOs: 1 to 3, respectively, is substituted with valine.

[0037] In preferred embodiments, amino acid sequences according to the above-mentioned preferred embodiments of the first aspect of the invention which share at least 82% sequence identity exhibit, 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: 10-12, and most preferably have 100% sequence identity to SEQ ID NOs: 10-12.

[0038] In particularly preferred embodiments of the first aspect of the present invention, the SoxB1 factor variant comprises or consists of the amino acid sequence of SEQ ID NO:13 or SEQ ID NO:14.

[0039] In alternative preferred embodiments, 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 most preferably is 100% identical to SEQ ID NO: 13 or 14, assuming the substitutions defined in a) are retained.

[0040] Furthermore, we found that other regions of Sox17, such as positions 24-28 of the Sox17 HMG domain, significantly reduced the reprogramming efficiency of Sox2. Therefore, it can be concluded that the reprogramming efficiency of Sox17 can be increased by substituting positions 24-28 of the Sox17 HMG domain with positions 24-28 of the Sox2 HMG domain.

[0041] Thus, in a second aspect, the present invention relates to a variant of Sox17 factor comprising: a) an HMG domain of the amino acid sequence of SEQ ID NO: 15, wherein amino acids 24 to 28 of SEQ ID NO: 15 are replaced by 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 defined in a), assuming that the substitutions defined in a) are retained.

[0042] 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 of positions 24 to 28 of the human wild-type HMG domain of Sox2.

[0043] In preferred embodiments, amino acid sequences according to the above-mentioned preferred embodiments of item b) of the second aspect of the invention, which share at least 82% sequence identity, exhibit, 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 defined in a), assuming that the substitutions defined in a) are retained.

[0044] Note that in this context, homologues of the human Sox17 HMG domain from other species, e.g., the HMG domain of Sox17 from mouse or cynomolgus monkey, rat, horse, pig, cow or other animal species, having a sequence identity level of at least 82% are also specifically included.

[0045] The definitions and preferred embodiments of the first aspect of the present invention apply mutatis mutandis to the second aspect of the present invention, insofar as they are acceptable in combination with the second aspect of the present invention. This also applies mutatis mutandis to further aspects of the invention described herein below. For example, the definitions and preferred embodiments of the first and second aspects apply mutatis mutandis to the third aspect of the present invention, insofar as they are acceptable in combination with the third aspect, etc.

[0046] Importantly, the inventors have surprisingly found that the SoxB1 factor variants of the present invention not only allow for the generation of iPSCs with significantly improved developmental potential, but also allow for reprogramming with otherwise deleterious Oct4 mutants, and with POU factors that normally do not allow for iPSC generation, such as Brn4, Oct2, Oct6 and Brn2.

[0047] Thus, in a third aspect, 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 a SoxB1 factor variant of the first aspect of the invention; or ii. a Sox17 or a Sox17 factor variant of the second aspect of the invention; wherein the HMG domain is selected from: iii. an HMG domain according to item a) or item b) of the first aspect of the invention; and wherein the POU factor is iv. an Oct4 or Oct4 variant; or v. an Oct2 or Oct2 variant; or vi. an Oct6 or Oct6 variant; or vii. a Brn2 or Brn2 variant; or viii. Brn4 or a Brn4 variant; or (ix) other natural or synthetic POU factors; the POU domain is selected from x. the POU domain of SEQ ID NO: 17 or 18 or a variant thereof that shares at least 82% sequence identity with SEQ ID NO: 17 or 18; preferably, the amino acids at positions 1 to 50, 78 and / or 82 of SEQ ID NO: 17 or 18, respectively, are retained.

[0048] SEQ ID NO: 17 corresponds to the amino acid sequence of the wild-type human Oct4 POU domain.

[0049] SEQ ID NO: 18 corresponds to the amino acid sequence of the wild-type mouse Oct4 POU domain.

[0050] The sequence identity of the amino acid sequence according to item x. of the third aspect of the present invention, with increasing preference, shares 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 SEQ ID NO: 17 or 18, respectively, and most preferably has 100% sequence identity with SEQ ID NO: 17 or 18, respectively.

[0051] The phrase "POU domain of SEQ ID NO:17 or 18," as used herein, means the amino acid sequence defined by the amino acid sequence set forth in SEQ ID NO:17 and 18, respectively.

[0052] The term "protein" as used herein is used interchangeably with the term polypeptide and refers to a polymer constructed of one or more chains of amino acid residues linked by peptide bonds. The group of "polypeptides" consists of molecules having more than 30 amino acids, separate from the group of peptides consisting of up to 30 amino acids. As used herein, the term "(poly)peptide" more generally refers to both "peptide" and "polypeptide." The term "(poly)peptide" also refers to a peptide or polypeptide that has been chemically or post-translationally modified. In general, the term applies to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids.

[0053] The term "fusion protein," as used herein, refers to a synthetic, semi-synthetic, or recombinant single poly(peptide) molecule that contains all or part 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 can further include a linker that connects the two or more different poly(peptides). Those skilled in the art will understand which linkers can be appropriately used to connect two or more different polypeptides.

[0054] In a preferred embodiment, the fusion protein of the third aspect of the present invention comprises a Sox factor and a POU factor, with the POU factor located at the N-terminus and the Sox factor located at the C-terminus within the fusion protein.

[0055] The term "POU factor," as used herein, refers to a transcription factor (TF) that contains a bipartite DNA-binding domain, termed the POU domain, flanked by N- and C-terminal transactivator domains (NTD and CTD). A bipartite POU domain consists of a POU-specific domain (POU) linked by a flexible, non-conserved linker domain that can be of variable length. S ) and POU homeodomain (POU HD The structure of the POU domain allows it to bind DNA and also participate in protein-protein interactions.

[0056] The term "other natural or synthetic POU elements," as used herein, generally encompasses (poly)peptides that contain the POU domains specifically described hereinabove. (Poly)peptides can include both naturally occurring products and products of recombinant DNA or other synthetic techniques.

[0057] In general, POU factors have different binding profiles and preferences for hetero- versus homo-dimerization (Jerabek et al., 2017; Malik et al., 2019; Mistri et al., 2015). Examples of POU factors include POU class 2 (e.g., Oct1 and Oct2), POU class 3 (e.g., Oct6, Brn1, Brn2, Brn4, etc.), and POU class 5 (e.g., Oct4) factors.

[0058] As used herein, the terms "POU factor," "POU family," or "Oct family" refer to a family of octamer ("Oct") transcription factors, which play an important role, inter alia, in maintaining pluripotency. POU5F1 (POU domain, class 5, transcription factor 1), also known as Oct4, is one representative of the POU family. Further examples include Oct1, Oct2, Oct6, Brn2, and Brn4. Exemplary Oct4 proteins are those encoded by the mouse Oct4 gene (all sequences deposited under NCBI reference sequence NM_013633, e.g., NM_013633.3) and the human Oct4 gene (all sequences deposited under NCBI reference sequence NM_002701, e.g., NM_002701.6).

[0059] The terms "Oct4," "OCT4," "Oct3 / 4," "Oct4 protein," "OCT4 protein," "POU5F1," "Oct3," "Oct3 / 4," and the like refer to either the naturally occurring form of the octamer 4 transcription factor, or a variant thereof that maintains Oct4 transcription factor activity of at least 10%, 30%, 50%, 80%, 90% or 100% activity, with increasing preference (for each value) compared to wild-type Oct4, as measured by methods known in the art, such as measuring the efficacy of iPSC generation in reprogramming experiments.

[0060] In some embodiments, variants have at least 90% amino acid sequence identity across their entire sequence compared to a naturally occurring Oct4 polypeptide. In other embodiments, the Oct4 protein is the protein identified by Genbank reference ADW77327.1, which corresponds to SEQ ID NO: 19, or the protein identified by SEQ ID NO: 20. Also specifically included are Oct4 variants that potentially do not retain Oct4 transcription factor activity but comprise or consist of the amino acid sequence of any of SEQ ID NOs: 21-24.

[0061] The terms "Oct2," "OCT2," "Oct2 protein," "OCT2 protein," "POU2F2," and the like refer to either the naturally occurring form of the octamer 2 transcription factor, or a variant thereof that retains at least 50%, 80%, 90%, or 100% of the Oct2 transcription factor activity, with increasing preference (for each value), compared to wild-type Oct2, as measured by methods known in the art, such as measuring the efficacy of iPSC generation in reprogramming experiments.

[0062] In some embodiments, variants have at least 90% amino acid sequence identity across their entire sequence compared to a naturally occurring Oct2 polypeptide. In other embodiments, the Oct2 protein is a protein identified by all sequences deposited under NCBI reference sequence NP_001193954, e.g., NP_001193954.1, or NP_001157027, e.g., NP_001157027.1, corresponding to SEQ ID NO: 25 or 26, respectively.

[0063] The terms "Oct6," "OCT6," "Oct6 protein," "OCT6 protein," "POU3F1," and the like refer to either the naturally occurring form of octamer 6 transcription factor, or a variant thereof that retains Oct6 transcription factor activity (e.g., at least within 50%, 80%, 90% or 100% activity, with increasing preference (for each value)) compared to wild-type Oct6, as measured by methods known in the art, such as measuring the efficacy of iPSC generation in reprogramming experiments.

[0064] In some embodiments, variants have at least 90% amino acid sequence identity across their entire sequence compared to naturally occurring Oct6 polypeptides. In other embodiments, the Oct6 protein is a protein identified by all sequences deposited under NCBI reference sequence NP_002690, e.g., NP_002690.3, or NP_035271, e.g., NP_035271.1, corresponding to SEQ ID NOs: 27 or 28, respectively.

[0065] The terms "Brn2," "BRN2," "Brn2 protein," "BRN2 protein," "N-Oct-3," "POU3F2," "Oct7," and the like refer to either the naturally occurring form of the Brn2 transcription factor or a variant thereof that retains at least 50%, 80%, 90% or 100% activity of Brn2 transcription factor activity, with increasing preference (for each value) compared to wild-type Brn2, as measured by methods known in the art, such as measuring the efficacy of iPSC generation in reprogramming experiments.

[0066] In some embodiments, variants have at least 90% amino acid sequence identity across their entire sequence compared to a naturally occurring Brn2 polypeptide. In other embodiments, the Brn2 protein is a protein identified by all sequences deposited under NCBI reference sequence NP_005595, e.g., NP_005595.2, or NP_032925, e.g., NP_032925.1, corresponding to SEQ ID NOs: 29 or 30, respectively.

[0067] The terms "Brn4," "BRN4," "Brn4 protein," "BRN4 protein," "POU3F4," "Oct9," and the like refer to either the naturally occurring form of the Brn4 transcription factor or a variant thereof that retains at least 50%, 80%, 90% or 100% of the Brn4 transcription factor activity, with increasing preference (for each value) compared to wild-type Brn4, as measured by methods known in the art, such as measuring the efficacy of iPSC generation in reprogramming experiments.

[0068] In some embodiments, variants have at least 90% amino acid sequence identity across their entire sequence compared to a naturally occurring Brn4 polypeptide. In other embodiments, the Brn4 protein is a protein identified by NCBI reference sequence NP_000298, e.g., NP_000298.3, corresponding to SEQ ID NO: 31 or 32, respectively, or all sequences deposited thereunder.

[0069] The term "domain" as used herein refers to the region / portion of an amino acid sequence that can autonomously adopt a specific structure and / or function.Therefore, "domain" refers to the functional or structural domain of a protein, such as a transcription factor.For example, the HMG domain involved in DNA binding is composed of approximately 75 amino acid residues that collectively mediate the DNA binding of chromatin-associated high mobility group proteins.

[0070] The term "variant" as used herein refers to a nucleic acid or amino acid sequence that differs in at least one base or amino acid residue, respectively, compared with the corresponding wild-type sequence. The difference can be artificially created or naturally occurring. In a preferred embodiment, a variant with respect to a reference (poly)peptide refers to an amino acid sequence variant of the corresponding wild-type amino acid sequence of said (poly)peptide. A variant retains or essentially retains the function of the wild-type form. Essentially, this means having an increased preference of at least 50%, at least 80%, at least 90%, and at least 95%.

[0071] Thus, in preferred embodiments, variants share, 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.

[0072] In a preferred embodiment of the third aspect of the present invention, the Sox factor may be a wild-type Sox factor or a Sox factor variant as defined above, preferably a Sox factor variant as defined in SEQ ID NO: 13 or 14.

[0073] In a fourth aspect, the present invention relates to a complex or 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, POU factor, HMG domain and POU domain are selected from the Sox factors, POU factors, HMG domains and POU domains defined according to the third aspect of the invention.

[0074] According to a fourth aspect of the present invention, the term "complex" refers to an association of two molecules that interact with each other through forces other than bonds and / or peptide bonds (e.g., van der Waals, hydrophobic, hydrophilic forces). The individual members of the complex may be linked by non-covalent interactions. A "complex" according to the fourth aspect of the present invention may also include an additional non-covalent interaction of at least one of the molecules defined in items a) to d) of the fourth aspect of the present invention with another macromolecule, such as a nucleic acid. It is understood that the complex may be multimeric. Each interacting molecule of a complex is referred to herein as an "individual member" or "member" of the complex.

[0075] The term "composition", as used herein, refers to a mixture comprising at least two (poly)peptides, also generally referred to herein as compounds, which may or may not be complexed.

[0076] In a fifth aspect, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: a SoxB1 factor variant according to the first aspect of the invention, a Sox17 factor variant according to the second aspect of the invention or a fusion protein according to the third aspect of the invention; and / or a complex or composition according to the fourth aspect of the invention; and / or - one or more additional reprogramming factor(s) The present invention relates to a nucleic acid molecule or a complex of nucleic acid molecules encoding the

[0077] The term "nucleic acid molecule" according to the present invention includes DNA, e.g., cDNA or double-stranded or single-stranded genomic DNA, and RNA, e.g., natural and modified DNA and RNA. In this context, "DNA" (deoxyribonucleic acid) refers to any chain or sequence of chemical building blocks, referred to as nucleotide bases, selected from adenine (A), guanine (G), cytosine (C), and thymine (T), typically linked together by phosphodiester bonds on a deoxyribose sugar backbone. DNA can have a single strand of nucleotide bases or two complementary strands that can form a double-helical structure. "RNA" (ribonucleic acid) refers to any chain or sequence of chemical building blocks, referred to as nucleotide bases, adenine (A), guanine (G), cytosine (C), and uracil (U), typically linked together by phosphodiester bonds on a ribose sugar backbone. RNA typically has a single strand of nucleotide bases, e.g., mRNA. Single-stranded and double-stranded hybrid molecules, i.e., DNA-DNA, DNA-RNA, and RNA-RNA, are also included. Nucleic acid molecules can also be modified by many means known in the art. Non-limiting examples of such modifications include methylation, "capping," substitution of one or more naturally occurring nucleotide analogs, and internucleotide modifications, such as uncharged linkages (e.g., methyl phosphate, phosphotriester, phosphoramidate, carbamate, etc.) and charged linkages (e.g., phosphorothioate, phosphorodithioate, etc.). Nucleic acid molecules, hereinafter also referred to as polynucleotides, can contain one or more additional covalently bound moieties, such as 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, metal oxides, etc.), and alkylating agents. Polynucleotides can be derivatized by forming methyl or ethyl phosphotriester or alkyl phosphoramidate linkages. Further included are nucleic acid mimetic molecules known in the art, such as synthetic or semi-synthetic derivatives of DNA or RNA and mixed polymers.Such nucleic acid mimic molecules or nucleic acid derivatives according to the present invention include phosphorothioate nucleic acids, phosphoramidate nucleic acids, 2'-O-methoxyethyl ribonucleic acids, morpholino nucleic acids, hexitol nucleic acids (HNA), peptide nucleic acids (PNA), and locked nucleic acids (LNA) (see Braasch and Corey, Chem Biol 2001, 8:1). LNA is an RNA derivative in which the ribose ring is terminated by a methylene bond between the 2'-oxygen and the 4'-carbon. Nucleic acids containing modified bases, such as thiouracil, thioguanine, and fluorouracil, are also included. Nucleic acid molecules typically carry genetic information, such as information used by cellular machinery to make proteins and / or polypeptides. Nucleic acid molecules of the present invention may further include promoters, enhancers, response elements, signal sequences, polyadenylation sequences, introns, 5'- and 3'-noncoding regions, etc.

[0078] In a preferred embodiment of the fifth aspect of the invention, the nucleic acid molecule is RNA, preferably modified RNA, more preferably modified mRNA (modRNA).

[0079] As used herein, the term "modified RNA" or "modified mRNA" refers to RNA or mRNA, respectively, that contains at least one modified nucleoside.For example, nucleoside uridine can be modified to pseudouridine or N1-methyl-pseudouridine, and / or nucleoside cytosine can be modified to 5-methylcytosine, which can change the secondary structure of RNA and reduce the recognition by the innate immune system while still allowing effective translation.

[0080] In a preferred embodiment of the fifth aspect of the present invention, the reprogramming factor is selected from the group comprising POU factors, Klf and Myc family members.

[0081] As used herein, the term "Myc family member" refers to a member of the transcription factor encoded by the myc proto-oncogene, which is related to cancer. c-Myc has been shown to be a transcription factor involved in the generation of mouse iPSCs and human iPSCs. Exemplary c-Myc proteins are those encoded by mouse c-myc genes (NCBI reference sequence NM_010849, for example, all sequences deposited under NM_010849.4) and human c-myc genes (NCBI reference sequence NM_002467, for example, all sequences deposited under NM_002467.6). n-Myc or l-Myc has also been used as a possible reprogramming factor to replace c-Myc. Therefore, the terms "c-Myc," "cMyc," "C-Myc," etc., as referred to herein include either the naturally occurring form of the c-Myc transcription factor or a variant thereof that retains c-Myc transcription factor activity (e.g., having at least 50%, 80%, 90% or 100% of the transcription factor activity, with increasing preference (for each value)) compared to wild-type c-Myc, as measured by methods known in the art, such as measuring the efficacy of iPSC generation in reprogramming experiments.

[0082] In some embodiments, variants have at least 90% amino acid sequence identity across their entire sequence compared to a naturally occurring c-Myc polypeptide. In other embodiments, the c-Myc protein is a protein identified by all sequences deposited under NCBI reference sequence NP_002458, e.g., NP_002458.2, or NP_001170823, e.g., NP_001170823.1, corresponding to SEQ ID NOs: 41 or 42, respectively.

[0083] Therefore, the terms "L-Myc," "LMyc," "L-Myc," etc., as referred to herein include either the naturally occurring form of the L-Myc transcription factor or a variant thereof that retains L-Myc transcription factor activity (e.g., having at least 50%, 80%, 90% or 100% activity (for each value) compared to wild-type L-Myc) when measured by methods known in the art, such as measuring the efficacy of iPSC generation in reprogramming experiments.

[0084] In some embodiments, variants have at least 90% amino acid sequence identity across their entire sequence compared to a naturally occurring L-Myc polypeptide. In other embodiments, the L-Myc protein is the protein identified by all sequences deposited under NCBI reference NP_001028253, e.g., NP_001028253.1, which corresponds to SEQ ID NO:43.

[0085] In a sixth aspect, the present invention relates to a vector or a combination of vectors comprising a nucleic acid molecule or a combination of nucleic acid molecules according to the fifth aspect of the invention.

[0086] The term "vector" according to the present invention preferably refers to a plasmid, cosmid, virus, bacteriophage, episome or another vector conventionally used in genetic engineering, carrying, for example, a nucleic acid molecule or a combination of nucleic acid molecules of the present invention. The nucleic acid molecule or combination of nucleic acid molecules of the present invention can be inserted, for example, into several commercially available vectors.

[0087] Nucleic acid molecules inserted into vectors can be synthesized, for example, by methods known in the art, or isolated from natural sources. Ligation of coding sequences to transcriptional regulatory elements and / or other amino acid coding sequences can also be carried out using established methods. Transcriptional regulatory elements (part of an expression cassette) ensuring expression in prokaryotic or eukaryotic cells are well known to those skilled in the art. These elements include regulatory sequences ensuring transcription initiation (e.g., a translation initiation codon, a promoter, e.g., a naturally associated or heterologous promoter and / or insulator; see above), optionally an internal ribosome entry site (IRES) (Owens, Proc. Natl. Acad. Sci. USA 98 (2001), 1471-1476), and optionally a poly-A signal ensuring transcription termination and stabilization of the transcript. Additional regulatory elements may include transcriptional and translational enhancers. Preferably, the polynucleotide encoding the SoxB1 factor variant, Sox17 factor variant, fusion protein, or component of the complex or composition of the present invention is operably linked to such an expression control sequence to enable expression in prokaryotic or eukaryotic cells. The vector may further include additional control elements, such as nucleic acid sequences encoding 5' and 3' UTRs that can extend the lifespan of mRNA. Such sequences are well known to those skilled in the art.

[0088] In preferred embodiments of the first to fifth aspects of the present invention, the Sox family TF, HMG domain, POU domain, POU factor, fusion protein, and / or composition complex or compound contains a nuclear localization signal (NLS) that targets the protein and composition complex or compound for nuclear localization. The NLS can be in the form of a single part or two separate parts, where a single part refers to a single cluster of amino acids and a two separate parts refers to two clusters of amino acids separated by several amino acids. In preferred embodiments of the present invention, the NLS is an NLS sequence contained in the amino acid sequence of a wild-type Sox family TF and / or POU domain and / or POU factor. For example, for wild-type human Sox2, the NLS sequence is a two-part NLS, with one part found at the beginning of the amino acid sequence SEQ ID NO:2 and the other part found at the end.NLS sequences can be obtained from NLS databases, e.g., NLSdb (https: / / rostlab.org / services / nlsdb / ; Nair, R., Carer, P., Rost, B., NLSdb: database of nuclear localization signals. Nucl Acids Res, 2003, 31:397-399), prediction algorithms, e.g., SeqNLS (http: / / mleg.cse.sc.edu / seqNLS / ; Lin JR, Hu J. SeqNLS: nuclear localization signal prediction based on frequent pattern mining and linear motif scoring. PLoS One. 2013 Oct 29;8(10):e76864. doi: 10.1371 / journal.pone.0076864. PMID: 24204689; PMCID: PMC3812174.), and NLStradamus (Nguyen Ba, AN, Pogoutse, A., Provart, N. et al. NLStradamus: a simple Hidden Markov Model for nuclear localization signal prediction. BMC Bioinformatics 10, 202 (2009)) or NoLS / NoD (http: / / www.compbio.dundee.ac.uk / www-nod / ; Scott MS, Troshin PV, Barton GJ. NoD: a Nucleolar localization sequence detector for eukaryotic and viral proteins. BMC Bioinformatics. 2011 Aug 3;12:317. doi: 10.1186 / 1471-2105-12-317. PMID: 21812952; PMCID: PMC3166288).

[0089] In preferred embodiments, the vector is an episome or other DNA vector, a self-replicating RNA, an unmodified or modified mRNA, or a viral vector.

[0090] The term "episomal vector" or "episome," as used herein, refers to a polynucleotide that is introduced into a cell, preferably a host cell, without integrating the vector or a portion of the vector into the chromosomal DNA (without integration). Specific episomal vectors, as used herein, are described in the Examples herein. Those skilled in the art will understand that the plasmid vector used to construct an episomal vector is not limited to those described herein, but can be selected by those skilled in the art as typically practiced in the art.

[0091] An episome is a polynucleotide capable of extrachromosomal replication within a host cell. The polynucleotide encodes an exogenous and / or endogenous polypeptide in the host cell. Episomes are advantageous over integrating vectors / plasmids due to their low chance of random integration and introduction of mutations into the host cell genome (see Van Craenenbroeck et al., 2000. Episomal vectors for gene expression in mammalian cells. Eur. J. Biochem. 267, 5665-5678). The episome(s) can remain in the cell. In this case, the introduced polynucleotide(s) encoding the protein(s) can be permanently, i.e., stably / constitutively expressed. In a more preferred embodiment of the fifth and sixth aspects of the present invention, the episome(s) is lost after several passages of the transformed / reprogrammed / reset cells, and the expression of the introduced polynucleotide(s) encoding the protein(s) is not permanent, i.e., transient.

[0092] Nucleoside-modified messenger RNA (modRNA) is a synthetic messenger RNA (mRNA) containing one or more natural or synthetic nucleoside analogs. mRNA can be introduced without being integrated into the chromosomal DNA of host cells. Modified mRNA is a non-viral RNA-based method for introducing polynucleotides into host cells, and offers advantages over DNA-based methods for introducing polynucleotides encoding exogenous polypeptides into the host cells. For example, the strength of protein expression is independent of promoter activity, preventing gene silencing, achieving independent expression of each mRNA-mediated gene from each other, resulting in more precise stoichiometric expression of multiple genes (Oh and Kessler, 2018. Design, Assembly, Production and Transfection of Synthetic Modified mRNA, Methods. 133:29-43). The modified mRNA can be delivered to the host cell by means known to those of skill in the art, preferably by means that protect the modified mRNA from ribonucleotides in the host cell without compromising the effectiveness of the introduction of the polynucleotide as the modified mRNA, such as transfection via lipofection, electroporation, nucleofection, delivery using lipids and lipid nanoparticles (LNPs) or polymer-based nanoparticles.

[0093] A non-limiting example of a method for protecting the modRNA from host cell ribonucleotides includes encapsulating the modRNA in liposomes for delivery and uptake into the host cell.

[0094] In a seventh aspect, the present invention relates to (a) a cell or (b) a cell derived from said cell, said cell comprising: (i) a SoxB1 factor variant according to the first aspect of the invention, a Sox17 factor variant according to the second aspect, a fusion protein according to the third aspect and / or a complex or composition according to the fourth aspect of the invention, and / or (ii) a nucleic acid molecule or combination of nucleic acid molecules of the fifth aspect; and / or (iii) A vector or vector combination according to the sixth aspect of the invention or (b) the cell is modified by the presence of the above in the cell; The cells of (b) preferably retain the phenotype of the cells of (a).

[0095] The term "cell," as used herein, is understood to refer to and include a single cell, a plurality of cells, or a population of cells, unless the context permits and unless otherwise specified.

[0096] The term "cell (b) derived from cell (a), wherein said cell (b) has been modified as described above" refers, according to a seventh aspect of the present invention, to a cell that comprises or previously comprised a SoxB1 factor variant of the first aspect of the present invention, a Sox17 factor variant of the second aspect, a fusion protein of the third aspect and / or a complex or composition of the fourth aspect, a nucleic acid molecule or combination of nucleic acid molecules of the fifth aspect of the present invention, or a vector or combination of vectors of the sixth aspect of the present invention, wherein said cell is modified in its characteristics according to the expression pattern of said nucleic acid molecule or combination of nucleic acid molecules of the fifth aspect of the present invention or said vector or combination of vectors of the sixth aspect of the present invention. For the purposes of this disclosure, a "cell" or a "cell derived from said cell" (also referred to as a cell derivative), as used herein, is preferably an isolated cell. Derivatives of a cell include progeny that may or may not exhibit the phenotype of the original cell. In particular, an enhanced developmental potential of the phenotype of the original cells carrying a SoxB1 factor variant of the first aspect or a Sox17 factor variant of the second aspect described herein, 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 naive pluripotency specific marker(s) selected from Klf17, Klf4, Sox2, Susd2, Argfx and Dnmt3l, an increased expression level of at least one primitive endoderm specific marker(s) selected from Gata6 and Sox17. Certain traits such as higher expression levels of a gene (a number of genes), an activated POU5f1 distal enhancer, a reactivated X chromosome in the female lineage, reduced DNA methylation, an increased ability to differentiate preferably into germ cells, and / or an increased ability to contribute to the development of the embryo(s) and / or animal(s), may be lost in the offspring or may be partially lost by at least 10%, by at least 20%, by at least 30%, by at least 40%, by at least 50%, by at least 60%, by at least 70%, by at least 80%, by at least 90% or by at least 95%.

[0097] In an eighth aspect, the present invention relates to a method for increasing the cooperativity between a Sox factor and a POU factor, the method comprising the step of increasing the average number and / or strength of interactions between the HMG domain of a Sox factor and the POU domain of a POU factor.

[0098] The term "cooperativity between Sox factors and POU factors" generally refers to the interaction between the aforementioned Sox factors and POU factors on their genomic targets, resulting in a higher stability of the Sox / Oct heterodimer on DNA compared to either of the monomers on the same DNA. Thus, 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: POU and HMG domains. Importantly, as can be seen in the examples herein below, the inventors have identified Sox2 as a co-regulatory factor for Sox2. A61V We found that replacing Sox / Oct with either Sox2-17 or Sox2-17 significantly increased Sox / Oct heterodimerization and the stability of the heterodimeric complexes on their target loci.

[0099] Therefore, the term "increasing the cooperativity between Sox factors and POU factors," as used herein, refers to increasing the propensity of Sox factors and Oct factors for heterodimerization and / or increasing the stability of the Sox factor / Oct factor complex on one of their genomic targets.

[0100] Interaction of transcription factors, transcription factor complexes, or compositions with DNA elements is measured by methods known to those skilled in the art, such as chromatin immunoprecipitation (ChIP), transcription factor footprinting analysis using assays for transposase-accessible chromatin sequencing (ATAC-seq), electrophoretic mobility shift assays (EMSA), fluorescence correlation spectroscopy (FCS), and molecular dynamics simulation (MDS).

[0101] Therefore, the cooperation of Sox and POU factors in the formation of heterodimers or the stability of complexes on one of their genomic targets can also be evaluated by ChIP, ATAC-seq, EMSA, MDS, FCS. Those skilled in the art will understand the steps required to perform these assays (see Zhang et al., Model-based analysis of ChIP-seq (MACS), 2008. Genome Biol 9.).

[0102] As noted, methods for assessing cooperativity between Sox and Oct factors are known in the art and include, for example, electrophoretic mobility shift assays (EMSAs) as shown in the Examples herein below and described in Ng et al., 2012, Huang et al., 2015, assays for transposable-accessible chromatin sequencing (ATAC-Seq), isothermal titration calorimetry (ITC), surface plasmon resonance (SPR), fluorescence correlation spectroscopy (FCS), nuclear magnetic resonance (NMR) spectroscopy, cryo-electron microscopy (cryo-EM), consecutive affinity-purification systematic evolution of ligands by exponential enrichment (CAP-SELEX), high-throughput SELEX (HT-SELEX), or Coop-seq as described in Chang et al., 2015.

[0103] Furthermore, computational molecular dynamics simulation (MDS), as described in the following examples, can be used to evaluate the average number and / or strength of interactions between Sox factor HMG domains and POU factor POU domains. Alternatively, Sox / Oct factor cooperativity can also be measured indirectly by evaluating their reprogramming efficiency or their ability to rescue non-cooperative variants in reprogramming experiments, with increased reprogramming efficiency corresponding to increased Sox / Oct factor cooperativity.

[0104] The differentiation and aging states of a cell are a continuous spectrum, with a terminally differentiated / aged state at one end and a dedifferentiated state (pluripotent state) at the other end. Therefore, the term "reprogramming," as used herein, refers to the process of altering or reversing the differentiation and / or aging state of a cell. Reprogramming includes complete or partial resetting of a given epigenetic state of a cell. That is, the term "reprogramming," as used herein, encompasses any transition of a cell's epigenetic state along a spectrum toward a less differentiated and / or less aged state. For example, reprogramming includes the reversal of a multipotent cell to a pluripotent cell, or the reversal of a terminally differentiated cell to either a multipotent or pluripotent cell, or the reversal of a cell's aging state to a less aged state. Therefore, the terms "less differentiated state" and "less aged" as used herein are relative terms and include a completely dedifferentiated or rejuvenated state and a partially differentiated or rejuvenated state. For example, a method for reprogramming fibroblasts into induced pluripotent stem cells (iPSCs) by ectopic expression of Oct4, Sox2 and c-Myc is described in Takahashi and Yamanaka, 2006.Methods for reprogramming-induced age reversal (rejuvenation) have been described in studies by Belmonte (e.g., Ocampo et al., In Vivo Amelioration of Age-Associated Hallmarks by Partial Reprogramming. Cell. 2016;167(7):1719-1733.e12), Sinclair (Lu et al., Reprogramming to recover youthful epigenetic information and restore vision. Nature. 2020;588(7836):124-129), Reik (Gill et al., Multi-omic rejuvenation of human cells by maturation phase transient reprogramming. 2022. Elife 11, 2021.01.15.426786) and other laboratories.

[0105] Thus, a "reprogramming factor" is a factor, most often a transcription factor, that can be used to reprogram a target cell. The term "reprogramming factor" further includes any similar molecule that mimics the function of the factor with respect to reprogramming ability.

[0106] The term "functionally mimics" as used herein means "retains" or "essentially retains function," where the latter means, with increasing preference, that at least 50%, at least 80%, at least 90% or at least 95% of the function is retained compared to the factor with respect to reprogramming ability.

[0107] As used herein, the term "reprogramming efficiency" refers to the number of cells in a sample that are successfully reprogrammed to a less differentiated state or pluripotency relative to the total number of cells in the sample. For newly engineered or designed factors and cocktails, efficiency can be measured by comparing them with conventional or wild-type reprogramming factors and cocktails. Reprogramming efficiency can be measured as a function of pluripotency markers. Such pluripotency markers include, but are not limited to, transgene reporters (e.g., endogenous Oct4-GFP), pluripotency-specific marker proteins (e.g., Oct4, Sox2, Nanog, Klf17, alkaline phosphatase, SSEA1, TRA-1-60, TRA-1-81, SUSD2, etc.) and mRNA expression, pluripotent cell morphology, and colony formation.

[0108] The interaction between the HMG domain and the POU domain according to the eighth aspect of the invention may include hydrophobic interactions, electrostatic interactions or or covalent bonds.

[0109] Thus, the term "increasing the cooperativity between Sox factors and POU factors" includes an increase in the average number and / or strength of interactions, preferably an increase in the average number of hydrophobic interactions, electrostatic interactions and / or the average strength of covalent bonds.

[0110] As shown in the Examples herein below, the present inventors found that substitution of the amino acid alanine at position 61 in the HMG domain of Sox2 significantly increased the cooperativity between Sox2 and POU factors such as Oct4 on the canonical SoxOct DNA motif.

[0111] Thus, in a preferred embodiment of the method of the eighth aspect of the present invention, the average number and / or strength of interactions is increased by substituting the amino acid at position 61 of the HMG domain of a Sox factor with a hydrophobic amino acid selected from valine, leucine, isoleucine, phenylalanine, methionine or tryptophan, preferably the amino acid at position 61 of the HMG domain is substituted with valine.

[0112] In a more preferred embodiment, the Sox factor is a SoxB1 factor or a SoxB1 factor variant according to the first aspect of the invention.

[0113] In a more preferred embodiment of the above preferred embodiment, the amino acid at position 61 is substituted with a hydrophobic amino acid selected from valine, leucine and isoleucine, preferably the amino acid at position 61 is substituted with valine.

[0114] In a ninth aspect, the present invention relates to a method of producing induced pluripotent stem cell(s) (iPSC(s)) from non-pluripotent cell(s), the method comprising culturing the non-pluripotent cell(s) under conditions suitable for reprogramming the non-pluripotent cell(s) into iPSC(s), wherein the conditions comprise administering to the non-pluripotent cell(s): a) a SoxB1 factor variant of the first aspect of the invention or a Sox17 factor variant of the second aspect of the invention and a POU factor; and / or b) a fusion protein of the third aspect of the invention or a complex or composition of the fourth aspect of the invention; and / or c) a SoxB1 factor variant comprising or consisting of the amino acid sequence of SEQ ID NO: 13 or 14, and a Klf family member; and optionally: - inhibitors of p53 function; and / or - one or more additional reprogramming factor(s) This includes increasing the level of

[0115] Culturing a cell according to the ninth aspect of the invention may comprise contacting the cell with a factor, fusion protein and / or complex or composition as defined in items a), b) or c) of the ninth aspect of the invention, wherein the respective factor, fusion protein and / or complex or composition is taken up by the cell, and transfecting or transducing the cell with a nucleic acid encoding the factor, fusion protein and / or complex and co-expressing the factor, fusion protein and / or complex.

[0116] Specific culture conditions are required for (a) culturing a cell(s). These conditions refer to the type of culture medium, temperature, and CO2 level. Mammalian cells are generally cultured at 37°C, with a 5% CO2 level, and using cell-type specific culture media containing various supplements. The type of basal medium is typically selected from the group including low-glucose DMEM, high-glucose DMEM, IMDM, DMEM / F12 medium, Neurobasal medium, etc.

[0117] Supplements include: fetal bovine serum (FBS), Knockout TM The inhibitor may be selected from the group comprising serum replacement (KSR), L-glutamine, penicillin, streptomycin, non-essential amino acids, sodium pyruvate, β-mercaptoethanol, growth factors (e.g. bFGF, EGF, Activin or LIF), N2 supplement mix, B27 supplement mix, small molecules (e.g. VPA, XAV939, PD0325901, CHIR99021, Y-27632 or Go6983 - each of HDAC, Wnt, Mek, GSK3, ROCK and PKC inhibitors).

[0118] Pluripotent stem cells are often cultured on a layer of inactive feeder cells, which are typically derived from mouse embryonic fibroblasts (MEFs), but can also be derived from other cell types.

[0119] Those skilled in the art will understand that culture conditions will be adjusted for the particular cell type being cultured.

[0120] For example, HEK293T cells can be cultured in low-glucose DMEM (Sigma) supplemented with 10% FBS (Capricorn Scientific), 1% Glutamax, 1% penicillin-streptomycin, and 1% non-essential amino acids (all from Sigma). Mouse, human, cynomolgus monkey, and porcine fibroblasts can be cultured in high-glucose DMEM (Sigma) supplemented with 15% FBS, 1% Glutamax, 1% penicillin-streptomycin, 1% non-essential amino acids (NEAA), 1% sodium pyruvate (Sigma), and 1% β-mercaptoethanol (Gibco). Bovine fibroblasts can be cultured in 50:50 DMEM / F12 (Gibco) with 15% FBS and the same supplements and IMDM (with HEPES, Cytiva). The addition of 5 ng / ml human bFGF (Peprotech) can be used to improve cynomolgus monkey, bovine, and porcine fibroblast cultures.

[0121] Mouse naive pluripotent stem cells (mESCs) can be grown on mitomycin C-inactivated C3H MEF feeder layers in high-glucose DMEM medium supplemented with KSR-based mouse embryonic stem cell (mESC) medium: 15% KSR (Invitrogen), 1% Glutamax, 1% NEAA, 1% penicillin-streptomycin, 1% β-mercaptoethanol, and 20 ng / ml human recombinant LIF (in-house purified). Mouse Gof18 GFP-E3 primitive ectodermal stem cells (EpiSCs) (Han et al., 2010) can be cultured on FBS-coated dishes in Stem Flex media (Gibco).

[0122] Human pluripotent cells can be cultured on Matrigel-coated dishes (Corning) or mitomycin C-inactivated CF1 MEF feeder layers in either hESC medium: DMEM / F12 supplemented with 15% KSR, 1% Glutamax, 1% NEAA, 1% penicillin-streptomycin, 1% β-mercaptoethanol, and 5 ng / ml bFGF, or in StemFlex medium (Gibco). Cynomolgus monkey iPSCs can be cultured on mitomycin C-inactivated CF1 MEF feeder layers in StemFlex medium. Bovine and porcine iPSCs can be induced and cultured on mitomycin C-inactivated CF1 MEF feeder layers in StemFlex medium supplemented with 2 μM XAV939 (Sigma) in a hypoxic 5% O2, 5% CO2 incubator at 37°C.

[0123] In a preferred embodiment, increasing the levels in said non-pluripotent cells of the SoxB1 factor variant or Sox17 factor variant defined in item a); the fusion protein or complex or composition defined in item b) and / or the SoxB1 factor variant defined in item c); and optionally an inhibitor of p53 function; and / or one or more further reprogramming factor(s) is achieved by (co)expressing in said non-pluripotent cell(s) the factor variant, POU factor, fusion protein, complex and / or Klf family member defined in items a) to c), and optionally an inhibitor of p53 function and / or one or more further reprogramming factor(s).

[0124] The terms "pluripotency" and "pluripotent," as used herein, refer to the ability of a cell to differentiate into cells of all three germ layers, i.e., endoderm, mesoderm, and ectoderm. Accordingly, the term "pluripotent cell" refers to an undifferentiated cell capable of differentiation under conditions that promote differentiation. These conditions include, but are not limited to, culturing cells in differentiation medium(s), treating pluripotent cells with differentiation factors, aggregating cells into spheroids / embryoid bodies or embryo-like structures using suspension droplets or specialized culture plates, such as AggreWell®, combining cells with embryos or embryo-like structures, and / or injecting cells in vivo to generate progeny that are derivatives of any of the three germ layers of the germline. Various types of embryonic cells, such as induced pluripotent stem cells (iPSCs), embryonic stem cells, and embryonic germ cells (EGCs), are included in the definition of pluripotent cells. In vivo pluripotency is a transient state acquired within the inner cell mass of developing preimplantation blastocysts during the separation of inner cell mass cells into primitive endoderm and pluripotent native preimplantation ectoderm. During the transition from preimplantation to early postimplantation, also referred to as the naive-to-primed transition, changes in the molecular and functional characteristics of these cells result in specific developmental restrictions with respect to their ability to self-organize, contribute to development, and differentiate into specific lineages, particularly the germ line. (Weinberger L, Ayyash M, Novershtern N, Hanna JH. Dynamic stem cell states: naive to primed pluripotency in rodents and humans. Nat Rev Mol Cell Biol. 2016 Mar;17(3):155-69. doi: 10.1038 / nrm.2015.28. Epub 2016 Feb 10. PMID: 26860365.)

[0125] The term "induced pluripotent stem cells (iPSCs)," as used herein, refers to a type of pluripotent stem cell artificially derived from a non-pluripotent cell, typically a somatic cell, such as a skin cell or a blood cell. iPSCs can be generated from somatic cells by epigenetic resetting of the somatic cell, for example, by retroviral transduction of somatic cells, such as fibroblasts, hepatocytes, or gastric epithelial cells, with transcription factors such as Oct3 / 4, Sox2, Klf4, and optionally c-Myc (Takahashi and Yamanaka, 2006).

[0126] Pluripotent cells can exist in multiple pluripotent states, the best known examples being naive and induced pluripotent cells, which resemble pre-implantation and post-implantation pluripotent states, respectively.

[0127] As used herein, the term "naive pluripotent cells" refers to pluripotent cells typically identified by one or more of the following characteristics: an active Oct4 distal enhancer (Oct4DE); expressing high levels of the pluripotency factors Sox2, Oct4, Nanog, Klf2, Klf4, Klf17, Rex1, Dnmt3L, Argfx, and / or Susd2; exhibiting a domed morphology, self-renewal in response to LIF, high clonogenicity, low levels of global genome methylation, and an XaXa X chromosome status, where Xa refers to a single active X chromosome. Naive cells contribute to development more efficiently than induced cells and are therefore more suitable for genetic engineering (especially multigene targeting), derivation of germline and extraembryonic lineages, developmental and disease modeling, and the like. However, different naive pluripotent cell lines exhibit differences in all of these properties, most importantly in developmental capacity, which the inventors found to correlate with the levels of endogenous Sox2 expression and Sox2 / Oct4 heterodimerization.

[0128] In a preferred embodiment, the "naive pluripotent cells" are further characterized by two or more features selected from the group comprising enhanced developmental potential, higher expression levels of Sox2, an activated POU5f1 distal enhancer, reactivation of the X chromosome in the female lineage, higher expression levels of naive pluripotency specific markers, reduced DNA methylation, preferably an enhanced ability to differentiate into germ cells, and / or an enhanced ability to develop into chimeric tissue(s), organ(s) or organism(s).

[0129] The term "high-grade naive pluripotent cells" refers to naive pluripotent cells with high developmental potential. Relative developmental potential can be measured by comparing the ability of a given pluripotent cell to develop into a chimeric animal upon aggregation with a morula or injection into a blastocyst for further intrauterine transfer or extrauterine development, and / or by its ability to generate whole PSC animals in tetraploid complementation assays and / or its ability to efficiently differentiate into all cell lineages, particularly germ line cell lines.

[0130] A "tetraploid complementation assay" generally involves first creating a tetraploid embryo by taking an embryo at the "two-cell stage" and fusing two cells, e.g., by applying an electric current. The fused cell is tetraploid and continues to divide, with all of its daughter cells being tetraploid. These tetraploid embryos can develop to the blastocyst stage and can be implanted in the uterus, but they cannot develop into viable fetuses. Because tetraploid embryos further develop into functional trophectoderm that gives rise to the placenta, they can carry the inner cell mass and serve as a surrogate for cultured pluripotent stem cells that can generate an entire fetus. For a tetraploid complementation assay, the tetraploid embryo is combined with diploid embryonic stem cells (ESCs) or iPSCs that can develop into a fetus, with the fetus derived exclusively from the ESCs or iPSCs and the extraembryonic tissues (e.g., placenta) derived from the tetraploid cells. Thus, the embryonic stem cells described in the exemplary tetraploid complementation assay develop into a whole ESC fetus. Instead of embryonic stem cells, other pluripotent stem cells, such as induced pluripotent stem cells, can be used, and the higher developmental potential of these pluripotent stem cells means a higher ability to generate whole iPSC animals in the above-mentioned tetraploid complementation assay. When used herein in accordance with the present invention, the tetraploid complementation assay is described in the following examples of this specification.

[0131] As used herein, the term "chimeric animal" or "animal chimera" refers to an animal composed of cells with different genetic backgrounds (or even different species, in the case of xenogeneic animal chimeras). Most commonly, in the current state of the art, chimeric animals are created for the purpose of animal genetic engineering by aggregating genetically edited cells with wild-type surrogate embryos (or surrogate embryos with different genetic backgrounds). The partially genetically engineered chimeric animal then produces a fully genetically edited animal as offspring. In the current state of the art, this is most commonly done with laboratory mice, but it could potentially be applied to all mammals. Thus, "chimeric molecule" or "chimeric polypeptide" refers to a molecule or polypeptide whose parts function as entities of different origins. For example, the Sox2-17 (super-SOX) shown herein functions as a single transcription factor (enhanced Sox2), but its distinct structural elements are derived from two transcription factors: Sox2 and Sox17. The structural elements of Sox17 were not added to Sox2, but rather some elements of Sox2 were replaced by corresponding non-conserved elements of its paralog, Sox17. Two different versions of Sox2-17: mouse and human (uppercase letters, SOX2-17) were prepared as described in the Examples.

[0132] The term "genetically engineered animal," as used herein, refers to any animal whose genome has been intentionally edited, for example, by introducing exogenous DNA into a cell, which becomes part of the genome of the animal derived from that cell.

[0133] The ability to contribute to the generation of chimeric animals can be determined, for example, by measuring the average percentage of cells within chimeric embryos at different developmental stages or postnatal stages that are derived from a given pluripotent cell in a morula aggregation / blastocyst injection assay. Differences in the ability to generate whole PSC animals can be determined by measuring the percentage of tetraploid embryos recruited by a given PSC that become full-term whole PSC animals, the percentage of tetraploid embryos recruited by a given PSC that are born alive, the percentage of tetraploid embryos recruited by a given PSC that initiate respiration, the percentage of tetraploid embryos recruited by a given PSC that survive to adulthood, etc. Differences in the ability to generate whole PSC embryos can also be assessed using in vitro artificial embryo assembly, as recently demonstrated by the Hanna and Zernicka-Goetz labs (Amadei et al. 2022). The ability to differentiate into germline cells can be determined by assessing the ability of a given PSC to differentiate into germline cells in vitro or in vivo, for example, by assessing the percentage of cells that activate germline markers, measuring the level of germline marker expression, or evaluating the functionality of the resulting germline. High-grade naive pluripotent cells have a higher developmental potential compared to low-grade naive pluripotent cells or induced pluripotent cells, which are low-grade by definition, as measured by one or several of the above-mentioned assays. The inventors also suggested that pluripotency grade can be determined or estimated by measuring the level of endogenous Sox2, which can heterodimerize with Oct4 in a given PSC, where an increased number of Sox2 / Oct4 heterodimers indicates a higher grade of pluripotency. This can be determined or estimated by methods such as Western blot, EMSA, qPCR, RNA-seq, ATAC-seq, ChIP-seq, immunostaining combined with microscopy or FACS, and reporter gene tracking.

[0134] Thus, as used herein, the term "low-grade pluripotent cells" refers to low-grade naive pluripotent cells and induced pluripotent cells such as induced pluripotent stem cells, embryonic stem cells or protoblast stem cells.

[0135] The term "induced pluripotent cells," as used herein, refers to cells typically identified by the following characteristics: absence of an active Oct4 distal enhancer, an active Oct4 proximal enhancer, flattened colony morphology, high levels of global genome methylation, low colony-forming ability, low developmental potential, insensitivity to Lif / Stat3; self-renewal in response to Fgf / Erk; and displaying an XaXi X chromosome activation state, where Xi is the inactive X chromosome (Nichols et al., 2009). Induced pluripotent cells are not equivalent across species; for example, human "normal" embryonic stem cells, although considered induced, have various naive characteristics and are distinct from induced mouse ectodermal stem cells. In general, induced pluripotent cells are considered to be more developmentally restricted and non-differentiating than naive pluripotent cells, such as mouse ectodermal stem cells; however, they downregulate certain pluripotency genes, such as Sox2, Klf2, Klf5, Nanog, Rex1, and Esrrb, and upregulate lineage arrest factors, such as Otx2 and Zic2, to induce specific future differentiation (Weinberger, L., Ayyash, M., Novershtern, N. et al. Dynamic stem cell states: naive to primed pluripotency in rodents and humans. Nat Rev Mol Cell Biol 17, 155-169 (2016). https: / / doi.org / 10.1038 / nrm.2015.28).

[0136] As used herein, the term "low-grade naive pluripotent cells" refers to naive pluripotent cells that exhibit lower developmental potential compared to high-grade naive pluripotent cells. For example, naive pluripotent cells that cannot generate whole PSC animals are low-grade compared to high-grade naive pluripotent cells that can generate whole PSC animals. "Low-grade" and "high-grade" are relative terms, not absolute. Theoretically, most high-grade PSCs can generate healthy adult whole PSC animals from 100% of tetraploid aggregate embryos. Theoretically, other types of PSCs may benefit from low- to high-grade conversion methods. The inventors have shown that low-grade PSCs have lower expression of endogenous Sox2 or / and a lower ability to form Sox2 / Oct4 heterodimers on the canonical SoxOct DNA motif compared to high-grade PSCs (see Example 5 herein below).

[0137] The term "non-pluripotent cells," as used herein, refers to any non-pluripotent cell that is partially or fully differentiated, such as a cell in culture, a cell explanted from a subject, or a cell within a subject. The non-pluripotent cells can be from any animal species, preferably mammalian, such as mouse, human, non-human primate, domestic animal, cat, dog, etc.

[0138] In a preferred embodiment of the ninth aspect of the invention, the non-pluripotent cells are selected from fibroblasts, keratinocytes, blood cells, urine-derived cells and / or any other somatic cell type.

[0139] As used herein, the term "Krueppel-like factor (Klf) family" refers to the Klf gene, which was first identified as a factor for generating mouse iPSCs and has also been shown to be a factor for generating human iPSCs. As used herein, members of the "Klf family" include, for example, Klf1, Klf2, Klf4, Klf5, Klf17, or other Klf factors from humans or other animal species. Exemplary Klf4 proteins are those encoded by the mouse Klf4 gene (all sequences deposited under NCBI reference sequence NM_010637, e.g., NM_010637.3) and the human klf4 gene (all sequences deposited under NCBI reference sequence NM_004235, e.g., NM_004235.6). As referred to herein, the terms "Klf4," "KLF4," "Klf-4," and the like include either the naturally occurring form of the Klf4 transcription factor or a variant thereof that retains Klf4 transcription factor activity (e.g., within at least 50%, 80%, 90%, or 100% activity) relative to wild-type Klf4 as measured by methods known in the art, such as measuring the efficiency of iPSC generation or measuring the efficiency of naive resets in reprogramming experiments.

[0140] In some embodiments, variants have at least 90% amino acid sequence identity across their entire sequence compared to a naturally occurring Klf4 polypeptide. In other embodiments, the Klf4 protein is a protein identified by NCBI reference sequence NP_001300981, e.g., NP_001300981.1, or NP_034767, e.g., NP_034767.2, or all sequences deposited under SEQ ID NO: 33 or 34, respectively.

[0141] As used herein, "p53 function inhibitor" can be any drug as long as it can inhibit either (a) the function of p53 protein or (b) the expression of p53 gene (TP53). This includes not only drugs that directly act on p53 protein to inhibit its function and drugs that directly act on p53 gene to inhibit its expression, but also drugs that act on factors involved in p53 signal transduction to inhibit the function of p53 protein or the expression of p53 gene, and these are also included in the scope of "p53 function inhibitor" described herein. Factors involved in p53 signal transduction include, but are not limited to, MDM2, MDMX, SIRT1, CRM1, FACT, E6 ubiquitin ligase, etc. Drugs that act on these factors include, for example: Nutilin-3a (MDM2), XI-006 (MDMX), Tenovins (SIRT1), Letomycin B (CRM1), Quanacrine (FACT), and RITA (E6) (see Sanz et al., Inhibition of p53 inhibitors: progress, challenges, and perspectives. J Mol Cell Biol. 2019. 11(7):586-599).

[0142] Exemplary inhibitors of p53 function include, but are not limited to, siRNA, shRNA, miRNA, antisense nucleic acid molecules, aptamers or ribozymes against p53, chemical inhibitors of p53, antibodies or antibody mimetics, anti-p53 antagonist antibodies or nucleic acids encoding same, and lure nucleic acids comprising consensus sequences for p53 response elements.

[0143] Preferably, the functional inhibitor of p53 is a substance that inhibits the expression of the p53 gene, more preferably an expression vector encoding an siRNA or shRNA against p53.

[0144] According to the present invention, the term "small interfering RNA (siRNA)," also known as short interfering RNA or silencing RNA, refers to a class of double-stranded RNA molecules 18-30, preferably 19-25, most preferably 21-23, or even more preferably 21 nucleotides in length that play a variety of roles in biology. Most notably, siRNAs are involved in the RNA interference (RNAi) pathway, in which siRNAs interfere with the expression of specific genes. In addition to their role in the RNAi pathway, siRNAs also act in RNAi-related pathways, for example, as an antiviral mechanism or in shaping the chromatin structure of the genome.

[0145] Naturally occurring siRNAs have a well-defined structure: a short double-stranded RNA (dsRNA) with a 2-nt 3' overhang at either end. Each strand has a 5' phosphate group and a 3' hydroxyl (-OH) group. This structure is the result of processing by Dicer, an enzyme that converts either long dsRNA or small hairpin RNA into siRNA. siRNAs can be exogenously (artificially) introduced into cells to result in specific knockdown of a gene of interest. Essentially any gene whose sequence is known can thus be targeted based on sequence complementarity with an appropriately tailored siRNA. Double-stranded RNA molecules or their metabolic processing products can mediate target-specific nucleic acid modification, particularly RNA interference and / or DNA methylation. While exogenously introduced siRNAs may lack overhangs at their 3' and 5' ends, it is preferred that at least one RNA strand possesses a 5'- and / or 3'-overhang. Preferably, one end of the duplex has a 3' overhang of 1 to 5 nucleotides, more preferably 1 to 3 nucleotides, and most preferably 2 nucleotides. The other end can be blunt or have a 3' overhang of up to 6 nucleotides. Generally, any RNA molecule suitable for acting as siRNA is contemplated in the present invention. The most effective silencing to date has been achieved using siRNA duplexes composed of 21-nt sense and 21-nt antisense strands, paired in a manner with a 2-nt 3' overhang. The sequence of the 2-nt 3' overhang contributes little to the specificity of target recognition, which is limited to the unpaired nucleotide adjacent to the first base pair (Elbashir et al. 2001, Nature; 411(6836):494-8). 2'-deoxynucleotides in the 3' overhang are as efficient as ribonucleotides, but are often cheaper to synthesize and can be more nuclease-resistant.

[0146] Short hairpin RNA (shRNA) is a sequence of RNA that creates a narrow hairpin turn and can be used to silence gene expression through RNA interference. shRNA uses a vector introduced into cells and utilizes a U6 promoter to ensure that the shRNA is always expressed. This vector is usually passed on to daughter cells, making gene silencing heritable. The shRNA hairpin structure is cleaved into siRNA by the cellular machinery and then bound to the RNA-induced silencing complex (RISC). This complex binds to and cleaves the target mRNA. The si / shRNA used in the present invention is preferably chemically synthesized using appropriately protected ribonucleoside phosphoramidites and a conventional DNA / RNA synthesizer. Suppliers of RNA 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). Most conveniently, siRNA or shRNA can be obtained from commercial RNA oligo synthesis companies that sell RNA synthesis products of different quality and cost. Generally, the RNA that can be applied in the present invention can be synthesized conventionally and easily provided with quality suitable for RNAi.

[0147] The following types of molecules also carry out RNAi: microRNA (miRNA) and antisense nucleic acid molecules. miRNA molecules are single-stranded RNA molecules. Endogenously existing miRNA molecules bind to complementary mRNA transcripts and induce the degradation of said mRNA transcripts through a process similar to RNA interference, thereby controlling gene expression.Therefore, exogenous miRNA can be used as an inhibitor of p53 function after being introduced into respective cells.

[0148] The term "antisense nucleic acid molecule" as used herein refers to a nucleic acid that is complementary to a target nucleic acid.Antisense molecules according to the present invention can interact with target nucleic acids, more specifically, can hybridize with target nucleic acids.Due to the formation of hybrids, the transcription of target gene(s) and / or the translation of target mRNA is reduced or blocked.Standard methods for antisense technology have been described (see, for example, Melani et al., Cancer Res. (1991) 51:2897-2901).

[0149] The antisense molecule, siRNA and shRNA of the present invention are preferably chemically synthesized using 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).

[0150] Antisense molecules, siRNAs, and shRNAs may contain modified nucleotides, such as locked nucleic acids (LNAs). The ribose moiety of an LNA nucleotide is modified with an extra bridge connecting the 2' oxygen and 4' carbon. The bridge "locks" the ribose in the 3'-end (North) conformation, often found in A-form duplexes. LNA nucleotides can be mixed with DNA or RNA residues in oligonucleotides whenever desired. Such oligomers are chemically synthesized and commercially available. The locked ribose conformation enhances base stacking and backbone preorganization, significantly increasing the hybridization properties (melting temperature) of the oligonucleotide.

[0151] Aptamers in the art are selected to bind to nucleic acids, proteins, small organic compounds, and even whole organisms. An aptamer database is maintained at http: / / aptamer.icmb.utexas.edu / . More specifically, aptamers can be classified as DNA or RNA aptamers or peptide aptamers. The former consist of (usually short) strands of oligonucleotides, while the latter consist of short variable peptide domains attached at both ends to a protein backbone. Nucleic acid aptamers are nucleic acid species engineered through repeated rounds of in vitro selection or equivalently, SELEX (Systematic Evolution of Ligands by Exponential Enrichment) to bind to various molecular targets, such as small molecules, proteins, nucleic acids, and even cells, tissues, and organisms. Aptamers are useful in biotechnology and therapeutic applications, as they offer molecular recognition properties that rival those of commonly used biological molecules, particularly antibodies. In addition to their discriminatory recognition, aptamers offer advantages over antibodies because they can be fully engineered in vitro, are easily produced by chemical synthesis, have desirable storage properties, and exhibit little or no immunogenicity in therapeutic applications. Unmodified aptamers are rapidly removed from the bloodstream, with half-lives of minutes to hours, primarily due to nuclease degradation and clearance from the body by the kidneys as a result of the aptamer's inherent 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 and polyethylene glycol (PEG) linkages, are available, which can easily increase the half-life of aptamers to the timescale of days or even weeks.

[0152] Ribozymes (derived from ribonucleic acid enzymes, also called RNA enzymes or catalytic RNAs) are RNA molecules that catalyze chemical reactions. Many natural ribozymes catalyze either their own cleavage or the cleavage of other RNAs, but they have also been found to catalyze the aminotransferase activity of ribosomes. Non-limiting examples of well-characterized small self-cleaving RNAs include hammerhead, hairpin, hepatitis delta virus, and in vitro-selected lead-dependent ribozymes, while group I introns are examples of larger ribozymes. The principle of catalytic self-cleavage has been well established in recent years. Hammerhead ribozymes are the best characterized RNA molecules with ribozyme activity. It has been shown that hammerhead structures can be incorporated into heterologous RNA sequences, thereby transferring ribozyme activity to these molecules. Therefore, catalytic antisense sequences for almost any target sequence can be generated, assuming the target sequence contains a potential compatible cleavage site. The basic principle for constructing hammerhead ribozymes is as follows: a region of interest containing a GUC (or CUC) triplet is selected. Two oligonucleotide strands, each typically 6–8 nucleotides long, are employed, and the catalytic hammerhead sequence is inserted between them. Best results are usually obtained using short ribozymes and target sequences.

[0153] A recent development that is also useful according to the present invention is the combination of an aptamer that recognizes small compounds with a hammerhead ribozyme. The conformational change induced in the aptamer upon binding to the target molecule can control the catalytic function of the ribozyme.

[0154] Examples of chemical inhibitors of p53 include, but are not limited to, p53 inhibitors typified by pifithrin (PFT)-α and -β, as disclosed in WO 00 / 44364, PFT-μ, as disclosed in Storm et al., 2006, its analogs and salts (e.g., acid addition salts, such as hydrochloride and hydrobromide salts), and the like. Among these, PFT-α and its analogs [2-(2-imino-4,5,6,7-tetrahydrobenzothiazol-3-yl)-1-p-tolyl-ethanone, HBr (product name: Pifithrin-α) and 1-(4-nitrophenyl)-2-(4,5,6,7-tetrahydro-2-imino-3(2H)-benzothiazolyl)ethanone, HBr (product name: Pifithrin-α, p-nitro)], PFT-β and its analogs [2-(4-methylphenyl)imidazo[2,1-b]-5,6,7,8-tetrahydrobenzothiazole, HBr (product name: Pifithrin-α, cyclic) and 2-(4-nitrophenyl)imidazo[2,1-b]-5,6,7,8-tetrahydrobenzothiazole (product name: Pifithrin-α, p-nitro, cyclic)], and PFT-μ. [Phenylacetylenylsulfonamide (product name: Pifithrin-μ)] is commercially available from Merck.

[0155] As used in the present invention, the term "antibody" includes, for example, polyclonal or monoclonal antibodies. Furthermore, derivatives or fragments thereof that still retain binding specificity to a target are also included in the term "antibody." The isotype of the antibody is not particularly limited, and is preferably IgG, IgM, or IgA, particularly preferably IgG. Antibody fragments or derivatives include, in particular, Fab or Fab' fragments, Fd, F(ab')2, Fv or scFv fragments, single-chain domains VH or V-like domains, such as VhH or V-NAR domains, as well as multimeric forms such as minibodies, diabodies, tribodies or triplebodies, tetrabodies or chemically conjugated Fab'-multimers (see, for example, Harlow and Lane "Antibodies, A Laboratory Manual", Cold Spring Harbor Laboratory Press, 198; Harlow and Lane "Using Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory Press, 1999; Altshuler EP, Serebryanaya DV, Katrukha AG. 2010, Biochemistry (Mosc.), vol. 75(13), 1584; Holliger P, Hudson PJ. 2005, Nat Biotechnol., vol. 23(9), 1126). In particular, multimeric forms include bispecific antibodies that can simultaneously bind to two different types of antigens. Non-limiting examples of bispecific antibody forms are Biclonics (bispecific, full-length human IgG antibodies), DART (dual affinity retargeting antibodies), and BiTE (composed of two single-chain variable fragments (scFvs) of different antibodies) molecules (Kontermann and Brinkmann (2015), Drug Discovery Today, 20(7):838-847).

[0156] The term "antibody" also includes embodiments such as chimeric (human constant domain, non-human variable domain), single chain and humanized (human antibody minus the non-human CDRs) antibodies.

[0157] Various techniques for producing antibodies are well known in the art and are described, for example, in Harlow and Lane (1988) and (1999) and Altshul et al., 2010, loc. cit. Thus, polyclonal antibodies can be obtained from the blood of animals after immunization with an antigen mixed with additives and adjuvants, and monoclonal antibodies can be produced by any technique that provides antibodies produced by continuous cell line culture. Examples of such techniques include the hybridoma technique first described in Koehler and Milstein, 1975, trioma technique, human B cell hybridoma technique (see, for example, Kozbor D, 1983, Immunology Today, vol. 4, 7; Li J, et al., 2006, PNAS, vol. 103(10), 3557), and EBV hybridoma technique for producing human monoclonal antibodies (Cole et al., 1985, Alan R. Liss, Inc., 77-96). Furthermore, recombinant antibodies can be obtained from monoclonal antibodies or prepared de novo using various display methods such as phage, ribosome, mRNA, or cell display. Suitable systems for the expression of recombinant (humanized) antibodies can be selected from, for example, bacteria, yeast, insect, mammalian cell lines or transgenic animals or plants (see, for example, U.S. Pat. No. 6,080,560; Holliger P, Hudson PJ. 2005, Nat Biotechnol., vol. 23(9), 11265). Surface plasmon resonance, such as that used in the BIAcore system, can be used to increase the efficiency of phage antibodies.

[0158] As used herein, the term "antibody mimetic" refers to a compound that can specifically bind to an antigen in a manner similar to an antibody, but is structurally unrelated to antibodies. Antibody mimetics are artificial peptides or proteins, typically with a molar mass of approximately 3-20 kDa. For example, the antibody mimetic can be selected from the group consisting of affibodies, adnectins, anticalins, DARPins, avimers, nanofitins, affilins, Kunitz domain peptides, Fynomers®, trispecific binding molecules, and probodies. These polypeptides are well known in the art and are described in further detail herein below.

[0159] The term "affibody" as used herein refers to a family of antibody mimics derived from the Z domain of Staphylococcus aureus protein A. Structurally, affibody molecules are based on a triple-helical bundle domain that can also be incorporated into fusion proteins. By themselves, affibodies have a molecular mass of approximately 6 kDa and are stable at high temperatures and under acidic or alkaline conditions. Target specificity is achieved by randomizing 13 amino acids located in two α-helices involved in the binding activity of the parent protein domain (Feldwisch J, Tolmachev V.; (2012) Methods Mol Biol. 899:103-26).

[0160] The term "Adnectin" (also referred to as "monobody"), as used herein, refers to molecules based on the tenth extracellular domain of human fibronectin III (10Fn3), which adopts a 94-residue Ig-like β-sandwich fold with two to three exposed loops but lacks a central disulfide bridge (Gebauer and Skerra (2009) Curr Opinion in Chemical Biology 13:245-255). Adnectins with desired target specificities can be genetically engineered by introducing modifications into specific loops of the protein.

[0161] The term "anticalin" as used herein refers to a genetically engineered protein derived from lipocalin (Beste G, Schmidt FS, Stibora T, Skerra A. (1999) Proc Natl Acad Sci US A. 96(5):1898-903; Gebauer and Skerra (2009) Curr Opinion in Chemical Biology 13:245-255). Anticalins have an eight-stranded β-barrel that forms a highly conserved core unit within lipocalins and naturally forms a binding site for ligands with four structurally different loops at the open end. Although anticalins are not homologous to the IgG superfamily, they exhibit characteristics previously considered typical of antibody binding sites: (i) high structural plasticity as a result of sequence variation and (ii) high conformational flexibility, which allows induced fit to targets with different shapes.

[0162] As used herein, the term "DARPin" refers to the ankyrin repeat domain (166 residues) that typically provides a rigid interface resulting from three repeated β-turns. DARPins usually have three repeats corresponding to an artificial consensus sequence, where six positions per repeat are randomized. As a result, DARPins lack structural flexibility (Gebauer and Skerra, 2009).

[0163] The term "avimer," as used herein, refers to a class of antibody mimics consisting of two or more peptide sequences, each 30-35 amino acids long, derived from the A domains of various membrane receptors and connected by a linker peptide. Target molecule binding occurs via the A domains, and domains with the desired binding specificity can be selected, for example, by phage display technology. However, the binding specificities of different A domains contained in an avimer may not necessarily be identical (Weidle UH, et al., (2013), Cancer Genomics Proteomics; 10(4):155-68).

[0164] "Nanophytin" (also known as affitin) is an antibody-mimetic protein derived from the DNA-binding protein Sac7d of Sulfolobus acidocaldarius. Nanophytins typically have a molecular weight of approximately 7 kDa and are engineered to specifically bind to target molecules by randomizing the amino acids on the binding surface (Mouratou B, Beehar G, Paillard-Laurance L, Colinet S, Pecorari F., (2012) Methods Mol Biol.; 805:315-31).

[0165] The term "affilin" as used herein refers to an antibody mimic that is developed by using either gamma-B crystallin or ubiquitin as a scaffold and modifying the amino acids on the surface of these proteins by random mutagenesis. The selection of affilin with desired target specificity is carried out, for example, by phage display or ribosome display technology. Depending on the scaffold, affilin has a molecular weight of about 10 or 20 kDa. As used herein, the term affilin also refers to the dimerization or multimerization form of affilin (Weidle UH, et al., (2013), Cancer Genomics Proteomics; 10(4):155-68).

[0166] "Kunitz domain peptides" are derived from the Kunitz domain of Kunitz-type protease inhibitors, such as bovine pancreatic trypsin inhibitor (BPTI), amyloid precursor protein (APP), or tissue factor pathway inhibitor (TFPI). Kunitz domains have a molecular weight of approximately 6 kDA, 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).

[0167] As used herein, the term "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 are described, for example, in Grabulovski et al. (2007) JBC, 282, pp. 3196-3204, WO 2008 / 022759, Bertschinger et al. (2007) Protein Eng Des Sel 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).

[0168] The term "trispecific binding molecule," as used herein, refers to a polypeptide molecule that has three binding domains and can therefore specifically bind to, preferably, three different epitopes. The trispecific binding molecule is preferably TriTac. TriTac is a solid tumor T cell engager composed of three binding domains designed to have an extended serum half-life and be approximately one-third the size of a monoclonal antibody.

[0169] As used herein, the term "probody" refers to a protease-activatable antibody prodrug. Probodies consist of an authentic IgG heavy chain and a modified light chain. A masking peptide is fused to the light chain via a peptide linker that is cleavable by tumor-specific proteases. The masking peptide prevents the probody from binding to healthy tissues, thereby minimizing toxic side effects.

[0170] Anti-p53 antagonist antibodies can be produced by known antibody or antiserum production methods using p53 or its partial peptides as the antigen. Examples of known anti-p53 antagonist antibodies include PAb1801 (Oncogene Science Ab-2) and DO-1 (Oncogene Science Ab-6) (Gire and Wynford-Thomas, 1998). Nucleic acids encoding anti-p53 antagonist antibodies can be isolated by conventional methods from hybridomas producing anti-p53 monoclonal antibodies. The resulting H-chain and L-chain genes can be linked together to prepare nucleic acids encoding single-chain antibodies.

[0171] Yet another agent that inhibits the function of the p53 protein is a decoy nucleic acid containing the consensus sequence of a p53-responsive element (e.g., Pu-Pu-Pu-GA / TT / AC-Py-Py-Py (Pu: purine base, Py: pyrimidine base); SEQ ID NO: 35). Such a decoy nucleic acid is commercially available (e.g., p53 transcription factor decoy (GeneDetect.com)).

[0172] In a preferred embodiment of the method of the ninth aspect of the invention, the method is carried out as an in vitro or ex vivo method.

[0173] In contrast to the preferred embodiment of the ninth aspect of the invention described above, the method is carried out as an in vivo method.

[0174] In a tenth aspect, the present invention relates to a reprogramming method for rejuvenating aged cell(s), tissue(s), organ(s) or organism(s), comprising administering to said aged cell(s), tissue(s), organ(s) or organism(s): a) a SoxB1 factor variant of the first aspect of the invention or a Sox17 factor variant of the second aspect of the invention and a POU factor; and / or b) a fusion protein of the third aspect of the invention or a complex or composition of the fourth aspect of the invention; and / or c) a SoxB1 factor variant and a Klf family member sharing an amino acid sequence comprising or consisting of SEQ ID NO: 13 or 14; and optionally: - inhibitors of p53 function; and / or - one or more additional reprogramming factor(s) The method includes increasing the level of

[0175] Suitable reprogramming factors are described elsewhere herein.

[0176] In a preferred embodiment of the tenth aspect of the present invention, the method comprises combining a SoxB1 factor variant or a Sox17 factor variant and a POU factor as defined in item a); and / or a fusion protein or complex or composition as defined in item b); and / or a SoxB1 factor variant and a Klf family member as defined in item c); and optionally; - inhibitors of p53 function; and / or - one or more additional reprogramming factor(s) by (co)expression of a factor variant, POU factor, fusion protein, complex and / or Klf family member as defined in items a) to c) and optionally: an inhibitor of p53 function and / or one or more further reprogramming factor(s).

[0177] In a preferred embodiment, the "rejuvenated cell(s)" are preferably rejuvenated senescent cells.

[0178] The term "rejuvenating" refers to the process of erasing epigenetic modifications that lead to aging in cells, tissues, organs or organisms. Aging in cells, tissues, organs or organisms is characterized by the deletion of, among other things, the following markers: p53 / p21 in cells and tissues; CIP1 , pRb / p16 INK4A or other tumor suppressor pathway activation, arrested cells, shortening of telomere size in cells, increased expression of senescence-associated factors (e.g., β-galactosidase (SAβ-Gal)), specific chromatin modifications such as senescence-associated heterochromatin regions (SAHF), specific secretome, reduced / altered mitochondrial activity, accumulation of epigenetic changes, changes to gene expression patterns, depletion or loss of stem cells in tissues and organs, loss or partial loss of tissue and organ regenerative capacity, loss or partial loss of functionality, and the occurrence or increased tendency to age-related diseases. If one or more or all of these markers of aging are reduced in aged or senescent cells, aged tissues, aged organs, or aged organisms due to the rejuvenation process, the rejuvenation process is observed.

[0179] The term "senescent cells" refers to cells that exhibit cell cycle arrest induced by replication exhaustion due to telomere attrition or in response to stresses such as DNA damage, chemotherapeutic drugs, or aberrant expression of oncogenes. This arrest is primarily mediated by activation of p53 and the cyclin-dependent kinase (CDK) inhibitor p16. INK4A and p21 CIP1 This is achieved through the upregulation of p53 / p21 (Collado et al. 2007, Cell, 130: 223-233). "Senescent cells" have the following characteristics: CIP1 and pRb / pl6 INK4ASenescence can be characterized by at least one or more of the following: activation of tumor suppressor pathways (hereinafter referred to as senescence effectors), irreversible cell arrest in G1, shortened telomere size, expression of senescence-associated β-galactosidase activity (SAβ-Gal), specific chromatin modifications such as senescence-associated heterochromatin regions (SAHF), specific secretome, and reduced / altered overall mitochondrial activity. Irreversible cell arrest in G1 can be assessed by FACS. Shortened telomere size can be characterized by assessing mean terminal restriction fragment (TRF) length, for example, by Southern blot analysis. Methods for detecting the expression of senescence-associated β-galactosidase activity (SAβ-Gal) are known in the art. A method for detecting the expression of senescence-associated heterochromatin regions (SAHF) by indirect immunofluorescence is described in EP 2694642. Overall mitochondrial activity can be assessed by measuring the transmembrane potential generated by a proton gradient.

[0180] Aging cells are proliferative cells that exhibit one or more of the following characteristics: upregulation of tumor suppressors, reduced ability to proliferate, changes to DNA methylation patterns, accumulation of other epigenetic changes, misregulation of gene expression, and reduced or lost ability to perform their function. Aging markers can be observed using techniques known in the art, such as the epigenetic clock (Horvath, 2013).

[0181] In a preferred embodiment, the aged or senescent cells exhibit at least one or more (or all) of the following characteristics of an aging phenotype: upregulation of tumor suppressors, cells irreversibly arrested in G1, expression of senescence-associated β-galactosidase activity (SAβ-Gal), expression of senescence-associated heterochromatin regions (SAHF) and altered overall mitochondrial activity, changes to DNA methylation patterns, accumulation of other epigenetic changes, misregulation of gene expression, and a reduction or loss of the ability to perform its function.

[0182] Aging tissue is tissue that exhibits one or more of the following characteristics: accumulation of senescent or aged cells within the tissue, changes to DNA methylation patterns, accumulation of other epigenetic changes, misregulation of gene expression, depletion or loss of stem cells within the tissue, reduced or lost regenerative capacity, and reduced or lost ability to perform its function.

[0183] An aging organ is one that exhibits one or more of the following characteristics: accumulation of senescent or aged cells within the organ, changes to DNA methylation patterns, accumulation of other epigenetic changes, misregulation of gene expression, depletion or loss of stem cells within the organ, reduced or lost regenerative capacity, and reduced or lost ability to perform its functions.

[0184] An aging organism is an animal organism that exhibits one or more of the following characteristics: senescence or accumulation of 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; misregulation of gene expression in some or all of its cells and tissues; depletion or loss of stem cells in one or more of its tissues or organs; loss of regenerative capacity of some or more of its tissues and organs;

[0185] Aged or senescent cells can be derived from various tissues, for example, from aged human patients or non-human animals that require self-renewal therapy.Methods for obtaining samples from various tissues and for establishing progenitor cells are well known in the art (see, for example, Jones and Wise, Methods Mol Biol. 1997).Said aged or senescent cells include, but are not limited to, progenitor cells from blood, bone marrow, adipose tissue, nervous tissue, skin, skin appendages, internal organs, such as heart, intestine or liver, mesenchymal tissue, muscle, bone, cartilage or skeletal tissue.

[0186] Increasing the level of a factor as described in items a) to c) according to the tenth aspect of the present invention may be carried out by contacting an aging cell, senescent cell, aging tissue, aging organ or aging organism with a factor, fusion protein and / or complex or composition as defined in items a) to c) of the tenth aspect of the present invention, allowing the cell to take up the respective factor, fusion protein and / or complex or composition, and transfecting or transducing the cell with a nucleic acid encoding the factor, fusion protein and / or complex, and (co)expressing the factor, fusion protein and / or complex.

[0187] In a preferred embodiment of the tenth aspect of the invention, the method is carried out as an in vitro or ex vivo method.

[0188] Contrary to preferred embodiments of the above-mentioned tenth aspect of the invention, the method is an in vivo method.

[0189] In a preferred embodiment of the ninth or tenth aspect of the invention, the one or more further reprogramming factors are selected from: a Klf family member, a Myc family member, Lin28, Nanog and GLIS1.

[0190] The term "Lin28" refers to the protein 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 IGF-2 (insulin-like growth factor 2) mRNA and enhances its translation. Lin28 has also been shown to bind to let-7 pre-miRNA and block the production of mature let-7 microRNA in mouse embryonic stem cells. Yu et al. demonstrated that it is a factor, but not a requirement, for iPSC generation. Exemplary Lin28s are proteins encoded by the mouse gene (NCBI reference sequence NM_145833, e.g., all sequences deposited under NM_145833.1) and the human LIN28A gene (NCBI reference sequence NM_024674, e.g., all sequences deposited under NM_ NM_024674.6). The term "Lin28," as referred to herein, includes either the naturally occurring form of the Lin28 transcription factor or a variant thereof that retains Lin28 transcription factor activity (e.g., within at least 50%, 80%, 90%, or 100% activity) relative to wild-type Lin28 as measured by methods known in the art, such as measuring the efficacy of iPSC generation in reprogramming experiments.

[0191] In some embodiments, variants have at least 90% amino acid sequence identity across their entire sequence compared to a naturally occurring Lin28 polypeptide. In other embodiments, the Lin28 protein is a protein identified by all sequences deposited under NCBI reference NP_078950, e.g., NP_078950.1, or NP_665832, e.g., NP_665832.1, corresponding to SEQ ID NOs:36 and 37, respectively.

[0192] The term "at least 50%, 80%, 90% or 100% active" or similar terms used throughout the specification is intended to mean "at least 50%, at least 80%, at least 90% or 100% active" in all instances.

[0193] The term "Nanog" or "nanog" refers to a transcription factor that is critically involved in the self-renewal of undifferentiated embryonic stem cells. In humans, this protein is encoded by the NANOG gene. An exemplary nanog is a protein encoded by the mouse gene (NCBI reference sequence NM_028016, e.g., all sequences deposited under NM_028016.3) and the human Nanog gene (NCBI reference sequence NM_024865, e.g., all sequences deposited under NM_024865.4). Thus, the term "Nanog" or "nanog" as used herein includes either the naturally occurring form of the Nanog transcription factor, or its variants that retain Nanog transcription factor activity (e.g., within at least 50%, 80%, 90%, or 100% activity) compared to wild-type Nanog, as measured by methods known in the art, such as measuring the effectiveness of iPSC generation in reprogramming experiments.

[0194] In some embodiments, variants have at least 90% amino acid sequence identity across their entire sequence compared to a naturally occurring Nanog polypeptide. In other embodiments, the Nanog protein is a protein identified by all sequences deposited under NCBI reference NP_079141, e.g., NP_079141.2, or NP_082292, e.g., NP_082292.1, corresponding to SEQ ID NOs: 38 and 39, respectively.

[0195] The term "GLIS1" as used herein refers to a protein belonging to the GLIS protein family (similar to the GLI family) of zinc finger transcription factors. Forced expression of GLIS1 promotes iPSC formation (Maekawa et al., 2001). An exemplary GLIS1 protein is the protein encoded by the GLIS1 gene in humans (NCBI reference sequence NM_147193, e.g., all sequences deposited under NM_147193.4). The term "GLIS1" as used herein includes naturally occurring forms of the GLIS1 transcription factor (e.g., isoforms) or variants thereof that retain GLIS1 transcription factor activity (e.g., within at least 50%, 80%, 90%, or 100%) compared to wild-type GLIS1, as measured by methods known in the art, such as measuring the effectiveness of iPSC generation in reprogramming experiments.

[0196] In some embodiments, variants have at least 90% amino acid sequence identity across their entire sequence compared to a naturally occurring GLIS1 polypeptide. In other embodiments, the GLIS1 protein is the protein identified by all sequences deposited under NCBI reference NP_671726, e.g., NP_671726.2, corresponding to SEQ ID NO:40.

[0197] In a preferred embodiment of the SoxB1 factor variant as defined under item a) of the ninth or tenth aspect of the present invention, the SoxB1 factor variant comprises or consists of the amino acid sequence of SEQ ID NO: 13 or 14.

[0198] In a further preferred embodiment of the POU element as defined in item a) or b) of the ninth or tenth aspect of the invention, the POU element is selected from: Oct4, Oct1, Oct2, Oct6, Brn1, Brn2, Brn4 or other engineered or naturally occurring POU element that has a high propensity to cooperate with Sox on the canonical SoxOct motif.

[0199] In an even more preferred embodiment of the SoxB1 factor variant, Sox17 factor variant and POU factor as defined in item a) of the ninth or tenth aspect of the invention, the method comprises combining: (i) a SoxB1 factor variant of the first aspect of the invention or a Sox17 factor variant of the second aspect of the invention and Oct4; and / or (ii) a SoxB1 factor variant of the first aspect of the invention or a Sox17 factor variant of the second aspect of the invention and Oct4 and Klf4 or GLIS1; and / or (iii) a SoxB1 factor variant of the first aspect of the invention or a Sox17 factor variant of the second aspect of the invention and O and / or (iv) a SoxB1 factor variant of the first aspect of the invention or a Sox17 factor variant of the second aspect of the invention and Oct4, Klf4 and c-Myc; and / or (v) a SoxB1 factor variant of the first aspect of the invention or a Sox17 factor variant of the second aspect of the invention and Oct4, Klf4, c-Myc and Lin28 in the non-pluripotent cell(s), or increasing their expression in said aged cell(s), tissue(s), organ(s) or organism(s).

[0200] Those skilled in the art will understand that the reference sequences encoding the proteins / factors described herein are not limited to the NCBI reference sequences expressly set forth herein, but include each and every tracked NCBI reference sequence encoding said proteins / factors.

[0201] Those skilled in the art are aware that proteins / factors in two different mammalian species are coded by the sequences that are listed in different NCBI reference sequence numbers.Those skilled in the art understand that for the purposes of the disclosure described herein, proteins / factors are selected from the same species, preferably human.For example, when one or more reprogramming factors(s) are selected from the group of human POU factors, any additional reprogramming factors and / or other reprogramming factors selected from the group of Klf and / or Myc family are preferably selected from human.

[0202] The term "aged cell" is synonymous with senescent cell in this and the following embodiments as well. As used throughout this specification, the term "aged cell(s), tissue(s), organ(s) or organism(s)" or similar terms is intended to mean in all instances "aged cell(s)," "aged tissue(s)," "aged organ(s)," and "aged organism(s)."

[0203] In a preferred embodiment of the Klf family member as defined under item c) of the ninth or tenth aspect of the invention, the Klf family member is selected from: Klf4, Klf2, Klf17.

[0204] In a preferred embodiment of the SoxB1 factor variant and Klf family member defined in item c) of the ninth or tenth aspect of the present invention, the method comprises the step of (co)expressing: (i) a SoxB1 factor variant and Klf4 sharing an amino acid sequence comprising or consisting of SEQ ID NO: 13 or 14; or (ii) a SoxB1 factor variant, Klf4 and a Myc family member sharing an amino acid sequence comprising or consisting of SEQ ID NO: 13 or 14 in non-pluripotent cell(s) or increasing their expression in said aged cell(s), tissue(s), organ(s) or organism(s).

[0205] As used herein, the term "Myc family member" refers to a factor encoded by the myc proto-oncogene that is associated with cancer.

[0206] In a further preferred embodiment of the method of the ninth or tenth aspect of the present invention, the method does not include (co)expressing an inhibitor of p53 function. p53 inhibition increases reprogramming efficiency but can reduce the quality of iPSCs due to increased cell proliferation and reduced response to DNA damage. The highly effective Super-SOX demonstrated here allows for a reduction in the number of factors required for reprogramming. The increased efficiency allows for the generation of higher quality iPSCs by avoiding oncogenes such as Myc or p53 inhibitors.

[0207] In an eleventh aspect, the present invention relates to a method for the conversion of pluripotent cell(s) into high-grade naive pluripotent cell(s), said method comprising culturing said pluripotent cell(s) under conditions suitable for conversion of said pluripotent cell(s) into high-grade naive pluripotent cell(s), said conditions comprising the expression in said pluripotent cell(s) of: a) a SoxB1 factor or a SoxB1 factor variant of the first aspect of the invention and a Klf family member; and / or b) a SoxB1 factor variant sharing an amino acid sequence comprising or consisting of SEQ ID NO: 13 or 14; and optionally: - inhibitors of p53 function; and / or - one or more additional reprogramming factor(s) This includes increasing the level of

[0208] In a preferred embodiment of the eleventh aspect of the present invention, a SoxB1 factor or a SoxB1 factor variant as defined in items a) and b), respectively; and a Klf family member; and optionally: - inhibitors of p53 function; and / or - one or more additional reprogramming factor(s) Increasing the level of is achieved by (co)expression of a factor, factor variant and / or Klf family member as defined in items a) and / or b) and optionally: an inhibitor of p53 function and / or one or more further reprogramming factor(s). The further reprogramming factor(s) are defined herein above.

[0209] Culturing the cell(s) according to the eleventh aspect of the invention preferably comprises culture conditions that support / facilitate conversion of pluripotent cells to high-grade naive pluripotent cells, and preferably allow proliferation of the high-grade naive pluripotent cells.

[0210] Culturing cells can include naive medium, 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: HDAC, WNT, MEK, FGF, FGFR, GSK3, ROCK, and PKC, p38, JNK, BMP, ERK, TGFB), optionally including a feeder layer and / or specific conditions such as hypoxic conditions (5% O2).

[0211] Culturing a cell(s) according to the eleventh aspect of the present invention may comprise contacting a cell(s) with a SoxB1 factor variant and a Klf family member as defined in items a) and b) of the eleventh aspect of the present invention to allow the cells to take up the respective SoxB1 factor variant and Klf family member, and transfecting or transducing the cells 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.

[0212] As used in accordance with the eleventh aspect of the present invention, the term "pluripotent cell" refers to any pluripotent cell that has a lower differentiation potential than a high-grade naive pluripotent cell. In a preferred embodiment of the eleventh aspect of the present invention, the pluripotent cell is a low-grade pluripotent cell.

[0213] In another even more preferred embodiment, the method comprises administering to said pluripotent cell(s): a) a SoxB1 factor or a SoxB1 factor variant of the first aspect of the invention, and a Klf family member; and / or b) a SoxB1 factor variant sharing an amino acid sequence comprising or consisting of SEQ ID NO: 13 or 14; and optionally: - inhibitors of p53 function; and / or - one or more additional reprogramming factors This includes (co)expressing

[0214] In a preferred embodiment of the eleventh aspect of the present invention, the pluripotent cells are selected from the group consisting of induced pluripotent stem cells, induced pluripotent stem cells (iPSCs) and embryonic stem cells (ECSs).

[0215] Furthermore, the one or more further reprogramming factors according to the eleventh aspect of the present invention are preferably selected from: a POU factor, a Klf family member, a Myc family member, Lin28, Nanog and GLIS1.

[0216] In a preferred embodiment of the SoxB1 factor variant defined in item a) of the eleventh aspect of the present invention, the SoxB1 factor is Sox2, and the SoxB1 factor variant of the first aspect of the present invention is a SoxB1 factor variant sharing an amino acid sequence comprising or consisting of SEQ ID NO: 11 or 47 or a SoxB1 factor variant comprising or consisting of the amino acid sequence of SEQ ID NO: 13 or 14.

[0217] In another preferred embodiment of the Klf family member as defined under item a) of the eleventh aspect of the invention, the Klf family member is selected from Klf4, Klf2 or Klf17.

[0218] In a preferred embodiment of the SoxB1 factor variant and Klf family member defined in item a) of the eleventh aspect of the present invention, the method comprises (co)expressing in a pluripotent cell(s): (i) a SoxB1 factor variant and Klf4 sharing an amino acid sequence comprising or consisting of SEQ ID NO: 13 or 14; or (ii) a SoxB1 factor variant, Klf4 and c-Myc sharing an amino acid sequence comprising or consisting of SEQ ID NO: 13 or 14; or (iii) a SoxB1 factor variant, Klf4 and Oct4 comprising or consisting of the amino acid sequence SEQ ID NO: 13 or 14.

[0219] In a further preferred embodiment of the eleventh aspect of the invention, the sequences encoding the SoxB1 factor or SoxB1 factor variant of the first aspect of the invention and the Klf family member are combined into a single polycistronic cassette. An example is the SOX-P2A-Klf4 cassette shown by the inventors in Figures 6i-j (SEQ ID NO: 44). Further examples include the cassettes of SEQ ID NO: 45 or 46.

[0220] In a particularly preferred embodiment of the eleventh aspect of the present invention, the polycistronic cassette contains sequences encoding a SoxB1 factor or a SoxB1 factor variant, a Klf family member, and a fluorescent protein, the latter of which can be used to track cassette expression and loss. An example is the mCherry-T2A-SOX-P2A-KLF4 episomal vector shown by the inventors in the Examples herein below.

[0221] The term "fluorescent protein," as used herein, means a protein that exhibits low, moderate or strong fluorescence upon illumination with light of an appropriate excitation wavelength.

[0222] In a preferred embodiment of the eleventh aspect of the invention, the method is carried out as an in vitro or ex vivo method.

[0223] Contrary to the above-mentioned preferred embodiment of the eleventh aspect of the invention, the method is carried out as an in vivo method.

[0224] In preferred embodiments of the ninth to eleventh aspects of the present invention, the cells are mammalian cells.

[0225] In particularly preferred embodiments, the cells are selected from human, non-human primate, murine, porcine, bovine, equine, canine, feline or elephant cells.

[0226] In a twelfth aspect, the present invention relates to a kit of parts comprising or consisting of: a) a SoxB1 factor variant according to the first aspect of the invention; and / or b) a Sox17 factor variant according to the second aspect of the invention; and / or c) a fusion protein according to the third aspect of the invention; and / or d) a complex or composition according to the fourth aspect of the invention; and / or e) a nucleic acid molecule or combination of nucleic acid molecules according to the fifth aspect of the invention; and / or f) a vector or combination of vectors according to the sixth aspect of the invention; and / or a cell according to the seventh aspect of the invention; and optionally instructions for use of the kit.

[0227] The various components of the kit may be enclosed in one or more containers, such as one or more vials. In addition to the components, the vials may contain preservatives or buffers for storage. The kit may include instructions on how to use the kit.

[0228] In a thirteenth aspect, the present invention relates to a method for producing rejuvenated cell(s), tissue(s), organ(s) or organism(s), the method comprising increasing the levels of: a) a SoxB1 factor variant of the first aspect of the invention or a Sox17 factor variant of the second aspect of the invention, and a POU factor; and / or b) a fusion protein of the third aspect of the invention or a complex or composition of the fourth aspect of the invention; and / or c) a SoxB1 factor variant of the first aspect of the invention and a Klf family member; and optionally one or more further reprogramming factor(s); thereby producing rejuvenated cell(s), tissue(s), organ(s) or organism(s).

[0229] In a preferred embodiment of the thirteenth aspect of the present invention, increasing the levels of the factor variant and POU factor defined in item a), and / or the fusion protein or complex or composition defined in item b) and / or the SoxB1 factor variant and Klf family member defined in item c) and optionally one or more further reprogramming factor(s) in aged cell(s), tissue(s), organ(s) or organism(s) is achieved by (co)expressing the factor variant, POU factor, fusion protein, complex and / or Klf family member and optionally one or more further reprogramming factor(s) defined in items a) to c).

[0230] Culturing cells according to the thirteenth aspect of the present invention comprises contacting aged cell(s), tissue(s), organ(s) or organism(s) with a SoxB1 factor variant or a Sox17 factor variant, and a POU factor and / or a fusion protein or complex or composition and / or a SoxB1 factor variant and a Klf family member as defined in item a) of the thirteenth aspect of the present invention, and inducing the SoxB1 factor variant, Sox17 factor variant, POU factor, fusion protein, complex or composition and / or Klf family member to the aged cell(s). The method may comprise incorporating the SoxB1 factor variant, Sox17 factor variant, POU factor, fusion protein, complex and / or Klf family member into aged cells(s), tissue(s), organ(s) or organism(s), and transfecting or transducing the aged cells(s), tissue(s), organ(s) or organism(s) with a nucleic acid encoding the respective SoxB1 factor variant, Sox17 factor variant, POU factor, fusion protein, complex and / or Klf family member and (co-)expressing the respective SoxB1 factor variant, Sox17 factor variant, POU factor, fusion protein, complex and / or Klf family member.

[0231] Culture conditions may be the same as normal for a given cell type or may include specific factors, media supplements, nutrient supplements that facilitate the rejuvenation process.

[0232] In a preferred embodiment of the thirteenth aspect, the method further comprises optionally increasing the level of one or more additional reprogramming factor(s) and / or inhibitor(s), wherein the inhibitor is selected from the group of TGFβ inhibitors, Src inhibitors and other kinase inhibitors, which may be used in combination with epigenetic modifiers. These inhibitors are known in the art and are routinely used to support the reprogramming of aged cell(s), tissue(s), organ(s) or organism(s).

[0233] In a preferred embodiment of the SoxB1 factor variant as defined in item a) of the thirteenth aspect of the present invention, the method comprises the step of co-expressing in a pluripotent cell a SoxB1 factor variant comprising or consisting of SEQ ID NO: 13 or 14.

[0234] In a preferred embodiment of the method of the thirteenth aspect, the rejuvenated cells are rejuvenated senescent cells.

[0235] In a further preferred embodiment, the Klf family member is Klf4.

[0236] In a fourteenth aspect, the present invention relates to a method for producing high-grade naive pluripotent cell(s), the method comprising increasing the levels of: a) a SoxB1 factor or a SoxB1 factor variant of the first aspect of the invention or a Sox17 factor variant of the second aspect of the invention, and a Klf family member; and / or b) a fusion protein of the third aspect of the invention or a complex or composition of the fourth aspect of the invention and a Klf family member; and optionally one or more further reprogramming factor(s); thereby producing high-grade naive pluripotent cell(s).

[0237] Increasing the level of the factor, fusion protein or complex or composition as defined in items a) and / or b) according to the fourteenth aspect of the present invention may be carried out by contacting the low-grade pluripotent cell(s) with the factor, fusion protein and / or complex or composition as defined in items a) and / or b) of the fourteenth aspect of the present invention, wherein the respective factor, fusion protein and / or complex or composition is taken up by the cell(s), and by transfecting or transducing the cell(s) with a nucleic acid encoding the factor, fusion protein and / or complex and (co)expressing the factor, fusion protein and / or complex.

[0238] In a preferred embodiment of the fourteenth aspect of the present invention, increasing the level of the mentioned factor or protein is achieved by (co)expression of the mentioned factor or protein.

[0239] In a preferred embodiment of the SoxB1 factor variant and Klf family member defined in item a) of the fourteenth aspect of the present invention, the method comprises (co)expressing in a low-grade pluripotent cell: (i) a SoxB1 factor variant and Klf4 sharing an amino acid sequence comprising or consisting of SEQ ID NO: 13 or 14; or (ii) a SoxB1 factor variant, Klf4 and c-Myc sharing an amino acid sequence comprising or consisting of SEQ ID NO: 13 or 14; or (iii) a SoxB1 factor variant, Klf4 and Oct4 comprising or consisting of the amino acid sequence SEQ ID NO: 13 or 14.

[0240] In a preferred embodiment of the method according to the fourteenth aspect of the invention, the method further comprises the step of recovering the naive pluripotent cells produced.

[0241] In a preferred embodiment, the Klf family member is Klf4.

[0242] In a further preferred embodiment of the method of the fourteenth aspect of the invention, the low-grade pluripotent cell(s) are iPSC(s), and optionally: - the iPSC(s) are obtained or obtainable by a method according to the ninth aspect of the invention; - the method according to the fourteenth aspect further comprises, prior to step a), the step of a) providing iPSC(s) by carrying out a method according to the ninth aspect of the invention.

[0243] In a preferred embodiment of the method of any of the ninth, tenth, eleventh, thirteenth and fourteenth aspects of the invention, the (co)expression is integration-free (co)expression, preferably the integration-free (co)expression comprises: - a nucleic acid molecule of the fifth aspect of the invention; or - derived from a vector of the sixth aspect of the invention, which vector is preferably an episomal vector.

[0244] As used herein, the term "(co)expression" refers to the production of one or more (poly)peptides(s), which may be (i) permanent, i.e., achieved by constitutive (co)expression; or (ii) non-permanent, i.e., achieved by transient (co)expression.

[0245] The term "constitutive (co)expression," as used herein, refers to the permanent production of a (poly)peptide in a host cell, wherein the (poly)peptide(s) are encoded in a polynucleotide(s) contained in an exogenous gene and delivered to the host cell. Gene delivery to the host cell can be, for example, by transfection methods known to those skilled in the art, such as lipofection, electroporation, nucleofection, viral delivery (transduction).

[0246] Typically, constitutive (co)expression is achieved by integration of DNA into the genome of the host cell.

[0247] In another preferred embodiment of the method of any of the ninth, tenth, eleventh, thirteenth and fourteenth aspects of the invention, the (co)expression is constitutive (co)expression.

[0248] The term "transient (co)expression" as used herein refers to the non-permanent production of a (poly)peptide in a cell. The production of the (poly)peptide can be directly driven by exogenous polynucleotide(s) encoding the (poly)peptide.

[0249] In a more preferred embodiment of the above preferred embodiment, expression from an episomal vector, also referred to herein as an episome, is transient (co)expression.

[0250] As used herein, the term "exogenous gene" refers to any polynucleotide sequence that encodes a (poly)peptide, which is - is partly or wholly heterologous, i.e., foreign to the host cell into which it is introduced; or - heterologous to the endogenous genes of the host cell into which it is introduced, but inserted in such a manner that it affects the expression of other endogenously expressed (poly)peptides in the host cell (e.g., transcription factor proteins that control the expression of endogenous proteins). Introduction of an exogenous gene, or in other words, delivery of a polynucleotide encoding a (poly)peptide into a host cell, can be achieved by transfection. Those skilled in the art will understand the methods known in the art for transfecting host cells. Non-limiting examples include nucleofection, lipofection, electroporation, or viral delivery.

[0251] In another even more preferred embodiment of the above preferred embodiment, the nucleic acid molecule of the fifth aspect of the invention is RNA, preferably modified RNA, most preferably modified mRNA (modRNA).

[0252] In a further preferred embodiment of the method of the fourteenth aspect of the invention, the method comprises (i) delivery to a host cell of a Sox family TF, preferably a SoxB1 factor or a SoxB1 factor variant and a Klf family member according to the first to sixth aspects of the invention, and (ii) culturing the cell(s) as single cells under conditions suitable to generate high-grade naive pluripotent cells.

[0253] Cells may be cultured in naive medium, e.g., Rset (STEMCELL Technologies), PXGL (Bredenkamp, ​​et al., 2019b), or other formulations (containing one or more small molecule inhibitors of the following molecules / pathways: HDAC, WNT, MEK, FGF, FGFR, GSK3, ROCK, and PKC, p38, JNK, BMP, ERK, TGFB), optionally with a feeder layer, and / or specific conditions such as hypoxic conditions (5% O2).

[0254] In a further or more preferred embodiment of item (i) of the above embodiment, the Sox family TF, preferably a SoxB1 factor or a SoxB1 factor variant according to the first to sixth aspects of the invention and a Klf family member, is delivered to the host cell, preferably a cell according to the seventh aspect of the invention, as a protein, mRNA, modified RNA or a plasmid.

[0255] In another more preferred embodiment of item (ii) of the above embodiment, the conditions suitable for producing naive pluripotent cells, including high-grade and low-grade naive pluripotent cells, comprise a) optionally plating the cell(s) as single cells onto an inactivated feeder layer or in feeder-free conditions, and b) optionally supplementing the medium with small molecules that support naive reset, i.e., induced-to-naive conversion (upgrading), or using a cell culture medium that supports naive pluripotent cell culture.

[0256] Small molecules according to the above embodiments are, for example, LIF, FGF signaling inhibitors (such as MEK / ERK inhibitors, e.g., PD0325901, or / and FGFR inhibitors, e.g., PD173074), Wnt inhibitors, e.g., XAV939, GSK3β inhibitors, e.g., CHIR99021, PKC inhibitors, e.g., Go 6983.

[0257] The term "feeder", also understood as "feeder cells", as used herein, refers to one type of cell that is co-cultured with another type of cell to provide an environment in which the second type of cell can grow. Feeder cells can optionally be derived from the same or different species as the cells they support. According to a more preferred embodiment described above, feeder cells can be inactivated when co-cultured with other cells by means known to those skilled in the art, such as irradiation or treatment with antimitotic agents, such as mitomycin C, to prevent them from growing the cells they support.

[0258] The term "feeder-free," as used herein, refers to "feeder cell-free" conditions in which a culture or cell population has less than 5% (with decreasing preference) of the total cells in the culture, such as less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.1%, and less than 0.01%, being feeder cells.

[0259] The cell culture medium supporting the naive pluripotent cell culture according to the above-mentioned embodiments can be any cell culture medium typically used in the art and known to those skilled in the art. In certain embodiments with the above-mentioned embodiments, the cell culture medium is a cell culture medium such as in-house produced N2B27 or commercially available RSeT supplemented with some or all of the above-mentioned factors and small molecules. TM Culture medium, NaieveCult TM is selected from the group:

[0260] In preferred embodiments of the thirteenth and / or fourteenth aspects of the invention, the method is carried out as an in vitro or ex vivo method.

[0261] Contrary to the above-mentioned preferred embodiments of the thirteenth and fourteenth aspects of the invention, the method is an in vivo method.

[0262] In a preferred embodiment of the methods of any of the ninth, tenth, eleventh, thirteenth and fourteenth aspects of the invention, the non-pluripotent cells of the ninth aspect, the aged cells of the tenth and thirteenth aspects and the pluripotent cells of the eleventh and fourteenth aspects of the invention are mammalian cells.

[0263] In particularly preferred embodiments of the above preferred embodiments, the non-pluripotent cells of the ninth aspect of the invention, the aged cells of the tenth and thirteenth aspects, the eleventh pluripotent cells and the low-grade pluripotent cells of the fourteenth aspect are selected from human, non-human primate, murine, porcine, bovine, equine, canine, feline, elephant or other mammalian cells.

[0264] In a fifteenth aspect, the present invention relates to an induced pluripotent cell, a high-grade naive pluripotent cell or a rejuvenated cell, tissue or organ produced or producible by a method according to any of the ninth, tenth, eleventh, thirteenth and fourteenth aspects of the invention, respectively.

[0265] In preferred embodiments of the fifteenth aspect of the present invention, the induced pluripotent stem cells (iPSCs) are characterized by (i) the expression of at least one, preferably at least two, more preferably at least three pluripotency-specific marker(s) selected from Oct4, Sox2, Nanog, and Klf4; and / or (ii) the ability to differentiate into each of the three primitive germ layers.

[0266] In a more preferred embodiment according to expression as defined in item (i) of the above preferred embodiment of the fifteenth aspect of the present invention, the expression is detectable expression.

[0267] As used herein, the term " detectable expression " refers to the protein or gene expression that can be quantified relative or absolute in cell samples, or directly in said cells, that is, can be detected by methods known in the art.Non-limiting examples for detecting protein expression include Western blot, immunofluorescence staining, enzyme-linked immunosorbent assay (ELISA) and mass spectrometry; non-limiting examples for detecting gene expression include Northern blot, quantitative polymerase chain reaction (qPCR), DNA microarray and RNA-Seq.

[0268] In a more preferred embodiment of item (ii) of the above-mentioned preferred embodiment of the fifteenth aspect of the present invention, the three primordial germ layers are ectoderm, mesoderm and endoderm.

[0269] As used herein, the term "primordial germ layer" refers to the embryonic germ layer.

[0270] The term "capacity," when used interchangeably herein with the terms "ability," "potential," and "readiness to differentiate," refers to the ability of a stem or pluripotent cell to differentiate into a subset of more differentiated cells. The term "capacity" does not encompass moving backward along the differentiation spectrum to create a cell that contains greater differentiation potential than the parent cell. That is, the term "capacity" does not encompass reprogramming methods that shift cells toward a less differentiated state.

[0271] The ability to differentiate into all three primordial germ layers can be tested by methods known to those skilled in the art, for example, in vivo: a) teratoma assays in which pluripotent cells are injected into immunocompromised (SCID) mice. After several weeks to months, tumors containing tissues derived from ectodermal, mesodermal, and endodermal lineages form, as shown, for example, in Figures 10c, j, n, t, aa, and ab herein; b) embryo complementation assays in which cells are either aggregated with early animal embryos or injected into blastocysts, after which pluripotent cells can integrate into the blastocyst and contribute to the development of the animal or even form the entire body of the animal.

[0272] In vitro tests to determine the ability to differentiate include, for example, a) random differentiation upon withdrawal of growth factors (LIF or FGF) that support pluripotent stem cell growth in undifferentiated conditions, or exposure of pluripotent cells to differentiation-inducing media or small molecules (FCS, retinoic acid, etc.). Emergence of ectodermal, mesodermal, and endodermal lineages can be assessed by marker gene expression (e.g., qPCR, RNA-seq, single-cell sequencing, immunostaining), microscopy, functional testing, and b) directed differentiation, in which pluripotent cells are exposed to specific growth factors, cytokines, transcription factors, or other media components that direct specific differentiation into desired cell types.

[0273] In another preferred embodiment of the fifteenth aspect of the present invention, the high-grade naive pluripotent cells have, compared to corresponding non-naive pluripotent cells or low-grade naive pluripotent cells, at least: (i) increased developmental potential; (ii) 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 six naive pluripotency-specific markers selected from (but not limited to) Klf17, Klf4, Sox2, Susd2, Argfx, and Dnmt3l. (iii) higher expression levels of at least one primitive endoderm-specific marker(s) selected from Gata6 and Sox17; (iv) an activated POU5f1 distal enhancer; (v) a reactivated X chromosome in the female lineage; (vi) reduced DNA methylation; (vii) an enhanced ability to differentiate preferably into germ cells; (viii) an enhanced ability to contribute to the development of embryo(s) and / or animal(s); and / or (ix) a combination of two or more of (i)-(viii).

[0274] It will be appreciated by those skilled in the art that the markers for naive pluripotency described above are not an exhaustive list of markers.

[0275] The term "Klf17" as used herein refers to the name of a gene and protein or transcription factor from the Klf family that controls pre-implantation development in humans and other primates.An example for humans is the KLF17 gene, which is encoded by the NCBI reference sequence NM_173484, for example, all sequences deposited under NM_173484.4, and is described in Boroviak et al., 2017 (Development (Cambridge, England) vol. 144,2: 175-186. doi:10.1242 / dev.145177) and in the following examples.

[0276] The term "Susd2," as used herein above, is the name of a gene and protein that is a cell surface marker of preimplantation pluripotency in primates. An example for humans is the SUSD2 gene, encoded by all sequences deposited under GenBank reference AAH33107, e.g., AAH33107.1, and described in Bredenkamp et al. (2019a) and in the Examples below.

[0277] The term "Argfx" as used herein above refers to the gene and protein name for the arginine 50 homeobox transcription factor, which is a marker of naive pluripotency in humans and other animals.An example for humans is the ARGFX gene, which is encoded by all sequences deposited under NCBI reference sequence NM_001012659, such as NM_001012659.2, and is described in Boroviak et al., 2017 (Development (Cambridge, England) vol. 144,2: 175-186. doi:10.1242 / dev.145177) and in the following examples.

[0278] The term "Dnmt3l" as used herein above is the gene and protein name for DNA (cytosine-5-)-methyltransferase 3-like protein, which is responsible for CpG methylation - an epigenetic modification important in embryonic development. An example for humans is the DNMT3L gene, which is encoded by all sequences deposited under NCBI reference sequence NM_013369, e.g., NM_013369.4, and NM_175867, e.g., NM_175867.3, and is described in Bi et al. 2022 and in the Examples below.

[0279] The term "Gata6," as used herein above, is the gene and protein name for GATA-binding protein 6, a zinc finger transcription factor that plays a key role in development, particularly controlling primitive endoderm fate. An example for humans is the GATA6 gene, encoded by all sequences deposited under NCBI reference sequence NM_005257, e.g., NM_005257.6, and described in Plusa et al., 2008.

[0280] In a preferred embodiment of the above preferred embodiments, (ix) a combination of two or more of (i)-(viii) refers to at least two, preferably at least three, more preferably at least four, more preferably at least five, more preferably at least six, more preferably at least seven, or most preferably eight of the features of (i)-(viii).

[0281] In another even more preferred embodiment, the combination of at least two refers to a combination of all features of items (i) and (ii). In another preferred embodiment, the combination of at least two refers to a combination of features of items (i) and (iii). In another preferred embodiment, the combination of at least two refers to a combination of features of items (i) and (vii). In another preferred embodiment, the combination of at least two refers to a combination of features of items (ii) and (vii). In another preferred embodiment, the combination of at least two refers to a combination of features of items (iii) and (vii).

[0282] In another more preferred embodiment, the combination of at least three refers to the combination of features of items (i), (ii), and (iii). In another more preferred embodiment, the combination of at least three refers to the combination of features of items (i), (ii), and (vii). In another more preferred embodiment, the combination of at least three refers to the combination of features of items (i), (iii), and (vii).

[0283] In another preferred embodiment, the at least four combinations refer to the combination of features of items (i), (ii), (iii) and (iv). In another preferred embodiment, the at least four combinations refer to the combination of features of items (i), (ii), (iii) and (vii).

[0284] In another more preferred embodiment, the at least five combinations refer to combinations of features from items (i), (ii), (iii), (iv) and (v). In another more preferred embodiment, the at least five combinations refer to combinations of features from items (i), (ii), (iii), (iv) and (vii).

[0285] In another more preferred embodiment, the at least six combinations refer to combinations of features from items (i), (ii), (iii), (iv), (v) and (vi). In another more preferred embodiment, the at least six combinations refer to combinations of features from items (i), (ii), (iii), (iv), (v) and (vii).

[0286] In another more preferred embodiment, at least seven combinations refer to combinations of features from items (i), (ii), (iii), (iv), (v), (vi), and (vii). In another more preferred embodiment, eight combinations refer to combinations of features from items (i), (ii), (iii), (iv), (v), (vi), (vii), and (viii).

[0287] The term "corresponding" refers to cells derived from a comparable, similar origin, i.e., an animal, preferably a cell that is identical to the parent cell prior to reprogramming / conversion, according to the above-described preferred embodiment of the fifteenth aspect of the present invention. For example, if 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 induced pluripotent cells, iPSCs, and embryonic stem cells.

[0288] The developmental potential as defined in item (i) of the above embodiment can be assessed by a person skilled in the art by the means described herein.

[0289] In a more preferred embodiment, the expression levels of the naive pluripotency-specific marker(s) and primitive endoderm-specific marker(s) as defined in items (ii) and (iii) of the above-mentioned preferred embodiment of the fifteenth aspect of the present invention are gene expression and / or protein expression, preferably the expression of the marker is detectable expression of said marker protein.

[0290] The expression level according to the present invention can be quantified by any suitable means and methods available in the art. Generally, relative and absolute quantification means and methods can be used. In absolute quantification, a known standard or control is not required. The expression level can be directly quantified. As is well known in the art, absolute quantification can, in certain further embodiments, rely on a predetermined standard curve. In relative quantification, the expression level is quantified relative to a reference, such as a known control expression level. In addition, the expression level can be quantified relatively when, for example, comparing fluorescence intensity in the absence of a control.

[0291] As used herein, the term "higher expression level" or "increased expression level" refers to the quantification of an expression level, eg, the level of a pluripotency-specific marker, compared to a reference. The increase compared to the reference may be at least, with increasing preference, 5 times higher, at least 10 times higher, at least 20 times higher, at least 30 times higher, at least 40 times higher, at least 50 times higher, at least 60 times higher, at least 70 times higher, at least 80 times higher, at least 900 times higher, at least 100 times higher, at least 110 times higher, at least 120 times higher, at least 130 times higher, at least 140 times higher, at least 150 times higher, at least 160 times higher, at least 170 times higher, at least 180 times higher, at least 190 times higher, at least 200 times higher, at least 210 times higher, at least 220 times higher, at least 230 times higher, at least 240 times higher, at least 250 times higher, at least 260 times higher, at least 270 times higher, at least 280 times higher, at least 290 times higher, at least 300 times higher.

[0292] As used herein, the term "POU5f1 distal enhancer," also referred to as "Oct4 distal enhancer," refers to a regulatory region in the POU5F1 gene that specifically controls the transcription of Oct4. Those skilled in the art can determine the status of the regulatory region of a gene, i.e., activated or inactivated, by methods routinely used in the art.

[0293] According to the above-mentioned embodiment of item (iv), the reactivated X chromosome in the female lineage is also referred to herein as the "Xa chromosome," and the active state of both X chromosomes is referred to as "XaXa." X chromosome reactivation occurs on the inactivated X chromosome as an epigenetic process. During cell differentiation, inactivation of one X chromosome in the female lineage occurs to compensate for the difference in the potential amount of X-linked genes between female XX cells and male XY cells. Inactivation of one X chromosome in XX embryos occurs early in mammalian development, resulting in female naive pluripotent stem cells with an XaXa state and female induced pluripotent stem cells with an XaXi state. For example, mouse embryonic stem cells derived from the inner cell mass of blastocysts contain two active X chromosomes, one of which is inactivated upon exiting the naive state during in vivo or in vitro induction or differentiation. In human embryonic stem cells, the X chromosome is already inactivated as they exhibit an induced pluripotent state. In human iPSCs and ESCs, the inactivation state can be reversed. Reprogramming hiPSCs or ESCs to a more undifferentiated state, such as a naive state, results in reversal of inactivation, and at least one X chromosome is reactivated. (Guo, et al., 2009; Theunissen, et al., 2016)

[0294] As used herein, the term "embryo" refers to a fertilized oocyte or alternatively a zygote, and also to cells at all stages of development from a fertilized oocyte or zygote up to day 5 or 6 (blastocyst stage) and up to the start of the third month of pregnancy in humans. The origin of the embryo is preferably selected from the group including natural conception, in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), somatic cell nuclear transfer (SCNT) into naturally or in vitro derived eggs, and / or aggregation from cultured cells.

[0295] In a preferred embodiment of the above-mentioned preferred embodiments of item (viii), the enhanced ability to contribute to the development of embryo(s) and / or animal(s) further comprises an enhanced ability to contribute to the development of embryo-like structures.

[0296] The term "embryonic structure," as used herein, refers to a synthetic embryo that, in contrast to a typical embryo as defined above, does not require fertilization of an oocyte. In other words, a synthetic embryo does not require oocytes and sperm cells, but relies on an artificial assembly from cultured cells or a combination of embryonic and cultured cells to mimic the natural structure that occurs in early development.

[0297] In another even more preferred embodiment of the above preferred embodiment of item (viii), the embryo(s) and animal(s) are chimeric.

[0298] The term "chimeric animal" or "chimeric embryo" is defined herein above.

[0299] In a sixteenth aspect, the present invention provides a method for producing a composition comprising: - as medicine; - in regenerative medicine; and / or - induced pluripotent cells (iPSCs), high-grade naive pluripotent cells or rejuvenated cells, tissues or organs of the fifteenth aspect of the invention for use in the treatment or prevention of age-related diseases.

[0300] In a preferred embodiment of the sixteenth aspect of the invention, the treatment or prevention of age-related diseases comprises delaying, inhibiting or reversing symptoms or delaying, inhibiting or reversing the onset of progression of these symptoms.

[0301] The term "age-related disease" as used herein preferably refers to age-related tissue degeneration, including muscle, nerve and / or skin tissue.

[0302] The term "age-related disease" or "age-related condition," as used herein, also refers to any condition, disease, or disorder associated with aging, including, but not limited to, neurodegenerative diseases (e.g., Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, dementia, and stroke), cardiovascular and peripheral vascular diseases (e.g., atherosclerosis, peripheral arterial disease (PAD), hematoma, calcification, thrombosis, embolism, and aneurysms), eye diseases (e.g., age-related macular degeneration, glaucoma, cataracts, dry eye, diabetic retinopathy, vision loss), skin diseases (skin atrophy and thinning, elastosis and skin wrinkling, sebaceous gland hyperplasia or hypoplasia, senile lentigines and other pigmentary abnormalities, graying of hair, hair loss or thinning, and chronic skin ulcers), autoimmune diseases (e.g., polymyalgia rheumatica (PMR), giant cell arteritis (GCA), rheumatoid arthritis (RA), and arthritis recurrence syndrome (ARS). arthritis (RA), crystalline arthropathy, and spondyloarthritis (SPA)), endocrine and metabolic dysfunction (e.g., adult hypopituitarism, hypothyroidism, apathetic thyrotoxicosis, osteoporosis, diabetes mellitus, adrenal insufficiency, various forms of hypogonadism, and endocrine malignancies), musculoskeletal disorders (e.g., arthritis, osteoporosis, myeloma, gout, Paget's disease, fractures, bone marrow failure syndromes, ankylosis, generalized idiopathic osteoarthritis, hematogenous bone disease, It may refer to diseases of the digestive system (e.g., cirrhosis, liver fibrosis, Barrett's esophagus), respiratory diseases (e.g., pulmonary fibrosis, chronic obstructive pulmonary disease, asthma, chronic bronchitis, pulmonary embolism (PE), lung cancer, and infections), and any other diseases and disorders associated with aging.

[0303] In a seventeenth aspect, the present invention relates to the use of induced pluripotent cells (iPSCs), high-grade naive pluripotent cells or rejuvenated cells, tissues or organs of the fifteenth aspect of the invention for the generation of differentiated cell(s), tissue(s), organ(s) or organism(s).

[0304] As used herein, the term "differentiated" or "differentiation" with respect to a cell(s), tissue(s), and / or organ(s) refers to the loss of pluripotency, as defined herein, in the process of cell differentiation into a specific cell type lineage. For example, a "differentiated cell" refers to a cell of a specialized cell type lineage derived from a cell of a less specialized cell type. Consistently, a differentiated cell is a non-pluripotent cell.

[0305] In a preferred embodiment of the use according to the seventeenth aspect of the invention, the differentiated cell(s), tissue(s), organ(s) or organism(s) is / are: (i) a transplant; (ii) an organoid; or (iii) cultivated meat.

[0306] As used herein, the term "transplant," also commonly known as a "graft," refers to free (non-adherent) cell(s), tissue(s), or organ(s) that are transferred from their site of origin to a recipient and become integrated into the recipient's bodily fluids, tissues, organs, or organisms, or into corresponding bodily fluids, tissues, organs, or organisms of the recipient. The term transplant also includes the transferred cell(s), tissue(s), or organ(s).

[0307] In a more preferred embodiment of item (i) of the above preferred embodiments, the implant is a skin graft.

[0308] In another more preferred embodiment of item (i) of the above preferred embodiments, the transplant is a replacement cell, preferably a replacement cell for damaged and / or diseased cells.

[0309] As used herein, the term "replacement cells" refers to cells applied in "cell replacement therapy" whereby the originally existing cells are replaced with alternative, ie, replacement, cells.

[0310] As used herein, term " organoid " refers to the in vitro three-dimensional proliferation of specific type of cell(s), which maintains the characteristics of tissue(s) and / or organ(s) that are composed of specific type of cells in vivo.In other words, organoid comprises the same specific cell type(s) that are found in natural tissue and / or organ, with the difference that organoid cell is grown in vitro.Organoid is used in research settings, preferably in drug screening, toxicity assay and regenerative medicine, instead of natural tissue(s) and / or organ(s).

[0311] As used herein, the term "drug screening" refers to the identification of compounds or drugs that are useful as treatments for pathological conditions of specific cells, tissues, or organs. Differentiated cells, tissues, and / or organs can be used to artificially induce pathological conditions or conditions that can be treated by potential drugs or compounds, thereby determining the effectiveness of potential new drugs or compounds.

[0312] The term " toxicity assay " refers to the assay commonly used in the art to test the effect of a compound by incubating test samples, such as cells, tissues, organisms, with the compound and determining the survival and viability of the sample.See, for example, adherent cell differentiation and cytotoxicity assay (ACDC assay) (see Barrier et al., (2010) Evaluation of a mouse embryonic stem cell adherent cell differentiation and cytotoxicity (ACDC) assay. The Toxicologist 114: 358-9).

[0313] The term "regenerative medicine" or "regenerative therapy" refers to promoting the regenerative capacity of cells, tissues, and / or organs. Regenerative medicine encompasses cell and / or tissue genetic engineering to replace, genetically engineer, or regenerate cells, tissues, and / or organs and / or to repair or improve one or more biological functions of dysfunctional or impaired cells, tissues, and / or organs; as well as tissue genetic engineering and organ regeneration.

[0314] As used herein, "regenerative capacity" refers to the conversion of cells, e.g., adult somatic cells, into dividing progenitor cells (cells that can differentiate into specific cell types) and differentiated tissue-specific cells. Regenerative capacity may also or alternatively refer to the ability of cells, tissues, and / or organs to reproduce, grow, restore, and / or regenerate function.

[0315] In a preferred embodiment of the use with cultured meat as defined in item (iii) of the above preferred embodiment of the seventeenth aspect, the cultured meat is a food product for animal or human food consumption.

[0316] In a more preferred embodiment of the use of the seventeenth aspect of the invention, the cultured meat is cultured beef and the differentiated cells are derived from bovine induced pluripotent cells, naive pluripotent cells or rejuvenated cells, tissues or organs.

[0317] In another more preferred embodiment of the seventeenth aspect of the invention, the cultured meat is cultured pork and the differentiated cells are derived from porcine induced pluripotent cells, naive pluripotent cells, or rejuvenated cells, tissues, or organs.

[0318] As used herein, the term "cultured meat" with respect to the present invention refers to cultured meat that is not grown as a natural component of a live animal; in other words, "cultured meat" is not obtained directly from the slaughter of a live animal.

[0319] As used herein, the term "food product" in relation to the present invention refers to a food supplement and / or composition that is safe for human or animal consumption.

[0320] In a more preferred embodiment of the use according to the seventeenth aspect, the production of the differentiated cell(s), tissue(s) or organ(s) is preferably an in vitro production.

[0321] In an eighteenth aspect, the present invention relates to the use of high-grade naive pluripotent cell(s) obtained by the method of the fourteenth aspect of the invention for producing embryo(s) or animal(s), preferably chimeric embryo(s) or chimeric animal(s).

[0322] In a preferred embodiment of the eighteenth aspect of the invention, the embryo is an "early embryo." The term "early embryo," as used herein, refers to an embryo at the zygote, cleavage, morula, or blastocyst stage.

[0323] In a nineteenth aspect, the present invention relates to a method of inducing high-grade naive pluripotency in embryo(s) and thereby enhancing the viability and / or developmental competence of said embryo(s), said method comprising increasing in said embryo(s) the level(s) of: a) a SoxB1 factor variant of the first aspect of the invention or a Sox17 factor variant of the second aspect of the invention, and optionally a Klf family member and / or a POU factor; and / or b) a fusion protein of the third aspect of the invention or a complex or composition of the fourth aspect of the invention, and optionally a Klf family member.

[0324] Induction of pluripotency according to a nineteenth aspect of the present invention comprises increasing the levels of a SoxB1 factor variant, a Sox17 factor variant, optionally a Klf family member and / or a POU factor as defined in item a), and / or a compound of the fusion protein, complex or composition as defined in item b), and optionally a Klf family member and / or a POU factor. According to the nineteenth aspect of the present invention, increasing the levels can be carried out by contacting an embryo with the respective factor, fusion protein and / or complex or composition as defined in items a) and / or b), allowing the respective factor, fusion protein and / or complex or composition to be incorporated into the embryo, and by transfecting or transducing a nucleic acid encoding the factor, fusion protein and / or complex and co-expressing the factor, fusion protein and / or complex or composition.

[0325] In a preferred embodiment, the Klf family member is Klf4.

[0326] In another preferred embodiment, the embryo is a non-human embryo.

[0327] In a further preferred embodiment of the SoxB1 factor variant as defined in item a) of the nineteenth aspect of the present invention, the method comprises the step of (co)expressing in an embryo: (i) a SoxB1 factor variant and Klf4 sharing an amino acid sequence comprising or consisting of SEQ ID NO: 13 or 14; or (ii) a SoxB1 factor variant, Klf4 and c-Myc sharing an amino acid sequence comprising or consisting of SEQ ID NO: 13 or 14; or (iii) a SoxB1 factor variant, Klf4 and Oct4 comprising or consisting of the amino acid sequence SEQ ID NO: 13 or 14.

[0328] In a preferred embodiment of the SoxB1 factor variant and Klf family member defined in item a) of the eleventh aspect of the present invention, the method comprises the step of co-expressing in a pluripotent cell: (i) a SoxB1 factor variant and Klf4 sharing an amino acid sequence comprising or consisting of SEQ ID NO: 13 or 14; or (ii) a SoxB1 factor variant, Klf4 and c-Myc sharing an amino acid sequence comprising or consisting of SEQ ID NO: 13 or 14; or (iii) a SoxB1 factor variant, Klf4 and Oct4 comprising or consisting of the amino acid sequence SEQ ID NO: 13 or 14.

[0329] In another preferred embodiment of the nineteenth aspect, the method refers to inducing pluripotency in totipotent cells of mammalian embryo(s) into high-grade naive pluripotent cells of the embryo(s). In other words, high-grade naive pluripotency is induced in the embryo(s) to assist the natural process of inducing naive pluripotency, thereby compensating for the low natural Sox2 expression of the natural embryo(s).

[0330] The viability of the embryo(s) according to the nineteenth aspect can be determined by those skilled in the art by methods routinely used in the art, such as non-invasive measurement of amino acid turnover, assessing the depletion / appearance of amino acids in the culture medium: turnover of three amino acids, Asn, Gly and Leu, has been significantly correlated with clinical pregnancy and live birth (see Brison DR, et al. Identification of viable embryos in IVF by non-invasive measurement of amino acid turnover. Hum Reprod. 2004 Oct;19(10):2319-24. doi: 10.1093 / humrep / deh409. Epub 2004 Aug 6. PMID: 15298971).

[0331] In a twentieth aspect, the present invention provides a method for developing a culture medium for culturing or inducing naive pluripotent stem cells. a) a variant SoxB1 factor according to the first aspect of the invention; and / or b) a Sox17 factor variant according to the second aspect of the invention; and / or c) a fusion protein according to the third aspect of the invention; and / or d) a complex or composition according to the fourth aspect of the invention; and / or e) a nucleic acid molecule or combination of nucleic acid molecules according to the fifth aspect of the invention; and / or f) a vector or vector combination according to the sixth aspect of the invention; and / or g) Cells of the seventh aspect and / or cells of the fifteenth aspect Regarding the use of.

[0332] In a twenty-first aspect, the present invention provides a method for the development of differentiation media for inducing germline and other cell types and tissues. a) a variant SoxB1 factor according to the first aspect of the invention; and / or b) a Sox17 factor variant according to the second aspect of the invention; and / or c) a fusion protein according to the third aspect of the invention; and / or d) a complex or composition according to the fourth aspect of the invention; and / or e) a nucleic acid molecule or combination of nucleic acid molecules according to the fifth aspect of the invention; and / or f) a vector or vector combination according to the sixth aspect of the invention; and / or g) Cells of the seventh aspect and / or cells of the fifteenth aspect Regarding the use of.

[0333] In a preferred embodiment of the twentieth or twenty-first aspect, one or more additional reprogramming factors can be further used to develop a medium for culturing or inducing naive pluripotent stem cells or a differentiation medium. The reprogramming factors are described above.

[0334] In a twenty-second aspect, the present invention relates to a germline differentiation medium comprising cells of the seventh and / or fifteenth aspect and a molecule that blocks DNA binding or expression of several Oct family members.

[0335] In a preferred embodiment, some Oct family members may be selected from the group of Oct2 and Oct4.

[0336] In a more preferred embodiment of the above-mentioned preferred embodiment, the culture medium for the naive set further comprises a nucleic acid encoding an Oct family member monomer blocker selected from the group consisting of siRNA, shRNA, miRNA, and antisense nucleic acid molecules; an Oct family member aptamer or ribozyme; a chemical inhibitor of an Oct family member; an antibody or antibody mimetic; an anti-Oct family member antagonist antibody or a decoy nucleic acid comprising the consensus sequence of the antibody and an Oct family member responsive factor. The antibody mimetic is selected from the group consisting of affibodies, adnectins, anticalins, DARPins, avimers, nanophytins, affilins, Kunitz domain peptides, Fynomers®, trispecific binding molecules, and probodies. These polypeptides are described above.

[0337] In a twenty-third aspect, the present invention relates to a method of developing or optimizing a culture medium, comprising testing whether a cell of the seventh aspect and / or a cell of the fifteenth aspect retains at least one marker of its phenotype or genotype over at least one cell cycle.

[0338] Alternatively, the test is carried out over at least one passage.

[0339] In a preferred embodiment of the twenty-third aspect, the developed or optimized medium is produced. Production may follow conventional methods of medium production.

[0340] In a twenty-fourth aspect, the present invention relates to a method of developing or optimizing a differentiation medium, preferably a germline differentiation medium, comprising testing whether a cell of the seventh aspect or a cell of the fifteenth aspect displays at least one marker representative of a germline or a cell type further differentiated therefrom.

[0341] In a preferred embodiment of the twenty-fourth aspect, the developed or optimized medium is produced. Production may follow conventional methods of medium production.

[0342] In a preferred embodiment of the twenty-third aspect, the at least one cell cycle or passage is at least 4, 5, 6, 7, 8, 9 or 10 cell cycles or passages.

[0343] In a preferred embodiment of the twenty-third or twenty-fourth aspect, the at least one marker representative of the induced pluripotent stem cells of the fifteenth aspect is: (i) expression of at least one, preferably at least two, more preferably at least three pluripotency-specific marker(s) selected from Oct4, Sox2, Nanog, and Klf4; and / or (ii) the ability to differentiate into each of the three primitive germ layers is selected from.

[0344] In another preferred embodiment of the twenty-third or twenty-fourth aspect, at least one marker representative of the high-grade naive pluripotent cells of the fifteenth aspect compared to a corresponding non-naive pluripotent cell or a low-grade naive pluripotent cell: (i) Enhanced generative capacity; (ii) higher expression levels 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 six naive pluripotency-specific marker(s) selected from Klf17, Klf4, Sox2, Susd2, Argfx, and Dnmt31; (iii) higher expression levels of at least one primitive endoderm-specific marker(s) selected from Gata6 and Sox17; (iv) activated POU5f1 distal enhancer; (v) a reactivated X chromosome in the female lineage; (vi) reduced DNA methylation; (vii) enhanced ability to differentiate, preferably into germ cells; (viii) an enhanced ability to contribute to the development of embryo(s) and / or animal(s); and / or (ix) A combination of two or more of (i) to (viii); is selected from.

[0345] In a preferred embodiment of the twenty-third or twenty-fourth aspect, retaining at least one marker of the phenotype of a cell of the seventh aspect comprises enhanced developmental potential, higher expression levels 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 naive pluripotency specific marker(s) selected from Klf17, Klf4, Sox2, Susd2, Argfx and Dnmt3l, Gata6 and Sox17. the cells of origin carrying the first aspect SoxB1 factor variant or the second aspect Sox17 factor variant retain at least one of the following: a higher expression level of at least one selected primitive endoderm-specific marker(s), an activated POU5f1 distal enhancer, a reactivated X chromosome in the female lineage, reduced DNA methylation, an enhanced ability to differentiate preferably into germ cells, and / or an enhanced ability to contribute to the development of embryo(s) and / or animal(s) of the phenotype of the original cells carrying the first aspect SoxB1 factor variant or the second aspect Sox17 factor variant.

[0346] In a twenty-fifth aspect, the invention relates to a cell culture medium made or preparable by the method of the twenty-third aspect.

[0347] In a twenty-sixth aspect, the invention relates to a germline differentiation medium produced or producible by the method of the twenty-fourth aspect.

[0348] The present invention also relates to methods for developing or optimizing naive stem cell induction or maintenance media, comprising assessing the levels and activity of Sox2 / Oct4 heterodimers, which can be done by conventional methods such as EMSA, Western blot and ATAC-seq.

[0349] The present invention also relates to a method for developing or optimizing a differentiation medium (e.g. for germline differentiation) comprising treating cells to be differentiated with a SoxB1 factor variant of the present invention or a cocktail composed of a SoxB1 factor or SoxB1 factor variant, a Klf factor, preferably KLF4, and optionally one or more further factors.

[0350] With respect to the embodiments characterized in this specification, particularly in the claims, singular terms are intended to be plural unless the context clearly dictates otherwise. For example, a cell also means a plurality of cells and is interchangeable with the term "cell(s)."

[0351] With respect to the aspects characterized in this specification, and particularly in the claims, each aspect recited in a dependent claim is intended to be combined with each aspect of each claim (independent or dependent) from which the dependent claim depends. For example, in the case of independent claim 1 reciting three alternatives A, B and C, dependent claim 2 reciting three alternatives D, E and F, and claim 3 dependent on claims 1 and 2 and reciting three alternatives G, H and I, unless expressly stated otherwise, it will be understood that the specification expressly discloses embodiments corresponding to the combinations A,D,G;A,D,H;A,D,I;A,E,G;A,E,H;A,E,I;A,F,G;A,F,H;A,F,I;B,D,G;B,D,H;B,D,I;B,E,G;B,E,H;B,E,I;B,F,G;B,F,H;B,F,I;C,D,G;C,D,H;C,D,I;C,E,G;C,E,H;C,E,I;C,F,G;C,F,H;C,F,I.

[0352] Similarly, and even when an independent and / or dependent claim does not cite alternatives, if a dependent claim refers back to more than one preceding claim, it is understood that any combination of the subject matter covered therein is considered to be clearly disclosed. For example, in the case of independent claim 1, dependent claim 2 which refers back to claim 1, and dependent claim 3 which refers back to both claims 2 and 1, the combination of the subject matter of claims 3 and 1 is understood to be clearly and unambiguously disclosed, as is the combination of the subject matter of claims 3, 2, and 1. If there is a further dependent claim 4 which cites any of claims 1-3, then the combinations of the subject matter of claims 4 and 1, claims 4, 2, and 1, claims 4, 3, and 1, and claims 4, 3, 2, and 1 are understood to be clearly and unambiguously disclosed. [Brief explanation of the drawings]

[0353] The drawings show: [Figure 1]Figure 1 | Reprogramming screen of the Sox2-Sox17 chimeric TF library. a, Bright-field and Oct4-GFP merged overview image (scale = 1 mm) showing retroviral reprogramming of MEFs harboring the Oct4-GFP (OG2) reporter on 21 dpi. b, Schematic representation of the Sox2 and Sox17 structures and chimeric transcription factors (TFs) created by exchanging non-conserved residues from Sox17 with Sox2. Sox2-derived sequences are in blue, and Sox17-derived sequences are in red. c, Protein sequence alignment of the DNA-binding domains of mouse and human Sox2, Sox17, and the most definitive chimeric Sox factor from this study. d-g, Reprogramming of Oct4-GFP MEFs with Klf4, a Sox2-Sox17 chimeric TF, and retroviral vectors carrying wild-type Oct4 (d), the Oct4L80A linker mutant (e-f), or Brn4 (g). Error bars represent SD; n = 3. Statistical significance was calculated by Student's t-test. h, Representative phase-contrast and Oct4-GFP merged microscopy images (scale = 200 μm) of primary iPSC colonies generated by retroviral vectors carrying different POU factors combined with Sox2AV and Klf4. i, Cell proliferation assay in which 2 × 10 MEFs were transduced with the indicated tet-inducible polycistronic constructs in a 96-well plate. Error bars indicate SD; n = 3. Cells were counted after 2, 4, and 6 dpi. Statistical significance was calculated by Student's t-test, comparing 61V with 61A for each construct. [Figure 2]Figure 2 | Molecular dynamics simulations reveal the SL configuration of Sox / Oct4. a-b, Models of the Sox2 / Oct4 vs. Sox2AV / Oct4 heterodimer (a) and the Sox2 / Oct6 vs. Sox2AV / Oct6 heterodimer (b) in the POUS (S) configuration on the HoxB1 DNA motif. Only the DNA-binding domains are shown. Oct4 / Oct6 are in yellow, and Sox2 / Sox2AV are in blue. c-d, Computational molecular dynamics simulations (MDS) of the Sox / Oct heterodimer on the HoxB1 motif. The plots show the coordination number (number of contacts) between residue 61 in Sox2 (blue) or Sox2A61V (red) with either the pre-DNA-binding domain of the Oct4 (dark) or Oct6 (light) molecule (c) or residue 21I in Oct (d). Calculations were performed during 4.8 μs of MDS for each ternary Sox / Oct / DNA complex. Four independent 1.2 μs-long simulations were performed using two different starting structure models (two simulations per model). To ensure randomness, each simulation was initiated using a different distribution of atomic velocities. The distance threshold for two-atom contact was 4.5 Å. (e) Model of Sox2AV / Oct4 binding in the POUS+linker (SL) configuration, where V61 interacts with the helical core of POUS at residue G24, and two salt bridges are formed between E78 and E82 of the Oct4 linker and between K57 and R50 of the Sox2 HMG, respectively. (f) Model of Sox2 / Oct4 binding in the distant POUS (DS) configuration on the Fgf4 motif; residue 61 of Sox2 is not included. [Figure 3]Figure 3 | Enhanced Sox / Oct cooperativity rescues nonfunctional POU factors during reprogramming to pluripotency. a–c, OSK reprogramming of Oct4-GFP MEFs using monocistronic retroviral vectors harboring Oct4 domain deletion mutants combined with wild-type Sox2 or Sox2-Sox17 chimeric factors, in which the linker domain was replaced with synthetic poly-glycine linkers (GL) of different lengths (3–30 residues) (a), the N- or C-terminal transactivator domain (NTD or CTD) of Oct4 was deleted (b), and the POUS or POUHD (excluding the NLS) was deleted (c). Error bars represent SD; n = 3. Statistical significance was calculated by Student's t-test. d, Western blot of whole cell lysates from HEK293 was used in Figure 3e. e, Electrophoretic mobility shift assay (EMSA) of whole-cell lysates of HEK293 cells transfected with Sox2 (S, blue), Sox2AV (SAV, bright red), and wild-type or mutant Oct4 with deleted POUS or POUHD domains on the Nanog promoter and HoxB1 enhancer SoxOct DNA elements labeled with Cy5. White arrowheads indicate nonspecific bands (ns), and black arrowheads indicate free DNA or DNA bound to Oct4, Sox2, or the heterodimer. f, Representative kinetic off-rate EMSA of whole-cell lysates from HEK293 cells overexpressing Oct4, Sox2 (blue), Sox2A61V (bright red), or Sox243-47,61,65-86c17 (Sox2-17, S*, dark red) on the Oct4 distal enhancer (Oct4DE), Nanog promoter, or Cy5-labeled Fgf4 enhancer DNA elements. White arrowheads indicate nonspecific bands (ns), and black arrowheads indicate free DNA or DNA bound to Oct4 (O / DNA), Sox (S / DNA), or Oct4 / Sox heterodimers (O / S / DNA). Error bars on the graph indicate SD; n = 3. g, Representative kinetic off-rate EMSA using purified proteins bound to Cy5-labeled Utf1 enhancer and Nanog promoter loci.After the binding reaction, excess unlabeled Nanog factor was added to determine half-lives at the indicated times. Error bars on the quantification graphs indicate SD, n=3. Ternary complex half-life t1 / 2. h, Schematic diagram of the enabling effect of highly cooperative Sox2AV mutants or non-enabling Oct4 mutants on reprogramming with tissue-specific POU factors. [Figure 4]Figure 4 | Highly cooperative Sox2AV improves the developmental competence of mouse OSKM iPSCs. a, Kinetic off-rate EMSA of purified wild-type Sox2 or Sox2AV in complex with Oct4 bound to the Widom 601 nucleosomal sequence, which matches the SoxOct motif at supercoil position (SHL) +6. After the binding reaction, ternary complex stability was determined at the indicated times by adding excess unlabeled Nanog factor. Black arrowheads mark free nucleosomes or the indicated nucleosome-protein complexes. b, Heatmap and read pileup plots of Sox2 and Oct4 ChIP-seq for 2 dpi tetO-OKS MEF reprogramming samples comparing Sox2AV vs. wild-type Sox2. c, Boxplots of quantile-normalized ChIP-seq peaks for Oct4 and Sox2 for 2 dpi OKS and KS reprogramming samples. The midline indicates the median, boxes indicate upper and lower quartiles, and whiskers indicate the 1.5-fold interquartile range. d, Fraction of binding sites containing both SoxOct, MORE, and motifs or excluding the motif among 2-dpi OKS reprogramming samples, where O is either Oct4 or Oct6, and S is either Sox2 or Sox2AV. e, Genome browser track of Oct4 and Sox2 ChIP-seq peaks for selected pluripotency-specific loci. f, Venn diagram showing several ESC-specific enhancers (Shen et al., 2012) bound by Sox2 versus Sox2AV on 2-dpi tetO-OKS reprogramming samples. g, Percentage of tetraploid (4N) aggregate embryos derived from the indicated tet-inducible or non-integrated episomal iPSCs that gave rise to full-term pups, pups that started breathing, pups that survived for at least 48 h during foster-nursing, and pups that survived to adulthood (at least 3 months). Bars represent the mean across all tested lines for each cocktail. Error bars represent SEM. Statistical significance was determined by the Mann-Whitney test. h, Adult tetO-OSAVKM all-iPSC mice (9 months).i, PCR genotyping of tetO-OSAVKM all-iPSC mouse offspring. [Figure 5]Figure 5 | Sox2-17 promotes reprogramming in five species. a, Schematic representation of a time-course Tet-induced lentiviral reprogramming experiment. b, Time-course programming of Oct4-GFP MEFs induced in OSKM or SKM with either Sox2 (S) or Sox2-17 (S*) for the indicated number of days; iPSC colonies were counted at 9 dpi. c, Representative bright-field and Oct4-GFP merged overview images of MEFs induced in OSKM or OS*KM for 4 days. Images were taken at 9 dpi; scale = 2 mm. d, Reprogramming of Oct4-GFP MEFs with episomal OKS (pCXLE-Oct4-P2A-Klf4-IRES-Sox) carrying either wild-type Sox2 or Sox2-17. 1.5x105 cells were transfected with Fugene6. e, Whole iPSC pups generated by a tetraploid (4N) complementation assay using epi-OKS* iPSC#1. Twenty aggregates were transplanted into two pseudopregnant CD-1 (white) females. f, The percentage of 4N aggregate embryos that gave rise to healthy adult mice (survived to at least 3 months) is based on previously published results (Velychko et al., 2019a). Only XY lineages are plotted. Data are expressed as the mean of all tested lineages. Error bars represent SEM. g, Reprogramming of human fetal fibroblasts (CRL-2097) with monocistronic retroviral OSKM harboring either wild-type Sox2 or Sox2 (SOX2-17, S*). The volume of viral supernatant was adjusted according to qPCR titration. 10 transduced cells were plated onto a feeder layer in a 6-well plate. TRA1-60+ colonies were counted 2 weeks after infection. Error bars represent SD; n = 3. h, Representative whole-well scan of (g). i, Two-factor (OS) reprogramming of human fibroblasts with monocistronic retroviral vectors. TRA1-60+ colonies were counted 4 weeks after infection. Error bars represent SD; n = 3. j, Representative whole-well scan of (i).k, TRA1-60 staining of human fetal (CRL-2097) fibroblasts reprogrammed with a self-replicating RNA (VEE) vector carrying either the OKSiG or OKS*iG reprogramming cassette. l–o, Whole-well scan of alkaline phosphatase (AP) staining for episomal reprogramming of (l) 25-day-old human male skin fibroblasts, (m) 25-day-old cynomolgus monkey (Macaca fascicularis) fibroblasts, (n) 21-day-old bovine fetal fibroblasts, and (o) 21-day-old porcine fetal fibroblasts after nucleofection. p-q, Hierarchical clustering analysis of integration-free iPSC lines derived from human ESCs and human neonatal foreskin (young, Y) or 56-year-old human skin (old, O) fibroblasts using episomal OSKML (pCXLE-OCT4+shP53, L-MYC-F2A-LIN28, and SOX-P2A-KLF4 harboring SOX2, SOXAV, or SOX2-17) based on global gene expression (RNA-seq), TPM ≥ 1 (p), or global methylome (RRBS) (q). Clustering was based on Euclidean distance. r, Comparison of the number of genes that lost imprinting in 31 DMRs using RRBS data. s, Model for the beneficial effect of highly cooperative Sox factors on cell fate resetting. [Figure 6]Figure 6 | Sox / Oct cooperativity in high vs. low-grade pluripotency. a, Spearman correlation of time-course ATAC-seq reads for naive-to-induced differentiated mouse ESC samples (Yang et al., 2019). b-c, TOBIAS footprinting analysis of (a) using the MEME TF motif database for ESC vs. day 1 EpiLC samples (b) and day 1 vs. day 2 EpiLC samples (c). d, EMSA of whole-cell lysates showing endogenous levels of Sox2 and Oct4 that can bind to Cy5-labeled Nanog promoter DNA elements. Arrowheads indicate DNA bound to Oct4 (yellow), Sox2 (blue), or the heterodimer (green). e, Western blot of the lysates used in Figure 6d. f, Supershift assay using anti-Oct4 and anti-Sox2 antibodies to confirm the identity of protein / DNA complexes in the whole-cell lysate EMSA. g, Endogenous EMSA of six PSC lines (Velychko et al., 2019a), including epi-OKSM1 and 3, which failed to generate whole iPSC mice. Lines were grown in 2iLIF on gelatin-coated plates and then split into either KSR-LIF medium (left panel) or 2iLIF medium again (right panel). h, Western blot of the lysate used in Figure 6g. i, Immunostaining for the naive pluripotency marker KLF17 of human iPSCs converted from induced to naive 6 days after transduction with a constitutive lentiviral vector (pHAGE2-EF1α). j, Endogenous EMSA of integration-free clonal mEpiSC lines converted from induced to naive using episomal mCherry-T2A-SOX-KLF4 vectors carrying wild-type SOX2, SOX2AV, or SOX2-17. Error bars represent SEM, and statistical significance was calculated by Student's t test between the top three of six strains transformed by each cocktail. k, SoxOct model of high-grade developmental resetting. [Figure 7]Figure 7 (related to Figure 1). a, OSK reprogramming of Oct4-GFP reporter MEFs with retroviral monocistronic Sox2 or Sox17EK in combination with Klf4 and Oct4L80A linker mutants. Error bars represent SD; n = 3. Statistical significance was calculated using Student's t-test. b-c, qPCR titration of retroviral vectors from Figure 1d-g. [Figure 8]Figure 8 (related to Figure 3). a, Western blot of whole-cell lysates from HEK293 cells overexpressing the flagged POU factors used in (b). b, EMSA of whole-cell lysates from (a) on the Cy5-labeled Nanog promoter locus. White arrowheads indicate nonspecific bands (ns), and black arrowheads indicate free DNA or DNA bound to Oct (O / DNA), Sox (S / DNA), or both (O / S / DNA). c-d, Representative bright-field and Oct4-GFP merged overview images showing MEFs reprogrammed with Oct4 mutants lacking the C- (c, ΔCTD) or N- (d, ΔNTD) terminal transactivator domains, 21 dpi, scale = 1 mm. e, Primary iPSC colonies generated with Oct4 mutants in which the POUHD domain (but not the NLS) was deleted, scale = 100 μm. f, PCR genotyping confirms the identity of two Oct4ΔPOUHD / Sox2AV / Klf4 iPSC lines. g, PCR genotyping of chimeric mice generated by aggregation of Oct4ΔPOUHD / Sox2AV / K-derived iPSCs. h, Bright-field and Oct4-GFP merged images of gonad-dissected 13.5-day-old embryos from the chimeric embryos derived from (g). i, Coomassie-stained SDS-polyacrylamide gel of mouse Sox2, Sox2AV, and Oct4 derived from the insect cells used in Figure 3g and Figure S2j. j, EMSA of insect cell-purified Sox2 (S, blue), Sox2AV (SAV, bright red), and wild-type Oct4 on Cy5-labeled Nanog promoter, Utf1, and Fgf4 enhancer DNA elements. Arrowheads indicate free DNA or DNA bound to Oct4 (O / DNA), Sox2 (S / DNA), or heterodimers (O / S / DNA). k, Representative kinetic off-rate EMSA using whole-cell lysates overexpressing full-length Oct4, Oct4L80A, Oct4GL19, or Brn4 combined with Cy5-labeled Sox2 vs. Sox2AV lysates on the Nanog promoter locus. After the binding reaction, half-lives were determined by adding excess unlabeled Nanog elements for the indicated times. White arrowheads indicate nonspecific bands (ns), and black arrowheads indicate free DNA or DNA bound to the POU / Sox heterodimer. [Figure 9] Figure 9 (related to Figure 4). a-b, HOMER (Heinz et al., 2010) de novo (a) and known SoxOct (b) motif analysis showing enrichment P values ​​for Oct4, Oct6, and Sox2 ChIP-seq. c, Sox2 and Oct4 ChIP-seq signal heatmap for MEFs reprogrammed with tet-inducible OKS at 2 dpi at loci containing Sox2 / Oct4 footprints in open chromatin of ESCs vs. MEFs as determined by TOBIAS footprint analysis of ATAC-seq data (Li et al., 2017). [Figure 10]Figure 10 (related to Figure 5). a, qPCR titration of tet-inducible lentiviral vectors from Figure 5b-c 24 hours after induction. Error bars represent SD; n = 3. b, Bisulfite sequencing analysis of DNA methylation at Oct4, Nanog, and Col1a1 promoters in MEFs and iPSC lines generated by 24-hour induction of tetO-OS*KM. c, H&E staining of a teratoma section generated by 24-hour OS*KM iPSCs, showing three germ layers (ectoderm—Ec: keratinized epithelium; mesoderm—M: striated muscle; endoderm—En: cuboidal epithelium). d, Bright-field and Oct4-GFP merged image of the gonad from an E13.5 24-hour OS*KM iPSC chimeric embryo. e, Whole iPSC pups generated by a tetraploid (4N) complementation assay using the 24-hour OS*KM iPSC#1 line. Twelve aggregates were transferred into pseudopregnant CD-1 (white) females. f, Representative bright-field and Oct4-GFP merged overview image showing Oct4-GFP MEFs reprogrammed with the tet-inducible lentiviral Sox-T2A-Klf4 vector harboring wild-type Sox2, Sox2c17, Sox17EK, or Sox2-17, 21 dpi, scale = 1 mm. g, Phase-contrast and Oct4-GFP merged microscopy image of the two-factor mouse S*K iPSC clonal line generated in (f) over three passages, scale = 100 μm. h, PCR genotyping of two mouse S*K iPSC lines derived from (f–g). i, Immunostaining of mouse S*K iPSC lines for the pluripotency markers Nanog and SSEA-1. Nuclei were stained with Hoechst 33342, scale = 100 μm. j, H&E staining of teratoma sections generated using the S*K mouse iPSC line, which displays three germ layers (ectoderm-Ec: keratinized epithelium; mesoderm-M: striated and smooth muscle; endoderm-En: cuboidal epithelium). k, Western blot of whole-cell lysates from HEK293T cells transfected with pCXLE-Oct4-P2A-Sox-T2A-Klf4-E2A-cMyc, pCXLE-Oct4-P2A-Klf4-IRES-Sox episomal vectors harboring mouse Sox2, Sox2AV, or Sox2-17.l, Phase contrast microscopy image of a two-factor human iPSC line generated using the monocistronic retroviruses OCT4 and SOX2-17 at two passages, scale = 200 μm. m, Immunostaining of a human OS* iPSC line for pluripotency markers NANOG and TRA1-81. Nuclei were stained with Hoechst 33342, scale = 100 μm. n, H&E staining of a teratoma section generated using an OS* human iPSC line with representation of three germ layers (ectoderm - Ec: neural rosette; mesoderm - M: cartilage, bone, endothelium; endoderm - En: intestinal and lung epithelium). o, Western blot of whole-cell lysates from HEK293T cells transfected with the original episomal pCXLE-SOX2-F2A-KLF4 construct and generated in this study in the P2A vectors: pCXLE-SOX2-P2A-KLF4, pCXLE-SOX2AV-P2A-KLF4, and pCXLE-SOX2-17-P2A-KLF4. p, PCR genotyping of episomal iPSC lines generated from cynomolgus fibroblasts at passage 3. q, Chromosome spread of two integration-free cynomolgus iPSC lines. r, Phase-contrast image of integration-free cynomolgus iPSC #11 from panel o at passage 7; scale = 200 μm. s, Immunostaining of cynomolgus integration-free iPSC lines for NANOG and OCT4. Nuclei were stained with Hoechst 33342; scale = 100 μm. t, H&E staining of a teratoma section generated by a cynomolgus monkey integration-free iPSC line with representation of three germ layers (ectoderm - Ec: neural rosettes; mesoderm - M: cartilage, smooth muscle; endoderm - En: cuboidal epithelium). u, Representative whole-well scan of alkaline phosphatase (AP) staining for episomal reprogramming of bovine fetal fibroblasts 21 days after nucleofection with episomal OSKML (omitting P53 knockdown). v, PCR genotyping of an episomal OSKML bovine iPSC line derived from (u) at passage 6. w, Phase contrast image of an integration-free bovine iPSC line derived from (v) at passage 8, scale = 200 μm. x, Representative chromosome spread of an integration-free bovine iPSC line derived from (v). y, Immunostaining of a bovine integration-free iPSC line for Sox2 and OCT4.Nuclei were stained with DAPI; scale = 200 μm. z, Representative image of episomal S*K-mediated biPSC resetting 6 days after nucleofection; scale = 200 μm. Cells were plated on feeders in KSL-LIF+XAV939 medium. aa, Teratomas generated by subcutaneous injection of S*K-reset biPSC lines into SCID mice, 5 weeks after injection. Untransfected biPSCs did not generate teratomas in three injection attempts. ab, H&E staining of teratoma sections generated by biPSCs (aa), showing representation of three germ layers (ectoderm—Ec: neural rosettes, epithelium, quamous epithelial cells; mesoderm—M: smooth muscle, connective tissue; endoderm—En: intestinal epithelium). ac, PCR genotyping of episomal iPSC lines generated from aged female skin fibroblasts (AG04148) at passage 3. ad–ae, karyotyping of human integration-free iPSC lines generated from neonatal foreskin fibroblasts (young, Y) or 56-year-old male fibroblasts (old, O) using chromosome spread (ad) or e-karyotyping based on RNA-seq data (ae). [Figure 11]Figure 11 (related to Figure 6). a) FACS of mEpiSCs transfected with the episomal mCherry-T2A-SOX-KLF4 vector using Lipofectamin Stem reagent on day 2. b) Representative phase-contrast / epi-mCherry / Oct4-GFP merged overview image of mEpiSCs transformed from clonally induced to naive cells using the episomal mCherry-T2A-SOX-P2A-KLF4 vector grown in KSR-LIF medium on a C3H feeder layer for four passages (scale = 500 μm). Equal numbers of cells were plated for each line. At least 100 colonies were quantified from three random overview images for each line. Error bars represent SEM. Statistical significance was calculated using a Student's t-test. c) Western blot of the lysates used in Figure 6j. d, Quantification of Gof18+ and Gof18- colonies generated by mEpiSC reset using episomal mCherry-SK or -S*K vectors at passage 4, grown in KSR-LIF medium on feeders. Each point represents the average percentage of Gof18+ colonies for one of six randomly selected lines for each construct. The same number of cells was plated for each line. >100 colonies were quantified from three randomized overview images for each line. Error bars represent SEM, and statistical significance was calculated using a Student's t-test. e, Method for human integration-free naive reset using episomal mCherry-S*K. A representative image of day 7 S*K-reset hiPSCs (female episomal line A18945, Gibco) stained for the human naive marker SUSD2 is shown on the right. RSeT medium and 5% CO2 were used starting from day 2. Scale = 50 μm. No SUSD2+ cells were detected in the control nucleofection. f, Representative image of day 7 S*K-reset hiPSCs (A18945, Gibco) stained for the human naive marker KLF17, scale = 50 μm. The majority of transformed cells were mCherry negative. No KLF17+ cells were detected in the control nucleofection.Episomal resetting of two hPSC lines (OS*KML O-hiPSC#1 and H9 hESC) showed identical results. g, Representative image of day 6 S*K-reset hiPSCs (A18945, Gibco) stained for human naive markers SUSD2 and KLF17; scale = 50 μm. Cells were grown in induction medium, and KLF17+ or SUSD2+ cells were detected in control nucleofections. Results were reproduced for two other hPSC lines. h, SoxOct model of high-grade developmental resetting. i, Two-step protocol for the derivation of high-grade pluripotent cells across species. [Figure 12]Figure 12. (Related to Figures 5 and 6). Figure 12 provides further details about the results shown in Figures 10 and 11. a, FACS for the primate-specific naive marker SUSD2 of human iPSCs (A18945, Gibco) nucleofected with episomal pCXLE-mCherry-T2A-SOX-KLF4 and grown in Rset medium 7 days after nucleofection and plating on feeders (Figure 12a). Error bars represent SD; n = 3; statistical significance was calculated using Student's t-test. b, RT-qPCR gene expression analysis of bulk day 7 reset samples (whole well lysates). Naive markers were also significantly upregulated in induction medium, but more so in Rset medium. WPRE expression confirmed elimination of the episomal vector in S*K samples. Error bars represent SD; n = 3; statistical significance was calculated using Student's t test. c, Cross-species human / mouse morula aggregation using sorted day 7 SUSD2+ hiPSCs (A18945, Gibco) with constitutive RFP expression (CRISPR-mediated knock-in) and reset with pCXLE-S*K (without mCherry). The majority of the aggregated embryos contained hiPSCs localized in the ICM. Similar levels of recombination were achieved using hand-picked domed colonies without sorting. d, Immunostaining of a representative cross-species chimeric E4 embryo from Figure 11f using human-specific SUSD2 and mouse-specific Oct4 antibodies. Microscopic images show robust integration of human S*K-reset cells into the embryo's ectoderm region, while mouse cells appear to contribute exclusively to the primitive endoderm. e, Model of interspecies cell competition hypothesis based on Zheng et al., 2021. Rodent PSCs win the competition with primate PSCs, with bovine PSCs being the ultimate losers. We hypothesize that these results may be explained by the "induced-to-naive" grading by which cells of a given species are stabilized in culture. It is conceivable that S*K resetting may confer a "winner" state to PSCs across species. f, Schematic representation of a tetraploid complementation experiment to assess the possibility of enhancing the developmental potential of already naive mouse ESCs.g, Representative phase-contrast image of day 5 S*K-reset mESCs on a C57BL / 6J background. The domed, naive-like morphology was not different between pCXLE-mCherry-S*K and pCXLE-mCherry mock control samples. h, Percentage of 4N aggregate embryos derived from ESCs derived from g that gave rise to full-term pups, pups that started breathing, pups that survived for at least 48 h when nursed, and pups that survived to adulthood (at least 3 months). Bars represent the average survival of all transplanted embryos; numbers are shown at the top. Eight-fold more pups were produced from pCXLE-mCherry-S*K-nucleofected cells compared with pCXLE-mCherry controls. i, All-iPSC pups were generated by tetraploid complementation assay e using S*K-reset female mESCs. j, Schematic simplified results in e–j. DETAILED DESCRIPTION OF THE INVENTION

[0354] The present invention will now be illustrated by examples. [Example]

[0355] Example 1 - Materials and Methods mouse All mice used were bred and housed in the mouse facility of the Max Planck Institute in Münster. Animal care complied with the MPI animal care guidelines.

[0356] Vector construction The pMX-Sox2 / Sox17 chimeric transcription factor vector was based on Addgene ID 13367 ( Takahashi and Yamanaka, 2006 ), and the tet-inducible pHAGE2-tetO-Oct4-P2A-Sox2-17-T2A-Klf4-E2A-cMyc (OS*KM) and pHAGE-tetO-Sox2-17-T2A-Klf4-E2A-cMyc (S*KM) vectors were based on Addgene IDs 136551 and 136541, respectively ( Velychko et al., 2019a ).

[0357] The self-replicating RNA vector T7-VEE-OKS*iG was based on Addgene ID 58974 ( Yoshioka et al., 2013 ). Mouse episomal vectors pCXLE-Oct4-P2A-Klf4-IRES-Sox2 (OKS) and pCXLE-Oct4-P2A-Klf4-IRES-Sox2-17 (OKS*), as well as human episomal vectors pCXLE-SOX2-P2A-KLF4 (SK), pCXLE-SOX2 AV -P2A-KLF4 (S AV K), pCXLE-SOX2-17-P2A-KLF4 (S*K), and pCXLE-mCherry-E2A-SOX2-P2A-KLF4 were based on Addgene ID 27078 (Okita et al., 2013), but the incompetent self-cleavage peptide F2A was replaced with P2A to avoid protein fusion. pCXLE plasmids showed fairly good yields when grown in Stbl2-competent E. coli (Invitrogen).

[0358] Mouse and human Sox2 A61V and the protein sequence of Sox2-17, where the HMG-box domain is in uppercase and the Sox17 portion is in bold, as follows: [Table 1] [Table 2] [Table 3] [Table 4]

[0359] All relevant constructs are available on Addgene.

[0360] cell culture HEK293T cells were cultured in low-glucose DMEM (Sigma) supplemented with 10% FBS (Capricorn Scientific), 1% Glutamax, 1% penicillin-streptomycin, and 1% non-essential amino acids (all from Sigma). Mouse, human, cynomolgus monkey, and porcine fibroblasts were cultured in high-glucose DMEM (Sigma) supplemented with 15% FBS, 1% Glutamax, 1% penicillin-streptomycin, 1% non-essential amino acids (NEAA), 1% sodium pyruvate (Sigma), and 1% β-mercaptoethanol (Gibco). Bovine fibroblasts were cultured in 50:50 DMEM / F12 (Gibco) and IMDM (with HEPES, Cytiva) with 15% FBS and the same supplements. 5 ng / ml human bFGF (Peprotech) was used to enhance cynomolgus monkey, bovine, and porcine fibroblast cultures.

[0361] Mouse naive pluripotent stem cells (mESCs) were grown on mitomycin C-inactivated C3H MEF feeder layers in KSR-based mouse embryonic stem cell (mESC) medium: high-glucose DMEM supplemented with 15% KSR (Invitrogen), 1% Glutamax, 1% NEAA, 1% penicillin-streptomycin, 1% β-mercaptoethanol, and 20 ng / ml human recombinant LIF (purified in-house). For 4N-complementation experiments, KSR-LIF medium was supplemented with 2i (1 mM PD0325901 and 3 mM CHIR99021) for one passage. Mouse Gof18 GFP- E3-present ectodermal stem cells (EpiSCs) (Han et al., 2010) were cultured in Stem Flex medium (Gibco) on FBS-coated dishes.

[0362] Human pluripotent cells were cultured in either hESC medium: DMEM / F12 supplemented with 15% KSR, 1% Glutamax, 1% NEAA, 1% penicillin-streptomycin, 1% β-mercaptoethanol, and 5 ng / ml bFGF, or StemFlex medium (Gibco) on Matrigel-coated dishes (Corning) or on mitomycin C-inactivated CF1 MEF feeder layers. Cynomolgus monkey iPSCs were cultured on mitomycin C-inactivated CF1 MEF feeder layers in StemFlex medium. Bovine and porcine iPSCs were derived and cultured on mitomycin C-inactivated CF1 MEF feeder layers in StemFlex medium supplemented with 2 μM XAV939 (Sigma) at 37°C in a hypoxic 5% O2, 5% CO2 incubator; other cells were cultured under normoxic conditions. They were separated on feeder-free FBS-coated dishes for karyotyping.

[0363] Pluripotent stem cells of all five species were passaged using Accutase (Sigma). 10 μM Rho-associated kinase inhibitor (Y-27632, Abcam) was added during the first 24 h (extended to 48 h for mouse EpiSCs) after passaging of induced pluripotent cells of all five species. Cells were routinely tested for mycoplasma contamination with negative results.

[0364] iPSC generation Mouse reprogramming experiments were performed as previously described (Velychko et al., 2019a, 2019b). Briefly, for retrovirus production, monocistronic pMX-Oct4, Sox, and Klf4 vectors were cotransfected with pCL-Eco (Addgene ID 12371) (Naviaux et al., 1996) in HEK293T cells using FuGENE6 (Promega) with a low-volume transfection protocol (Steffen et al., 2017). For lentivirus production, pHAGE2-tetO vector was cotransfected with PAX2 and VSV. After 2 and 3 days, viral supernatants were harvested, filtered (Millex-HV 0.45 μm; Millipore), aliquoted, and stored at -80°C. For reprogramming, Oct4-GFP MEFs (OG2 or Rosa26TA-Gof18) were cultured at 3x10 per well in fibroblast medium. 4 Cells were plated onto gelatin-coated 12-well plates. After several hours, the cells were infected with titered volumes of each viral supernatant supplemented with 6 μg / ml (final concentration) of protamine sulfate (Sigma). Two days later, the medium was replaced with mouse ESC medium.

[0365] For human retroviral reprogramming, 48 h post-infection, transduced cells were plated at 10 per 6-well plate. 4 After one week, the fibroblast medium was replaced with hESC medium.

[0366] For mouse episomal reprogramming, 5 Oct4-GFP (Rosa26TA-Gof18) MEFs were plated overnight on gelatin-covered 6-well plates and transfected with 1.5 μg of pCXLE-OKS or OKS* combined with 0.5 μg of pCXWB-EBNA1 (Addgene ID 37624) using FuGENE6.

[0367] Human self-replicating RNA system reprogramming was performed as previously described (Yoshioka et al., 2013). Briefly, the T7-VEE construct was digested with MluI and then in vitro transcribed using the RiboMAX Large Scale RNA Production System Kit (Promega). The transcripts were 2'-O-methylated, capped, and poly(A)-tailed using the respective CELLSCRIPT kit according to the manufacturer's protocol. For reprogramming, 1 μg of RNA replicon was transfected into 10 wells on a 6-well plate using RiboJuice (Sigma) in the presence of 100 ng / ml B18R (Promega). 5 fibroblasts were transfected with TRA-1-60. The medium was supplemented with 0.5 mM VPA and 5 μM EPZ to promote highly incompetent RNA-based reprogramming. Reprogramming was more efficient when puromycin selection was not used. After two weeks, cells were preserved on TRA-1-60 and plated on CF1 feeder layers in human ESC medium without B18R.

[0368] Human and cynomolgus monkey episomal reprogramming was performed as previously described (Kime et al., 2015). Briefly, 5x10 5 Human neonatal foreskin fibroblasts (young, Y) (Shahbazi et al., 2016), 56-year-old male skin fibroblasts (old, O, AG04148), or cynomolgus monkeys (MHH Hannover) were nucleofected with 3 μg of the following plasmid DNA mix: pCXLE-SOX2-P2A-KLF4 or pCXLE-SOX2-17-P2A (generated for this study), pCXLE-L-MYC-F2A-LIN28 (ML, Addgene ID 27080), pCXLE-hOCT4-shTP53 (Addgene ID 27077), and pCXWB-EBNA1 using the Lonza NHDF Nucleofector kit (U-23 program) and plated at different densities on CF1 feeder layers in ROCKi-containing fibroblast medium.

[0369] Livestock (bovine) reprogramming, 10 6 Fetal bovine fibroblasts (GOF 451-1) (Wuensch et al., 2007) or fetal porcine fibroblasts (Nowak-Imialek et al., 2011) were nucleofected with 6 μg of the following plasmid DNA mix: pCXLE-SOX2-P2A-KLF4 or pCXLE-SOX2-17-P2A, pCXLE-L-MYC-F2A-LIN28, pCXLE-hOCT4 (Addgene ID 27076), pCXLE-p53DD (Addgene ID 41859), and pCXWB-EBNA1 using the human protocol. For bovine reprogramming, pCXLE-p53DD could be omitted.

[0370] Viral supernatant volume was adjusted according to qPCR titration using generic WPRE or 3'UTR primers normalized to Rpl37a (Velychko et al., 2019a). All tetO lines were screened for promoter leakage, and only those with minimal leakage were selected for characterization. Newly generated iPSC lines (mouse, human, cynomolgus monkey, and bovine) were karyotyped using DAPI staining of metaphase spreads, and only lines with the correct chromosome count were selected for characterization. As previously reported (Velychko et al., 2019a), no differences in the occurrence of chromosomal aneuploidy were observed between the different cocktails. As with other studies, we only tested the quality of male iPSCs for this work.

[0371] Except for bovine iPSCs (biPSCs), which did not produce teratomas in SCID mice in our two injection attempts, iPSCs were characterized as previously described. For the third attempt, we injected control biPSCs or S*K biPSCs (nucleofected with episomal pCXLE-SOX2-17-P2A-KLF4 1 week prior) into the left and right sides of the same mice. Teratomas developed only on the right side 1 month later (Figure 10z-ab).

[0372] Induced to naive conversion For induced-to-naive conversion (pluripotency upgrade), human iPSCs were transduced with monocistronic or polycistronic pHAGE2-EF1α lentiviral vectors carrying reprogramming factors. Two days later, cells were plated at low density (10 per 24-well plate) on inactivated C3H feeder layers supplemented with ROCKi and in mESC medium with or without 2i. 3 Cells were passaged in mESC medium (2i). After 24 h, the medium was changed to mESC medium without ROCKi, with or without 2i. After 6 days, cells were fixed and stained for KLF17 (HPA024629, ATLAS, 1:500). PD0325901, but not CHIR99021 or PD0325901 + CHIR99021 (2i), increased the number of KLF17 colonies.

[0373] 3x10 for built-in versatility upgrade 5 GFP-negative Gof18 E3 mouse ectodermal stem cells (mEpiSCs) ( Han et al., 2010 ) cells were seeded onto FBS-coated 12-well plates in StemFlex+ROCKi medium and co-cultured with 2 μg of episomal pCXLE-mCherry, pCXLE-mCherry-T2A-SOX2-P2A-KLF4, or pCXLE-mCherry-T2A-SOX2. AV-P2A-KLF4 or pCXLE-mCherry-T2A-SOX2-17-P2A-KLF4 using 4 μL of Lipofectamine Stem Reagent (Invitrogen) according to the manufacturer's instructions; 48 h later, cells were sorted for mCherry and plated at 10 cells per 12-well plate in mouse ESC medium + ROCKi on an inactivated C3H feeder layer. 4 Approximately 30% of the sorted cells survived; of these, approximately 50% SK / S AV K / S*K transfected colonies grew domed and were GFP+ already on day 4 after passage. GFP+ colonies were selected and clonally expanded for further characterization.

[0374] Reset from non-integrated induction to naive No built-in reset, 3x10 5 GFP-negative Gof18 E3 mouse epiblast stem cells (mEpiSCs) 10 6 Cells were seeded onto FBS-coated 12-well plates in StemFlex+ROCKi medium and simultaneously transfected with 2 μg of episomal pCXLE-mCherry, pCXLE-mCherry-T2A-SOX2-P2A-KLF4, or pCXLE-mCherry-T2A-SOX2-17-P2A-KLF4 (Addgene IDs 193293, 193296, and 193294, respectively) using 4 μL of Lipofectamine Stem Reagent (Invitrogen) according to the manufacturer's instructions; 48 h later, cells were sorted for mCherry and plated at 10 cells per 12-well plate on inactivated C3H feeder layers in mESC medium+ROCKi. 4Approximately 30% of the sorted cells survived; of these, approximately 50% of SK / SAVK / S*K transfected colonies grew domed and were already GFP+ / mCherry- on day 4 after passage. GFP+ colonies were selected and clonally expanded for further characterization. Lipofection with RNA synthesized from T7-VEE-CFP-e2a-SOX2-p2a-KLF4 and T7-VEE-CFP-e2a-SOX2-17-p2a-KLF4 (Addgene IDs 193358 and 193360, respectively) yielded the same results (data not shown).

[0375] For human iPSC (hiPSC, Gibco episome A18945) or ESC (hESC, H9) naive sets, 0.5x10 6 The induced cells were transfected using Nucleofector 2b (program B-016) and Lonza Human Stem Cell Nucleofector TM Nucleofection was performed using Kit 1 (Cat#: VPH-5012) according to the manufacturer's protocol with 6 μg of pCXLE-mCherry-T2A-SOX2-17-P2A-KLF4 or pCXLE-mCherry control, mixed with pCXWB-EBNA1 (Addgene ID 37624) at a 3:1 ratio. For EMSA experiments, pCXLE-SOX2-17-P2A-KLF4 (Addgene ID 193290) versus control empty pCXLE plasmid was used. Nucleofected cells were plated on feeder- or FBS-coated dishes in StemFlex+ROCKi medium and cultured at 37°C in a hypoxic 5% O2, 5% CO2 incubator. On day 2, the medium was changed to StemFlex. Optionally, human naive medium (RSeT™, STEMCELL Technologies) was added on day 3. RSeT works well on feeders, and StemFlex expresses more KLF17 in feeder-free conditions. + and SUSD2 + Colonies were formed.

[0376] For mouse / human chimera experiments, the following plasmids were used to constitutively label the A18945 hiPSC line: AAVS1-Pur-CAG-mCherry (Addgene #80946), gRNA_AAVS1-T2 (Addgene #41818), and pX330-U6-Chimeric_BB-CBh-hSpCas9 (Addgene #42230). To assess the contribution of embryonic development, we aggregated S*K-reset hiPSCs with mouse morulae at E2.5 as previously described (Eakin et al., 2006). After 2 days of culture (E4.5), chimeric embryos were stained for human-specific SUSD2 (Biolegend 327401) and mouse-specific Oct4 (D6C8T, Cell Signaling). All experiments were performed in accordance with ISSCR guidelines.

[0377] Tetraploid (4N) complementation assay 1. Preparation of tetraploid embryos Superovulated B6C3 F1 females were mated with CD1 males. Two-cell stage E1.5 embryos were flushed from the oviduct and collected in M2 medium.

[0378] After equilibration in fusion solution (0.3 M D-mannitol, 50 μM CaCl2, 0.3% BSA (Sigma)), 50–75 embryos were placed between the electrodes of a 250 μm gap electrode chamber (BLS Ltd.) containing 0.3 M mannitol with 0.3% BSA and fused using a 0.5 mm Microfusion Slide (BTX-450) in a Cellfusion CF-150 / B apparatus (BLS Ltd.). An initial electric field of 2 V was applied to the embryos, followed by one peak pulse of 60 V for 50 μs. The embryos were then transferred to KSOM-aa medium and immediately returned to a 37°C incubator with 5% CO2. The embryos were observed for 15–60 min post-fusion. The fused tetraploid embryos were cultured under the same conditions for 24 h until the four-cell stage.

[0379] 2. iPSC aggregation using zona-free embryos (1) Preparation of aggregation plates for mouse embryo chimera production 1 h before aggregation: A 100 μl pipette filled with KSOM medium is used to create four rows of microdroplets (approximately 3 mm diameter) in a 35 mm dish (Falcon, Cat. No. 35-3001), with two droplets in the first and fourth rows and five droplets in the second and third rows. The entire plate is covered with paraffin oil. Sterilize a coagulation needle (BLS Ltd.) with 70% ethanol. Press the agglutination needle into the plastic through the paraffin oil and culture medium, moving in a circular motion to produce small depressions of approximately 300 µm diameter with transparent flat walls. Within each droplet, 6-10 holes can be created. (2) iPSCs are aggregated and cultured with denuded 4-cell stage mouse tetraploid embryos as described with slight modifications (Nagy et al., 1993): Loosely attached iPSC clumps (15–20 cells each) from short trypsinized day 2 iPSC cultures were selected and transferred to microdroplets of KSOM medium under mineral oil; each clump was placed in a microdroplet well. Meanwhile, batches of 30–50 embryos were briefly incubated in acidified Tyrode's solution until the embryonic zona pellucida dissolved (Hogan et al., 1986). Two embryos were placed on each iPSC clump. All clumps were assembled in this manner and cultured overnight at 37°C and 5% CO2. After 24 h of culture, the majority of the aggregates formed blastocysts. 10–14 embryos were transferred into one uterine horn of a 2.5-dpc pseudopregnant recipient. Adult CD-1 females weighing 30+ g were used as pseudopregnant dams.

[0380] Mammalian cell overexpression and whole cell lysate (WCL) production HEK293T cells cultured on 10 cm dishes were transfected with 10 μg of pLVTHM or pHAGE2 vectors containing wild-type or mutant versions of Oct4 or Sox2 under the control of the EF1α promoter by Fugene6 (Promega) using a low-volume protocol (Steffen et al., 2017). Three days after transfection, cells were detached from the plate using Accutase (Sigma), harvested, counted, and washed with PBS. WCLs were lysed in lysis buffer (20 mM HEPES-KOH pH 7.8, 150 mM NaCl, 0.2 mM EDTA pH 8, 25% glycerol, 1 mM DTT, and cComplete TM Pellets were resuspended in 12.5 μL per million cells in protease inhibitor cocktail (Merck) by five freeze-thaw cycles. After disruption, the lysates were spun at 14k RCF for 10 minutes at 4°C. After centrifugation, the pellets were discarded and the supernatants were transferred to new tubes for further analysis. Samples were diluted in 0.1% SDS solution and analyzed by A. 230 and A 260 Measure and use the formula: Conc.(μg / μL)=(0.183*A 230 -0.075*A 260 )*Dilution ratio was applied to estimate protein concentrations.

[0381] All samples were diluted to 1 μg / μL, aliquoted, flash-frozen, and stored at -80°C. Western blots were performed to compare expression levels between mutants. Expression was assessed by Quantity One® (v4.6.7, Bio-Rad) densitometry and adjusted for equal expression using WCL of untransfected cells, preserving total protein content where necessary.

[0382] Western blot analysis Five to ten micrograms of total protein were combined with Laemmli sample buffer, heated, and loaded onto a 12% mini SDS-polyacrylamide gel (SDS-PAG) using the Towbin buffer system (Towbin et al., 1979). The gel was first run at 15 V for 15 minutes to load the sample into the stacking gel, and then run at 50 V for 30–60 minutes to resolve the proteins of interest. The samples were transferred to an Immobilin®-FL PVDF membrane (Merck Millipore Ltd.) at 300 V for 2 hours at 4°C. The membrane was blocked for 1 hour in 5% skim milk (Sigma) dissolved in PBS with 0.1% Tween-20 (PBS-T) at room temperature and then incubated overnight at 4°C with rotation in primary antibodies diluted in blocking solution. The following day, the membrane was washed three times in PBS-T and then incubated for 1 hour at 25°C in secondary antibodies diluted in blocking solution. The following antibodies were used: polyclonal goat anti-Oct4 N-19 (sc-8628, Santa Cruz Biotechnology) or monoclonal mouse anti-Oct4 (611203, BD Biosciences), polyclonal goat anti-Sox2 (sc-17320, Santa Cruz Biotechnology), monoclonal mouse anti-α-tubulin (T6199, Sigma), 647-conjugated anti-goat (Alexafluor), and 647-conjugated anti-mouse (Alexafluor). Western blot signals were detected using a Fujifilm FLA-9000 fluorescent scanner (Fujifilm).

[0383] Insect cell expression and protein purification Full-length mouse Sox2 or Sox2 AVThe coding sequence of was cloned into the pCoofy27 plasmid with an N-terminal 6xHis tag using SLIC: forward primer 3C, reverse primer ccdB as previously described (Scholz et al., 2013). The plasmid was then transformed into DH10EMBacY (a gift from Dr. Imre Berger) for baculovirus plasmid DNA amplification (Trowitzsch et al., 2010). Bacmids were purified using Macherey-Nagel Xtra BAC100 (Dueren) and then grown in serum-free EX-CELL® 420 medium containing L-glutamine (Sigma). 0.8x10 6 A suspension of Sf9 cells at 1000 cells / mL was transfected and incubated at 26°C with shaking for virus production. Cells were monitored daily for an increase in cell size and GFP fluorescence. Once approximately 90% of the cells were GFP+, the virus suspension was spun down and then filtered through a 0.22 mm filter. The virus supernatant was expanded once and filtered aliquots were stored at -80°C before use in infections.

[0384] Optimal protein expression conditions were determined empirically. High Five in mid-logarithmic phase TM Insect cells, 10 in 2L 6 The cells were split into 1000 cells / mL and then infected with 10-12 mL of the P1 baculovirus from the previous step per L of cells. After 96 h of incubation at 28 °C with shaking, the cell pellet was collected by centrifugation. The pellet was then lysed in lysis buffer (20 mM HEPES pH 7.5, 300 mM NaCl, 30 mM imidazole, 5% glycerol, 0.1% Triton X-100, complete). TMThe pellets were resuspended in protease inhibitor cocktail (Merck) and 1 mM DTT, freeze-thawed once, and then sonicated at 4°C using a probe sonicator (Bandelin Sonopuls, Bandelin Electronics). The pellets were washed with inclusion body wash buffer (20 mM HEPES pH 7.5, 200 mM NaCl, 1 mM EDTA, 1% Triton X-100, complete). TMThe pellets were resuspended in a protease inhibitor cocktail (Merck) and 1 mM DTT and subjected to four cycles of Dounce homogenization, followed by 20 min of centrifugation at 18k RCF and 4°C. They were then subjected to two rounds of inclusion body wash buffer and two rounds of buffer without Triton X-100. The final pellet was digested twice in DMSO and then incubated at 25°C for 30 min. Unfolding buffer (7 M guanidine hydrochloride, 20 mM Tris-HCl pH 7.5, 5 mM DTT) was added to the pellet and incubated at 25°C for 1 h with rotation. Nickel Sepharose slurry (GE Healthcare) was washed and equilibrated in binding buffer, and then the supernatant was added and incubated overnight at 4°C with rotation. Proteins were further eluted using unfolding buffer with 500 mM imidazole. Elution fractions were checked by SDS-PAGE, and relevant fractions were pooled. Using 7 kDa molecular weight cutoff (MWCO) dialysis tubing, the pooled fractions were dialyzed against respective volumes of refolding buffer (7 M urea, 20 mM Na acetate pH 5.2, 200 mM NaCl, 1 mM EDTA, and 5 mM DTT) at 4 °C for at least 6 h with three buffer exchanges. After centrifugation to remove any insoluble material, the supernatant was dialyzed in refolding buffer with decreasing amounts of urea (7 kDa MWCO): 1 h 6 M urea, 2 h 4 M, 2 h 2 M, and 1 h in size-exclusion chromatography (SEC) buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 5% glycerol). The eluate was centrifuged to remove any precipitate and then loaded onto a HiLoad 16 / 60 Superdex 200 SEC column (GE Healthcare).

[0385] The full-length Oct4 coding sequence from mouse was cloned into the pOPIN expression vector using the SLIC method and Phusion Flash high-fidelity PCR master mix (Finnzymes / New England Biolabs). The SLIC reagent was then added to the pOPIN expression vector. TM OmniMACTM The pOPIN-cHis-Oct4 construct was transformed into 2T1® chemically competent E. coli (ThermoFisher Scientific). After sequencing, the pOPIN-cHis-Oct4 construct was purified using flashBACULTRA TM Recombinant baculovirus was generated by co-transfection of Sf9 cells (ThermoFisher Scientific) with bacmid DNA (Oxford Expression Technologies) using Cellfectin II® (ThermoFisher Scientific). Virus was amplified using mid-logarithmic phase Sf9 cells. Suspension High Five TM Cells were infected with P3 virus at 27°C for 2 days with shaking at 120 rpm. After expression, crude lysates were purified on a HiTrap TALON column (GE Healthcare), cleaved on a column with 3C protease, and followed by size-exclusion chromatography (HiLoad Superdex 200, GE Healthcare). The final product was collected in 25 mM HEPES pH 7.8, 150 mM NaCl, 1 mM TCEP, and 5% glycerol, and approximately 95% purity was confirmed by SDS-PAGE. Fractions were checked by SDS-PAGE, pooled, and analyzed by Protein A using a NanoDrop spectrophotometer (ND-1000, ThermoFisher Scientific) and the specific molecular weight and extinction coefficient of either Sox2 or Oct4. 280 Final quantification was performed using a program. All chemicals were from Sigma-Aldrich unless otherwise indicated.

[0386] Electrophoretic mobility shift assay (EMSA) DNA probes were generated by annealing complementary 5'-labeled Cy5 oligos (Metabion International AG) and then purified from a 10% polyacrylamide gel. For the binding reaction, WCL (2-4 μg of total protein) or purified protein was incubated for 1 h at 37°C in binding buffer (25 mM HEPES-KOH pH 8, 50 mM NaCl, 0.5 mM EDTA, 0.07% Triton X-100, 4 mg / mL BSA, 7 mM DTT, and 10% glycerol) and 70 nM Cy5-dsDNA. Samples were then loaded onto a 6% native polyacrylamide gel (37.5 / 1 acrylamide / bis-acrylamide) containing 0.3x Tris-borate EDTA and 5% glycerol and run at 10 mA / gel in a running buffer of the same composition. Gels were imaged using a Fujifilm FLA-9000 fluorescence scanner (Fujifilm). The bound fraction was analyzed using Quantity One® (v4.6.7, Bio-Rad) and the following formula for specific bands: F B =DNA 結合 / (DNA 結合 +DNA 未結合 ) were determined by densitometry of the raw data and then normalized. Half-lives were calculated using fraction bound as a function of protein concentration from at least two independent experiments; error bars represent SD.

[0387] The equation for decay was determined using nonlinear regression in Prism 7 for Mac (version 7.0a) and R 2 The fit was only used if it was 0.95 or higher, as assessed by the value.

[0388] For competition experiments, preformed protein / DNA or protein / nucleosome complexes (see binding conditions above) were loaded onto native gels (t=0) and then incubated with unlabeled double-stranded DNA containing the Nanog locus. Protein separation was monitored by removing aliquots of the reaction at predetermined time points and loading them onto a running gel. Because protein complex stability is highly variable, conditions for competition assays were empirically determined and can be seen in the table below. [Table 5] [Table 6] [Table 7]

[0389] Nucleosome assembly The nucleosomal DNA sequence Widom+6 consists of the 147 bp established Widom 601 sequence (Lowary and Widom, 1998) with a Sox / Oct motif (CTTTGTTATGCAAAT (SEQ ID NO: 55)) at superhelical position +6 and nucleosome dyad 0 (Michael et al., 2021). DNA was double-stranded, purchased from IDT (Coralville), and labeled using Cy-5 conjugated primers via PCR as previously described (Michael et al., 2020). Nucleosomes were assembled with purified full-length Drosophila melanogaster histone octamers (Klinker et al., 2014) using a previously described salt gradient dialysis method (Luger et al., 1999) at DNA:octamer ratios ranging from 1:1.2 to 1:1.6. The final buffer composition was 10 mM HEPES pH 7.6, 50 mM NaCl, 1 mM EDTA, and 0.5 mM DTT. After dialysis, nucleosomes were heat-shifted at 37°C for 2 h. Nucleosome quality and concentration were assessed using native PAGE run with a histone-free DNA standard curve generated from parental DNA. Histone stoichiometry was checked by 22% SDS-PAGE followed by Coomassie staining (R-250; SERVA). Nucleosomes were stored in the dark at 4°C and used within 3 weeks of assembly.

[0390] Molecular Dynamics Simulation (MDS) A previously constructed model of the Oct4-Sox2 heterodimer bound to regulatory DNA elements from the Hoxb1 enhancer ( Esch et al., 2013 ) was used to model Sox2 / Oct4, Sox2 A61V / Oct4, Sox2 / Oct6, Sox2 A61VThe DNA fragments were used as templates to build new models for / Oct6. Using MODELLER (https: / / salilab.org / modeller / ), the sequences were matched, with each model of the Oct factors being extended by 4 and 8 residues at the N- and C-termini, respectively, and each model of the Sox factors being extended by 4 and 5 residues. One hundred models were built for each ternary complex using the "slow" optimization procedure, which included "slow" MD refinement, as specified in MODELLER. The models were ordered using energy scores ("DOPE") and two models selected for each complex for MD simulations. In each of these models, the DNA was extended by 16 and 18 base pairs at the 5' and 3' ends using the mouse Hoxb1 sequence (Aksoy et al., 2013b). The final sequences in the models were: 5'-AGAGTGATTGAAGTGTCTTTGTCATGCTAATGATTGGGGGGAGATGGAT-3' (SEQ ID NO: 56) It was.

[0391] The systems were then solvated in a truncated octahedron periodic box of SPCE water with a distance greater than 12 Å between any protein-DNA atom and the box edge. Seventy-three neutralizing Na+ ions and 150 mM KCl (225 K+ and 225 Cl- ions) were added. For ions, parameters developed by Li and Merz (Li et al., 2015) were used. The Amber-ff14SB (Maier et al., 2015) and Amber-parmbsc1 (Ivani et al., 2015) force fields were used for protein and DNA, respectively. Each system was energy minimized and equilibrated using a previously described protocol (Jerabek et al., 2017). For each model, two independent 1.2 μs-long MD simulations were performed by aligning different velocity distributions before equilibration (total of 4 x 1.2 μs = 4.8 μs per system). Periodic boundary conditions were applied in the isothermal-isobaric (NPT) ensemble with a time step of 2 fs. Temperature was maintained at 300 K using Langevin dynamics (damping coefficient of 0.1 ps-1). Pressure was maintained at 1 atmosphere using the Nose-Hoover-Langevin Piston method with period and damping of 1.2 and 1.0 ps, ​​respectively. Direct calculation of non-bonded interactions was truncated at 10 Å, and hydrogen chemical bonds were robustly maintained using the SHAKE algorithm. Long-range electrostatics were calculated using the particle mesh Ewald algorithm. All simulations were performed in NAMD (Phillips et al., 2005). Snapshots were selected for analysis every 10 ps.

[0392] The coordination number between two atom selections describes the number of contacts between the selections using a continuous switching function with a distance threshold for contact formation as implemented in the COLVAR module of NAMD. The formula is:

number

[0393] NGS and bioinformatics analysis For ChIP-seq experiments, Rosa26TA-Gof18 MEFs were incubated with a titrated volume of pHAGE2-tetO-Klf4-IRES-Sox2 / Sox2 with or without pHAGE2-tetO-Oct4 / Oct6. AV Cells were infected with HIV-1. After 48 h, the medium was replaced with fibroblast medium supplemented with doxycycline (dox). Samples were harvested 48 h after dox induction. RNA-seq and RRBS were performed on human iPSCs at passages 10–12, human ESCs at passages 35–36 grown on Matrigel in StemFlex medium, and human fibroblasts at passages 11–15 grown on gelatin-coated dishes in fibroblast medium. Sample processing and data analysis for RNA-seq, ChIP-seq, and RRBS were performed as previously described (Keshet and Benvenisty, 2021; Malik et al., 2019; Velychko et al., 2019a).

[0394] For footprinting analysis, briefly, publicly available data (ArrayExpress: E-MTAB-7207) were aligned to the mm10 genome using bowtie2 (Langmead and Salzberg, 2012) with the "--highly sensitive-X 2000-no mixing" options; mitochondrial and duplicate reads were removed, and reads were classified and indexed using samtools (Danecek et al., 2021); Spearman correlations were plotted using deeptools (Ramirez et al., 2016); peaks were called using macs2 with the "-g mm -f BAMPE --call-summits --cutoff-analysis --keep-dup all -B" options (Zhang et al., 2008); and the JASPAR MEME motif database (Castro-Mondragon et al., 2019) with the ENCODE blacklist (Amemiya et al., 2019), several additional custom motifs (Malik et al., 2019). al., 2022 ), and the output of macs2 was used for TOBIAS footprinting analysis ( Bentsen et al., 2020 ).

[0395] Example 2 - Defining the structural factors of Sox17 that benefit the induction of pluripotency Oct4 (Pou5f1) 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), whereas other members of the family, such as the ubiquitously expressed Oct1 (Pou2f1), neuronal 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 preferences for heterodimerization versus homodimerization (Jerabek et al., 2017; Malik et al., 2019; Mistri et al., 2015). In our studies to discover what makes Oct4 unique among POU factors, we found that it binds Sox17 but not wild-type Sox2. E57K We found that the POU mutant (Jauch et al., 2011) can be used in combination with Brn4 to efficiently generate iPSCs (Figure 1a). POU factors consist of a DNA-binding domain (POU domain) flanked by N- and C-terminal transactivator domains (NTD and CTD). The POU domain is divided into two parts, a POU-specific domain (POU) connected by a flexible, non-conserved linker. S ) and POU homeodomain (POU HD ) The linker of Oct4, but not Oct1, contains an α-helix near its N-terminus (Esch et al., 2013; Klemm et al., 1994). Substitution of the Oct4 linker with that from other POU factors or even a point mutation L80→A in the linker helix is ​​detrimental to the induction and maintenance of pluripotency or to supporting normal development (Chen et al., 2020a; Esch et al., 2013; Han et al., 2022a). Surprisingly, Sox17 EK Oct4 L80AThe reprogramming ability of the cells was also rescued (Fig. 7a).

[0396] To identify the structural factors responsible for the rescue phenomenon, we constructed a library of chimeric TFs in which non-conserved residues from Sox17 were exchanged with Sox2 (Fig. 1b-c). We used monocistronic retroviral supernatants for reprogramming of mouse embryonic fibroblasts (MEFs) harboring an endogenous Oct4-GFP reporter (OG2); the volume of the viral supernatant was adjusted based on qPCR titration (Fig. 7b-c). Initial screening results pointed to amino acids 61-62 of Sox17 as the most definitive (Fig. 1d). Sox17-CTD enhanced reprogramming by wild-type Oct4, but not Sox2, unless combined with the 61-62 region. 61-62 c17 chimera, Fig. 1d-e) Oct4 L80A (Sox2c17 chimera, Figure 1d-e). Two other Sox17 regions, 43-47 and 65-86, also accelerated reprogramming efficiency, especially when combined with 61-62 (Figure 1d-e). Other Sox17 factors (e.g., 24-28) significantly reduced the reprogramming efficiency of Sox2 (Figure 1d). A second screen revealed that a single A61V exchange in Sox2 significantly reduced the reprogramming efficiency of Oct4. L80A and Brn4, but L62Q was determined to be insignificant (Figure 1f-g). A61V Oct1 (Pou2f1) also enabled reprogramming by Oct2 (Pou2f2), Oct6, and Brn2 (Pou3f2 or Oct7) in MEFs (Fig. 1h). A61V Sox2 was the only POU factor tested that did not generate iPSC colonies when co-expressed with Klf4 (Fig. 1h). Because leucine is even more hydrophobic than valine, the A61L mutation was also tested. A61L performed better than wild-type Sox2, but not Sox2, particularly in rescuing Brn4. A61V (Future Sox2 AV) (Fig. 1f-g). Notably, a complex chimeric Sox factor, Sox2, which combines 16 beneficial Sox17 residues in the Sox2 HMG-box domain and a complete CTD exchange (Fig. 1b-c), 43-47,61,65-86 c17 (hereafter Sox2-17 or S*) is Sox2, Sox2c17, or Sox17 EK The reprogramming capacity of Sox2 was significantly increased (Figure 1f-g). Cell proliferation is essential for the induction of pluripotency; cMyc, GATA factors, and SV40 large T antigen increase reprogramming efficiency by accelerating cell proliferation (Rand et al., 2018; Velychko et al., 2019a). However, the A61V mutation has the opposite effect: Sox2 AV Four-factor induction with A61V or Sox2-17 resulted in significantly lower cell proliferation compared to their respective 61A variants (Figure 1i). The inhibitory effect on cell proliferation explains why A61V can rescue non-functional Oct4 mutants but does not increase reprogramming efficiency by itself with wild-type Oct4 (Figure 1d). The accelerated efficiency results solely from the synergistic effect between A61V and the more potent Sox17 transactivator (Figure 1d-g).

[0397] Example 3: Sox2 A61V enhances both Sox / Oct cooperativity and the developmental potential of iPSCs A61 is a POU heterodimer that binds to the canonical SoxOct motif. S It is located in the third helix of the high mobility group (HMG) domain of Sox2, facing the HMG domain (Fig. 2a-b). Compared to alanine, valine has two additional methyl groups, making it more hydrophobic. Molecular dynamics simulations (MDS) of the Sox / Oct heterodimer for the HoxB1 SoxOct factor showed that the exchange of A to V significantly enhances the interaction between Sox2-HMG and Oct4- or Oct6-POU. S The I21 residue of both Oct factors was shown to increase the average number of hydrophobic interactions between Sox2 and Oct3 (Fig. 2c). AVThe most common binding was with 61V, which could potentially increase the cooperativity of the Oct4-POU factor (Fig. 2d). The simulations also revealed a novel structural rearrangement of the Sox2 / Oct4 heterodimer that results in a higher Oct-Sox coordination number and therefore potentially higher cooperativity. This rearrangement was not observed when Oct4 was replaced with Oct6 (Fig. 2c). In this configuration, stabilized by A61V, residue 61 is the only residue in the Oct4-POU complex. S The POU interacts with the proximal core of G24 of the helix, and residues R50 and K57 of Sox2-HMG form salt bridges with E82 and E78 of the Oct4 linker (Fig. 2e). S Contrary to the S configuration, which only involves POU S This structure of the Sox2 / Oct4 heterodimer is termed the SL configuration because it involves both the Sox2 and Oct4 linkers. An additional configuration described for the Sox / Oct4 heterodimer on the rarer Fgf4 motif was also modeled, in which the Sox and Oct sites are spaced apart by a three-base-pair gap. Sox2 and Oct4 cooperate more distally on the Fgf4 motif, forming a distant S(DS) configuration that involves T78 and T80 of Sox2 rather than A61 (Figure 2f).

[0398] FLAG-tagged Oct1, Oct2, Oct4, Oct6, Brn2, and Brn4 were overexpressed in HEK293 cells to confirm their equivalent expression (Fig. 8a). Lysates were then used for electromobility shift assays (EMSA) against the Nanog promoter locus. POU monomer binding was comparable for all tested factors, except for Oct1, which reduced binding. Oct4, however, exhibited the strongest heterodimerization ability with Sox2 (Fig. 8b). Wild-type Sox2, Sox2 AV There was no difference in monomer binding between Sox2 and Sox2-17, but Sox2 AV Both Sox2 and Sox2-17 enhanced heterodimerization with all tested POU factors (Fig. 8b). AVTo discover which domains of Oct4 remain essential for generating iPSCs when combined with other chimeric Sox factors, we replaced all 17 residues of the Oct4 linker domain with synthetic polyglycine linkers of different lengths (GL3-30) (Fig. 3a). Such flexible linkers were detrimental to reprogramming with wild-type Sox2, but not with Oct4. GL15-30 The mutant Sox2 AV The efficiency was rescued by Oct4ΔNTD and further increased by Sox2-17 (Figure 3a). Second, we truncated both the NTD and CTD of Oct4, both of which have previously been shown to be crucial for reprogramming (Kim et al., 2020). Indeed, neither Oct4ΔNTD nor Oct4ΔCTD could generate iPSCs when combined with wild-type Sox2 (Figures 3b, S2c-d). However, Sox2 AV Alternatively, Sox2c17 can rescue Oct4ΔCTD, and Sox2 AV c17, Sox17 EK Sox2-17 or Sox2-17 rescued either of the transactivator-deficient mutants (Fig. 3b, 8c-d). These results suggest that increased Sox / Oct cooperativity, especially when combined with stronger Sox transactivators, can compensate for the loss of Oct4 transactivation during reprogramming. Third, neither chimeric Sox factor nor the Oct4-POU, which is directly involved in the Sox / Oct interaction, could be directly involved in the Sox / Oct interaction. S The deletion of the Sox2 domain could not be rescued (Fig. 3c). AV is POU HD Oct4ΔPOU, in which the DNA-binding domain is truncated, leaving the RKRKR peptide at its N-terminus, which functions as a nuclear localization signal HD Several GFP+ colonies bearing Oct4ΔPOU were also identified (Fig. 3c, Fig. 8e). PCR genotyping revealed two clonal Oct4ΔPOU HD / Sox2 AV The identity of the / Klf4 iPSC lines was confirmed ( Fig. 8f ), and they were able to contribute to germline-containing chimeric mice ( Fig. S2g ) ( Fig. 8h ).

[0399] Oct4 and Sox2 mutants were overexpressed in HEK293 cells, and equivalent expression was confirmed (Fig. 3d), followed by EMSA experiments. Again, monomer binding was observed in Sox2 on either HoxB1 or Nanog DNA factors. AV While 61V showed no difference between Oct4 and wild-type Sox2 (Fig. 3e), 61V significantly increased heterodimerization with wild-type Oct4, in accordance with the reprogramming results (Fig. 3c, Fig. 8e-h). HD The DNA binding of the Oct4 mutant was partially rescued (Fig. 3e). HD The rescue data are particularly interesting because of previously discussed reports on the role of the POU subdomain in the pioneering function of Oct4. While the nucleosome does not present a challenge for Sox2 (Dodonova et al., 2020), the POU S and POU HD POU binds to the opposite site of DNA, which inevitably collides with the histone core, thus preventing canonical Oct4 binding (Huertas et al., 2020; Michael et al., 2020; Soufi et al., 2015). This prompted speculation that Oct4 binds closed chromatin with just one of its domains (Soufi et al., 2015) or even that POU alone is involved in chromatin opening (Michael et al., 2020). On the other hand, Oct4 uses POU to recognize specific sequences on nucleosomes. S and POU HD While other domain scans have been shown to nonspecifically create barriers to nucleosome closure (MacCarthy et al., 2021), the data presented here suggest that POU HD We show that removing the POU disrupts both Oct4 DNA binding on the SoxOct motif (Fig. 3e) and reprogramming efficiency (Fig. 3c compared to Fig. 3a-b), demonstrating that even for highly cooperative Sox factors, HDThis highlights the importance of the binding of Oct4 to wild-type Sox2. It is highly unlikely that stable, and therefore eventual, Oct4 binding can be achieved by just one of its subdomains (Fig. 3e). To compare the stability of the Sox / Oct4 heterodimer relative to different natural regulatory SoxOct DNA factors, we performed off-rate EMSA experiments in which excess unlabeled DNA was added to preformed Sox / Oct / DNA complexes and the samples were loaded onto a gel over a time course. AV Both Oct4DE and Sox2-17 dramatically enhanced heterodimer stability on both the Oct4 distal enhancer (Oct4DE) and Nanog promoter elements, which were similar to wild-type Sox2 stability at the Fgf4 locus (Fig. 3f). With both Oct4DE and Nanog probes, a proportion of heterodimers disappeared almost immediately, whereas the remaining heterodimers were exceptionally stable (Fig. 3f), again suggesting that there may be two distinct Sox / Oct configurations, similar to our MDS-based results (Fig. 2). Oct4, Sox2, and Sox2 purified from insect cells AV (Fig. 8i) was used in EMSA against three natural SoxOct factors: the Nanog promoter, Utf1, and Fgf4 enhancer (Fig. 3g, Fig. 8j). Sox2 and Sox2 AV Monomer binding and heterodimerization of Fgf4 were similar, whereas heterodimerization of Nanog and Utf1 was enhanced by the A61V mutation (Fig. 8j). Off-rate EMSA for Nanog and Utf1 factors reminiscent of our whole-cell lysate experiments: A61V increased the stability of the Sox2 / Oct4 / DNA complex by 3-4 fold (Fig. 3g). Consistent with the MD simulations (Fig. 2), Sox2 AV also strongly increased heterodimer stability with Oct4 linker mutants and Brn4, which would otherwise be reduced (Figure S2k), explaining their rescue ability in reprogramming experiments (Figures 1f-h, Figure 3a). We conclude that POU factors and Oct4 mutants that normally cannot reprogram to pluripotency can be rescued by forced cooperation with Sox2 (Figure 3h).

[0400] Both Oct4 and Sox2 are pioneer factors that can bind to inaccessible chromatin and activate gene expression (Soufi et al., 2012; Teif et al., 2012). A modified Widom 601 nucleosome array, in which the SoxOct motif fits at superhelical position (SHL) +6, was assembled and used to resolve the Oct4 / Sox2 / nucleosome complex by cryo-electron microscopy (Michael et al., 2020). Sox2 AV dramatically enhanced the stability of the Oct4 / Sox2 / nucleosome complex (Figure 4a). Chromatin immunoprecipitation with sequencing (ChIP-seq) was then performed to identify binding sites at a very early stage of reprogramming—2 days after doxycycline (Dox) induction of KS or OKS in MEFs. HOMER motif enrichment analysis (Heinz et al., 2010) (Figure 9a-b) revealed that the A61V sample exhibited a significant increase in the stability of Oct4 / Sox2. AV / K and Oct6 / Sox2 AV The SoxOct motif was more enriched in both KS and OKS samples, whereas the Sox2 motif was not significantly different in the SoxOct ChIP. Overall, the Sox2 peak was more enriched in both Sox2 and Sox2 in either KS or OKS samples. AVThe binding profiles of Oct4 and Sox2 were not significantly different between the two (Figures 4b-c), suggesting that 61V does not alter the binding profile of Sox2 itself. On the other hand, Oct4 ChIP for either Oct4 or Oct6 showed increased intensity and number of SoxOct peaks (Figures 4b-c), significantly increasing the ratio of SoxOct to Oct and OctOct (MORE) for the OKS cocktail with the A61V mutant (Figure 4d). Using TOBIAS footprinting analysis of published ATAC-seq datasets for ESC vs. MEF samples (Li et al., 2017), we determined the relevant binding sites of Oct4 and Sox2 that lead to chromatin opening (Figure 9c). 61V did not alter Sox2 binding, but significantly increased Oct4 association with SoxOct sites that opened over the reprogramming course (Figure 9c). Oct4 / Sox2 compared to Oct4 / Sox2 AV Stronger binding by α-glucan is indicated by increased signal for both Oct4 and Sox2 ChIP at prominent pluripotency targets, such as Nanog, Pou5f1, Nr5a2, Gdf3, and Lefty2 (Fig. 4e). Consistent with our computational modeling (Fig. 2f) and EMSA results (Fig. 3f), binding to the Fgf4 locus was unaffected (Fig. 4e).

[0401] In ESCs, Oct4 and Sox2 cooperatively regulate genes; most pluripotency genes contain SoxOct motifs in their regulators (Chen et al., 2014), but binding of Oct4 alone increases cell division rates (Lee et al., 2010). Overexpression of Oct4 in somatic cells induces hyperproliferation (Hochedlinger et al., 2005). Accordingly, OSKM induction in MEFs caused a significantly stronger acceleration of cell proliferation compared with SKM induction (Figure 1i) (Velychko et al., 2019a). At the onset of reprogramming, when Oct4 and Sox2 are overexpressed in somatic cells, they attempt to bind to thousands of non-native genomic loci that are largely independently bound (Chen et al., 2016; Chronis et al., 2017; Li et al., 2019; Soufi et al., 2012). We hypothesized that increasing Sox / Oct cooperativity may improve the reprogramming process, as cooperativity between TFs increases their specificity (Von Hippel et al., 1996). Indeed, already on day 2 of OKS induction, Sox2 AV Oct4 bound to 511 ESC-specific super-enhancers compared with 378 for wild-type Sox2 (Fig. 4f). To assess the effect of enhanced Sox2 / Oct4 cooperativity on the developmental potential of iPSCs, we performed a multi-step analysis of iPSCs expressing either wild-type Sox2 or Sox2. AV Tetraploid (4N)-complementation assays were performed on iPSC lines generated using lentiviral tet-inducible or episomal delivery methods harboring polycistronic OSKMs. In both cases, Sox2 AV iPSC lines generated by Sox2 produced significantly more fully developed whole iPSC pups (greater than a two-fold difference) (Fig. 4g, Table 1). AVThe benefit of tetO-OSKM all-iPSC mice was particularly evident for their survival. As previously reported by us and others ( Buganim et al., 2014 ; Chen et al., 2015a ; Velychko et al., 2019a ), OSKM all-iPSC mice rarely survive to adulthood: none of the tetO-OSKM all-iPSC mice achieved maturity, and none of the five tetO-OSKM all-iPSC mice tested achieved maturity. AV Three of the KM iPSC lines gave rise to adult mice (Figure 4g-h, Table 1). AV KM all-iPSC mice were fertile; PCR genotyping confirmed the inheritance of the transgene in their offspring (Figure 4i). Episomal (epi-) vectors resulted in more moderate overexpression and yielded better-quality integration-free iPSCs, even in the presence of exogenous Oct4 (Velychko et al., 2019a). However, there was a 22.2% OS. AV Compared with KM whole-iPSC embryos, only 4.2% of transplanted epi-OSKM whole-iPSC embryos survived to adulthood. Thus, increasing Sox2 / Oct4 cooperativity significantly improves the developmental potential of mouse OSKM iPSCs.

[0402] Example 4: Genetically engineered super-SOX promotes reprogramming across species Our attention then shifted to the most effective chimeric Sox factor in this study, Sox2-17-Sox17, containing 61V along with three other structural factors (Figure 1b-c). Sox2-17 was cloned into a tet-inducible polycistronic OSKM or SKM reprogramming cassette, and comparable expression levels were confirmed using qPCR (Figure 10a). A time-course experiment with limited Dox induction (Figure 5a) showed that Sox2-17 dramatically enhanced the kinetics and efficiency of reprogramming (Figure 5b). While at least 3 days of OSKM induction was required to generate the first iPSC colonies, OS*KM with Sox2-17 generated iPSC colonies just 24 h after induction—the fastest reprogramming reported to date. Clonally expanded 24-h iPSC lines lost methylation of the Nanog and Pou5f1 promoters and acquired methylation of the fibroblast-specific Col1a1 promoter (Figure 10b). 24h iPSCs were able to differentiate into all three germ layers in teratoma assays (Figure S4c), contribute to chimeric mice (including germline) (Figure 10d), and successfully generate live-born whole-iPSC pups in 4N complementation assays (Figure 10e, Table 1). When induced for just 3-4 days, OS*KM gave rise to 10-200 times more colonies than OSKM, depending on the quality of fibroblasts and the level of overexpression (Figure 5b-c). Sox2-17 further enabled mouse two-factor iPSC generation with Klf4 alone (S*K cocktail), albeit at low efficiency, whereas Sox2, Sox2c17, or Sox17EK failed to do so (Figure 10f). Clonal S*K iPSC lines displayed normal morphology (Figure S4g), and PCR genotyping confirmed the identity of the two lines we tested (Figure S4h). S*K iPSCs stained positive for the pluripotency markers Nanog and SSEA-1 (Fig. 10i) and gave rise to three germ layers in a teratoma assay (Fig. 10j). These data suggested that Sox2-17 may require shorter times, lower doses, and reduced numbers of cofactors to successfully induce pluripotency, and may be particularly beneficial for the relatively low-efficiency integration-free reprogramming method.We generated episomal Myc-free polycistronic Oct4-P2A-Klf4-IRES-Sox vectors carrying either wild-type Sox2 or Sox2-17 (epi-OKS and epi-OKS*, respectively), and confirmed their correct expression by Western blot (Fig. 10k). Sox2-17 enhanced epi-OKS reprogramming of MEFs by up to 150-fold (Fig. 5d) and generated high-quality iPSCs that could generate whole-iPSC mice in a 4N complementation assay with an efficiency of up to 77% (Fig. 5e, Table 1). Remarkably, all 10 tested integration-free epi-OKS and OKS* iPSC lines gave rise to healthy adult whole-iPSC mice with high survival rates, similar to both Sox2 and Sox2-17 (Fig. 5f, Table 1).

[0403] The human version of SOX2-17 (Fig. 1c) was tested to reprogram human fetal fibroblasts by retroviral monocistronic OSKM delivery (Fig. 5g-h). Transduced cells were plated on inactivated feeders and stained for the human pluripotency marker TRA1-60 after 2 weeks. SOX2-17 generated 56-fold higher iPSC colonies compared to SOX2, with overall reprogramming efficiencies of 8.9% and 0.16%, respectively (Fig. 5g-h). SOX2-17 was found to be able to reprogram human cells even when combined with OCT4 alone (OS* cocktail), despite its low efficiency (Fig. 5i-j). While the 61V in SOX2-17 was shown to be crucial for enabling two-factor reprogramming (Fig. 5i), SOX17 EKThe OKS*iG vector, which encodes human self-replicating RNA encoding OCT4, KLF4, SOX2-17, and GLIS1 (Yoshioka et al., 2013), did not yield single human OS iPSC colonies (Figure 4i-j). Human clonal OS* iPSCs exhibited normal morphology (Figure 10l), expressed pluripotency markers NANOG and TRA1-81 (Figure 10m), and could differentiate into tissues of the three germ layers in a teratoma assay (Figure 10n). The OKS*iG vector, which encodes human self-replicating RNA encoding OCT4, KLF4, SOX2-17, and GLIS1 (Yoshioka et al., 2013), also yielded approximately 50-fold higher TRA1-60 colonies compared with wild-type SOX2 (Figure 5k). Furthermore, using the RNA-OKS*iG vector, integration-free iPSCs derived from Parkinson's disease patient skin fibroblasts were successfully generated and characterized, which failed to yield iPSCs using the original vector carrying wild-type SOX2 (Rosety et al., Sci Adv 2022, in press). SOX2-17 was also cloned into the episomal SOX2-F2A-KLF4 vector (Okita et al., 2011), replacing the original F2A self-cleavage peptide with P2A to reduce the formation of fusion polyproteins (Velychko et al., 2019b). Western blot analysis revealed that SOX2 / SOX2 AV We confirmed the equivalent expression and precise cleavage of SOX2-17 and KLF4 (Figure 10o). SOX2-17-P2A-KLF4 (S*K) in combination with the OCT4 / shTP53 and L-MYC / LIN28 (OS*KML) vectors showed significantly improved performance, particularly in reprogramming aged human dermal fibroblasts, where several reprogramming attempts using SOX2-carrying vectors resulted in a single alkaline phosphatase-positive (AP) cell. +) colonies (Figure 5l). To test whether superSOX could facilitate reprogramming for other species, the episomal vector was tested in generating iPSCs from cynomolgus monkeys (Macaca fascicularis), an important non-human primate model (Han et al., 2022b; Wunderlich et al., 2012, 2014), where ESCs were derived (Chen et al., 2015b; Fu et al., 2020), but no transgene-independent iPSCs were reported. While the wild-type episomal OSKML vector failed to generate any cynomolgus monkey iPSC (ciPSC) colonies during multiple attempts, the same cocktail containing SOX2-17 generated several putative APs. + They generated iPSC-like colonies (Figure 5m). While most hiPSC lines lost the episomal vector before the third passage, only three of the 11 generated ciPSC lines completely lost the transgene after three passages (Figure 10p). Two of the three lines with the correct chromosome number were characterized (Figure 10q). Integration-free ciPSCs showed similar morphology to hiPSCs, except that they tended to be more differentiated in the center of the colony and could be maintained only on feeder layers (Figure 10r). They expressed pluripotency markers NANOG and OCT4 (Figure 10s) and could differentiate into three germ layers in teratoma assays (Figure 10t). Reprogramming of bovine and porcine fibroblasts was also attempted using XAV939-containing bFGF-based medium, recently developed by the Smith lab to induce and maintain bovine ESCs (Kinoshita et al., 2021). Episomal reprogramming using wild-type SOX2 failed in both species, whereas the same cocktail containing SOX2-17 efficiently reprogrammed APs for both bovine (Figure 5n) and porcine (Figure 5o). +iPSC-like colonies were generated. Bovine iPSCs (biPSCs) could be generated without p53 inhibition, albeit with low efficiency (Figure 10u). We established 12 such epi-OS*KML biPSC lines (without P53DD), and all lost the episomal vector by passage 6 (Figure 10v). They could be passaged at least 11 times, maintained ESC-like morphology (Figure 10w) and accurate chromosome number (Figure 10x), and stained positive for SOX2 and OCT4 (Figure 10y). The biPSCs stained positive for SOX2 and OCT4 (Figure 10y) and could differentiate into three germ layers in teratoma assays (Figures 10z-ab). SOX2-17 subsequently enabled the first generation of integration-free, virus-free bovine iPSCs with potential applications in the cultivated beef industry, livestock genome editing, and beyond.

[0404] SOX2 for the accuracy of human reprogramming AV To investigate the effects of SOX2-17 and SOX2-17, we generated and characterized 30 hiPSC lines using episomal reprogramming of neonatal foreskin (young, Y) and 56-year-old male skin (old, O) fibroblasts. All selected clonal iPSC lines tested positive for integration (Figure S4aa-ab) and had normal genotypes (Figure S4aa-ab). Hierarchical clustering of global gene expression based on RNA-seq showed that all iPSCs clustered far from fibroblasts and near hESCs (Figure S4p). Differences in gene expression among the lines likely arose more from the cell source than from the SOX factors used. Reduced-representation bisulfite sequencing (RRBS) (Meissner, 2005) was performed to analyze the methylomes of the hiPSCs. All lines clustered far from fibroblasts and near hESCs (Figure S4q).

[0405] Loss of imprinting (LOI) is a common, potentially cancerous (Holm et al., 2005; Jelinic and Shaw, 2007) and irreversible (Hiura et al., 2013) epigenetic abnormality affecting both mouse and human iPSC technology (Bar et al., 2017; Carey et al., 2011; Keshet and Benvenisty, 2021; Takikawa et al., 2013). Multiple studies have reported a correlation between LOI and poor developmental outcomes of whole iPSC embryos in 4N complementation experiments (Buganim et al., 2014; Carey et al., 2011; Chen et al., 2015a; Stadtfeld et al., 2012; Takikawa et al., 2013; Velychko et al., 2019a). Thirty-one differentially methylated regions (DMRs) were analyzed, and all lines and fibroblasts of origin had different levels of LOI, with the younger fibroblast-derived SOX2 AV We found that hiPSCs and senescent fibroblast-derived SOX2-17 hiPSCs exhibited the lowest average levels of LOI among iPSC lines (Figure ​(Figure5r). 5r). We conclude that highly cooperative Sox factors can enable efficient, high-quality reprogramming across species (Figure ​(Figure5s). 5s).

[0406] Example 5: Sox / Oct heterodimerization is central to naive pluripotency ESC derivatives of mouse preimplantation inner cell mass and their iPSC counterparts grown in LIF-containing medium are referred to as "naive" (Nichols and Smith, 2009). Mouse naive lines exhibit the highest developmental potential of all cultured cells, can contribute to chimeric animals, and some, but not all, naive lines can generate all-PSC mice in 4N complementation assays. However, this is not true for "induced" PSCs of other species, including humans, which are more similar to mouse primitive ectodermal stem cells (mEpiSCs) (Tesar et al., 2007). Interestingly, Oct4DE is active in naive cells but not in induced pluripotent cells (Choi et al., 2016; Gafni et al., 2013; Yeom et al., 1996), and highly cooperative Sox factors dramatically increase the stability of the Sox2 / Oct4 heterodimer on Oct4DE (Figure 3f). Inspired by these results, we hypothesized that Sox / Oct cooperativity may be central to naive pluripotency. We analyzed a previously published time-course ATAC-seq dataset (Yang et al., 2019) of naive-to-induced transition samples generated for mouse ESCs upon exposure to bFGF medium. Spearman correlation of sequencing reads confirmed reproducibility between replicates and indicated that the most significant transition occurred between day 1 (d1) and day 2 (d2) of differentiation into primitive ectoderm-like cells (EpiLCs) (Figure 6a). Footprinting analysis using TOBIAS (Bentsen et al., 2020) showed that the most dominant depleted footprints on d1 were those of estrogen-related receptors (Esrr) and Klf factors (Figure 6b). This is not surprising, as members of both the Klf (Klf4) and Esrr (Essrb) families can convert mouse EpiSCs into naive ESCs (Adachi et al., 2018; Guo et al., 2009). d1 samples also had a significant depletion of Sox / Oct footprints (Figure 6b), which became the most depleted of all TF footprints between the d1 and d2 transitions (Figure 6c).Whole cell lysate EMSA was performed against SoxOct factors on the Nanog promoter, E14 mESC lines grown in KSR-LIF medium, and GFP carrying the Oct4-GFP reporter (Gof18) grown in bFGF-containing hESC medium. - E3 mEpiSC lines, induced to naive and sorted Oct4-GFP grown in the same medium containing KSR-LIF or 2i (Mek and GSK-3 inhibitors, PD0325901 and CHIR99021, respectively ( Ying et al., 2008 )). +We measured the heterodimerization levels of naturally expressed Sox2 and Oct4 in mEpiSCs and human iPSCs grown in conventional bFGF medium (Fig. 6d). Endogenous EMSA results echoed footprinting analysis: mEpiSCs had significantly lower levels of Sox2 and Oct4 capable of heterodimerization compared to mESCs, but heterodimerization in EpiSCs was restored to ESC levels upon conversion from induced to naive (Fig. 6d). Heterodimerization (as well as Oct4 monobinding) was further enhanced in the presence of 2i (Fig. 6d). hiPSCs, like mEpiSCs, had low Sox2 / Oct4 heterodimerization capacity but also higher levels of Oct4 monobinding. Western blot analysis indicated that the lack of Sox2 / Oct4 heterodimerization was due to lower Sox2 expression in both mouse and human induced cells; Oct4 expression levels were equal in mEpiSC and mESC-LIF samples but higher in 2iLIF samples and hiPSCs (Figure 6e). The identity of the Sox2, Oct4, and Sox2 / Oct4 bands was confirmed in all tested lines using antibody supershift assays (Figure 6f). To determine whether Sox2 / Oct4 heterodimerization could also explain the vast differences in developmental potential between iPSC lines, we tested six integration-free mouse PSC lines, which had been extensively characterized in a previous study (Velychko et al., 2019a). Two of the six iPSC lines (epi-OKSM#1 and epi-OKSM#3) failed to generate full-term whole iPSC pups (4N-off, "insufficient quality"), whereas the other four (epi-OKSM#2, epi-KSM#1, ESC#1, and ESC#2) were able to generate adult whole PSC mice (Velychko et al., 2019a). Cells were first adapted to feeder-free conditions with several passages in 2iLIF medium, then passaged either with or without 2i and harvested for endogenous EMSA experiments.Among cells grown in LIF-only medium, two developmentally incompetent iPSC lines had the lowest capacity for Sox2 / Oct4 heterodimerization (Figure 6g) and the lowest Sox2 expression (Figure 6h) among the six lines, a pattern similar to that of EpiSCs (Figure 6d-e). However, the same lines passaged in 2iLIF showed no differences in either Sox2 / Oct4 heterodimerization or Sox2 expression (Figure 6g-h). This suggests that pluripotent cell lines may be stabilized at different levels of Sox2 expression and Sox2 / Oct4 heterodimerization as their developmental competence gradually declines, from Sox-high "very naive" to different types of "moderately naive" and "induced" cultures (Figure 6a-h). Although we and others have previously described capturing different grades of pluripotency (Bernemann et al., 2011; Kinoshita et al., 2020), the distinct roles of Sox2 expression and Sox2 / Oct4 heterodimerization have not received significant attention. The widely used 2i medium appears to equalize heterodimer content among different grades of naive cells, but fails to stably reprogram cell fate: moderate Sox-low lines revert to moderate after 2i withdrawal (Figure 6g-h). Therefore, how can low-grade pluripotent cells, such as mouse EpiSCs or conventional pluripotent cultures, be stably "upgraded" to pluripotent state in most other species, including humans?

[0407] Mouse EpiSCs could be converted into naive ESCs by overexpression of Klf4 (Guo et al., 2009). Similar conversion was attempted with human cells, but Klf4 alone was found to be insufficient (Figure 6i). Screening of different combinations of Yamanaka factors showed that overexpression of Sox2 and Klf4 (SK) was sufficient to convert conventional hiPSCs into domed colonies positive for the human naive pluripotency marker KLF17, even in the absence of small molecule inhibitors (Figure 6i) (Guo et al., 2016; Kilens et al., 2018; Lea et al., 2021; Shahbazi et al., 2017). Similar to SKM reprogramming in mice (An et al., 2019; Velychko et al., 2019a), combining Sox2 and Klf4 in a single bicistronic vector enabled the conversion of hiPSCs into naive ESCs. + The number of colonies increased (Fig. 6i). Supplementing KSR-LIF medium with Mek inhibitor further enhanced the efficiency of conversion (Fig. 6i). For further experiments using Gof18 mEpiSCs as a model, we used the SK cocktail. The goal was to understand integration-free pluripotency upgrade and the role of Sox / Oct cooperativity in SK-based induced-to-naive conversion. mCherry was cloned into a human episomal reprogramming vector to express SOX2, SOX2 AV pCXLE-mCherry-T2A-SOX-P2A-KLF4 carrying SOX2-17 or SOX2-17 was generated. Lipofection was used to deliver the episomal Cherry-SK vector into mEpiSCs, and on day 2, mCherry + / Oct4-GFP - Cells were sorted for Oct4-GFP expression (Fig. 11a) and plated on inactivated feeders in KSR-LIF medium. The majority of surviving cells were already expressing Oct4-GFP four days after plating (six days after transfection). + and mCherry - Six individual Oct4-GFP cells formed dome-shaped colonies.+ / mCherry - Colonies were selected for each of the three cocktails and clonally expanded for further characterization (Figure 11b). Regardless of Sox version, SK-converted lines exhibited significant differences in endogenous Sox2 / Oct4 heterodimerization as determined by EMSA (Figure 6j), which correlated with differences in Sox2 expression as determined by Western blot (Figure 11c), likely displaying different grades of pluripotency. Transient overexpression of wild-type SK resulted in the expression of Oct4-GFP cells grown under the same conditions. - mEpiSCs reset Sox2 / Oct4 heterodimerization by up to 62% compared to controls (Fig. 6j). Both highly cooperative Sox factors appear to be highly potent in resetting Sox2 / Oct4 heterodimerization: six S AV Three of the S*K strains had a 120-140% increase in heterodimerization, and two of the six S*K strains had a remarkable 180% enhancement (Figure 6j). S*K-converted naive strains also showed significant increases in Oct4-GFP expression during passage. + They had a significantly lower tendency to spontaneously lose the state (Fig. 11b, d), suggesting that superSOX provides a more stable reset.

[0408] Episomal mCherry-S*K or mCherry-alone plasmid (Fig. 11e) was nucleofected into hiPSCs grown in induction medium (StemFlex), and optionally on day 2, the medium was changed to human naive medium (RSeT) (Fig. 11e). On day 7, cells were stained for the human naive pluripotency markers KLF17 and SUSD2. Under naive medium conditions, most colonies nucleofected with episomal mCherry-S*K expressed SUSD2. + , KLF17 +Domed colonies were generated that were mCherry- and mCherry-transfected, whereas control transfected cells were not positive for these markers (Fig. 11e-f). Similar to mEpiSC resetting, the mCherry-S*K plasmid was eliminated from the cells within a few days (Fig. 11e-g), whereas the mCherry-only plasmid remained (Fig. 11f). Surprisingly, transient S*K expression was partially suppressed by SUSD2, even in the feeder-free induction medium. + and KLF17 + These data suggest that reducing Sox2 / Oct4-driven chromatin opening may be responsible for the impaired developmental potential during the induction of pluripotent cells in early development (Fig. 11e), that SK reprogramming can reverse the process, and that highly cooperative Sox factors significantly promote this resetting (Fig. 11i).

[0409] Example 6: SOX2-17+KLF4 cocktail enhances the developmental potential of iPSCs and ESCs across species We further optimized the episomal S*K resetting protocol for human iPSCs (female episomal hiPSC line A18945, Gibco; Figures 11e and 12a). As described in Example 5 above (Figure 11e), hiPSCs were co-nucleofected with pCXLE-mCherry-S*K and pCXWB-EBNA1 (to increase the longevity of episomal expression) and plated onto high-density feeder layers in induction medium (StemFlex, Thermofisher). On day 2, the medium was changed to human naive medium (Rset, STEMCELL Technologies), and the cells were transferred to a low-oxygen incubator (5% O). By day 7, just one passage later, S*K-treated cells generated dome-shaped colonies that tested positive for the human naive pluripotency markers SUSD2 (Figures 11e, 12a) and KLF17 (Figure 11f) (Bredenkamp et al., 2019a; Wojdyla, et al., 2020; Bi, et al., 2022). On average, 18% of day 7 S*K hiPSCs were SUSD2 +and mCherry-, confirming the unprecedented resetting efficiency and transgene-independent state of the generated naive cells. Most of the S*K reset colonies were KLF17 + However, mCherry control transfected cells were not positive for either SUSD2 or KLF17 (Fig. 11f, 12a). Surprisingly, transient overexpression of S*K did not induce SUSD2 expression even under conventional feeder-free culture conditions in induction medium. + and KLF17 + hiPSC colonies were generated (Figure 11g). Naive resets in induction medium were not shown for other TF cocktails (Takashima et al., 2014; Liu et al., 2017; Theunissen et al., 2014; Hanna et al., 2010; Yamauchi et al., 2020, Qin et al., 2016).

[0410] RT-qPCR was performed to assess the expression of key naive pluripotency genes. S*K reset resulted in significant upregulation of DNMT3L, KLF17, and ARGFX in both induced and naive media, with stronger upregulation in naive media. On the other hand, Rset naive media alone did not increase naive gene expression, except for a six-fold upregulation of KLF4 (Figure 12b). Both FACS and qPCR data for WPRE confirmed that the mCherry-S*K plasmid was eliminated from cells by day 7, leaving only the mCherry-only plasmid (Figure 12a, b), suggesting that S*K reset may trigger the transgene silencing mechanism described for mouse naive cells (Yang et al., 2015). The results of the episomal S*K naive reset were replicated for two other human lines: H9 ESCs and OS*KML O-hiPSC#1 (generated during this study).

[0411] To test the developmental potential of our putative naive hiPSCs, we transfected sorted SUSD2 hiPSCs into hiPSCs at day 7 of aggregation with morula-stage (E2.5) mouse embryos as previously described. + We used S*K-reset cells (Eakin et al., 2006). hiPSC lines were marked with constitutive RFP expression to assess the level of chimeric contribution across species. Surprisingly, human cells were detected in the ICM of most aggregated embryos (Fig. 12c). Chimerism was confirmed by costaining E4.5 aggregated embryos with human-specific SUSD2 and mouse-specific Oct4 antibodies. Human SUSD2+ cells were incorporated into the ICM of 6 of 11 aggregated embryos tested; interestingly, in one case, immunostaining demonstrated dramatic occupation of the entire blastodermal region by S*K-reset hiPSCs (Fig. 12d). The results of the mouse / human chimera experiment were reproduced using another hiPSC line (OS*KML O-hiPSC#1, data not shown).

[0412] Although SuperSOX enabled the generation of episomal bovine iPSCs (biPSCs), they failed to produce teratomas in severe combined immunodeficiency (SCID) mice in several injection attempts. Similarly, cultured bovine ESCs do not readily produce teratomas (Jun Wu, pers. comm.). As described above in Example 4, to test whether S*K-resetting could enhance the developmental potential of biPSCs, we injected control biPSCs or S*K-reset biPSCs into the left and right flanks of the same mice. Five weeks later, teratomas developed only in the right flank, containing tissues representing all three embryonic germ layers (Fig. 10aa).

[0413] Finally, we investigated whether S*K resetting could improve the developmental potential of a "poor-quality" naive female mouse ESC line (mESC, C57BL / 6J background) grown in 2 iL of medium, which had failed to efficiently generate whole ESC mice in our previous tetraploid complementation experiments (Fig. 12f). Nucleofection of mESCs with either episomal mCherry-S*K or mCherry vectors yielded domed, naive-like colonies (Fig. 12g). Colonies manually selected 5 days after nucleofection were used for tetraploid complementation experiments (Fig. 12f). Strikingly, S*K-reset cells yielded eight times more full-term born whole ESC pups compared with controls (Fig. 12h-j). Three S*K-reset whole ESC pups survived nursing, while only the control whole ESC pups died shortly after birth (Fig. 12i). The in vivo evidence of enhanced developmental potential for three unrelated species presented in this section, most importantly the birth of S*K-reset all-ESC animals, provides compelling evidence for our proposed "heterodimer model" of pluripotency (Fig. 11h).

[0414] Consideration iPSC technology remains unsatisfactory, especially for non-mouse cells, and reprogramming often results in epigenetic abnormalities that result in inconsistent iPSC quality (Carey et al., 2011; Hiura et al., 2013; Keshet and Benvenisty, 2021; Takikawa et al., 2013). Several alternative cocktails have been shown to improve the fidelity of the reprogramming process in mice (Buganim et al., 2014; Chen et al., 2015a; Velychko et al., 2019a), but failed to reprogram human cells, which appear to have stronger epigenetic barriers (Kim et al., 2021). Perhaps the apparent barriers are simply evidence of the inadequacy of the wild-type factor-based reprogramming mechanisms we currently use.

[0415] Many studies have addressed the uniqueness of Oct4 among POU factors by swapping domains between Oct4 and other POU factors and introducing mutations (Jerabek et al., 2017; Kim et al., 2020; Van Leeuwen et al., 1997; Nishimoto et al., 2003; Roberts et al., 2021; Tapia et al., 2012; Velychko et al., 2019b). We and others have investigated the Oct4-POU S , POU HD We determined the importance and uniqueness of the linker and CTD domains. Here, we report that a residue exchange, A61V, at the Sox / Oct interface of Sox2 increases the stability of the Sox / Oct complex on the canonical SoxOct motif that controls pluripotency genes. Particularly when combined with the more potent Sox17 transactivator, 61V enhances the stability of the otherwise obstructive Oct4 linker, POU. HD For highly cooperative Sox factors, the Oct4-POU, which contains the Sox / Oct interface, can rescue deletion of the NTD or CTD domains. S The Oct4-POU domain (Figure 2) has been found to be most crucial for reprogramming (Figure 3a-c). A 17-residue linker peptide connects the two DNA-binding subdomains of the Oct4-POU domain; all resolved POU structures to date suggest that the linker is not directly involved in DNA binding (Figure 2a-b), but appears to be important for both reprogramming to pluripotency and development (Chen et al., 2020b; Esch et al., 2013; Han et al., 2022a). Until now, the effect of linker mutations on Sox / Oct stability has not been tested. Here, we show that Oct4 linker mutants, such as L80A or complete substitution with a flexible polyglycine peptide, reduced the stability of the Sox / Oct heterodimer as well as the highly cooperative Sox2. AV was found to partially rescue both the stability of the heterodimers and their reprogramming ability (Figs. 1f, 8k, 3a), similarly to Sox2 AVMDS enables Sox / POU heterodimerization and reprogramming with tissue-specific POU factors (Figure 1g-h, Figure 8b, k). MDS reveals a previously undescribed SL configuration of the Sox2 / Oct4 heterodimer on the canonical SoxOct motif, where Oct4 residues 78E and 82E form salt bonds with Sox2 residues 57K and 50R, respectively (Figure 2c, e). K57E The mutant was reported to be deleterious to iPSC generation and disrupts Sox2 / Oct4 heterodimerization on the canonical SoxOct motif, but not on Fgf4 (Jauch et al., 2011; Remenyi et al., 2003a). AV and Sox2-17 enhance cooperation with Oct4 at the Nanog promoter, Pou5f1 distal enhancer (Oct4DE), HoxB1, and Utf1 enhancer loci, all of which contain the canonical SoxOct motif ( Figure 3 , Figure 8 ); Sox2 AV We showed that SoxOct factors (Fig. 4a) enhanced heterodimerization in situ in early reprogramming samples (Fig. 4b-f). However, consistent with model predictions (Fig. 2f), Sox2 AV Neither Sox2-17 nor Sox2-17 altered cooperativity on the Fgf4 enhancer motif, which has three additional base pairs spaced between the Sox and Oct factors (Figures 3f, 8j, and 4e) (Remenyi et al., 2003b). Fgf4 controls cell proliferation and differentiation in the protoectoderm (Kunath et al., 2007; Niswander and Martin, 1992), and inhibiting the Fgf4 pathway with PD0325901 facilitates the maintenance of naive pluripotent cells (Kunath et al., 2007). Therefore, specific enhancement of the S and SL configurations, but not the DS, of the Sox2 / Oct4 heterodimer by A61V could be important for its greatly improved performance in reprogramming. Further enhancement of the SL configuration and / or targeted disruption of the DS binding on Fgf4 could further improve the developmental resetting technology of the super-SOX system.

[0416] The animal evolutionary tree suggests that Sox2 was the initiating member of the Sox2 / Oct4 pair. Both 50R and 57K were already present in sponges, where SoxB factors control early embryonic development (Fortunato et al., 2012); V at position 61 could also be found in Sox factors in these most primitive animals. 57K and 61A of Sox2 are conserved in hydrozoans, where SoxB genes are expressed in stem cells that give rise to the neuroectoderm (default) and germline (Bosch and David, 1987; Jager et al., 2011; Siebert et al., 2019). POU5 appeared much later in the evolutionary tree and is a vertebrate innovation that cooperates with Sox2 to control early development (Leichsenring et al., 2013; White et al., 2016a). Interestingly, although the linker is the least conserved part of POU factors, negative charges at positions 78 and 82 of the POU5 linker are already present in the jawless hagfish (Sukparangsi et al., 2022). Computer simulations and functional data on the A61V mutant reveal that the most significant feature distinguishing Oct4 from other POU factors is its ability to form a stable heterodimer with Sox2, which has been implicated in regulating early embryonic development in lower animals. Sox17, a master regulator of the germline, also cooperates with Oct4 but prefers a compacted rather than canonical SoxOct motif (Irie et al., 2015; Jauch et al., 2011; Merino et al., 2014). Germline and pluripotent fates share many characteristics that are highlighted by the possibility of inducing mouse pluripotent cells from germline stem cells (Kanatsu-Shinohara et al., 2004, 2008; Ko et al., 2009). The structural elements of Sox17 that facilitate the induction of pluripotency evolved independently of Sox2, making them relevant for identifying our most valuable lineages.

[0417] Although we and others have reported genetically engineered reprogramming factors that increased the efficiency of mouse reprogramming (Aksoy et al., 2013; Hirai et al., 2012; Tan et al., 2021a; Veerapandian et al., 2018), studies have not demonstrated substantial gains in the efficiency or fidelity of iPSC generation over mouse models. All alternative cocktails that improved mouse iPSC quality reduced reprogramming efficiency, making them impractical even when used in humans (Buganim et al., 2014; Chen et al., 2015a; Velychko et al., 2019a). We combined the Sox2 and Sox17 structural factors to construct a Sox2-17 chimeric TF that enhanced reprogramming in five tested species: mouse, human, cynomolgus monkey, cow, and pig (Figures 5 and 10). Remarkably, just 24 h of induction using our modified Yamanaka cocktail was able to generate mouse iPSCs that supported full-term development, demonstrating the most rapid TF-induced cell fate reset reported to date (Fig. 5a-b, Fig. 10a-e). Sox2-17 was shown to enhance integration-free three-factor reprogramming efficiency by 150-fold in mice without compromising the developmental potential of the resulting iPSCs (Fig. 5d-f, Table 1). A point mutation in Sox2, A61V, significantly increased the developmental potential of mouse OSKM iPSCs, as indicated by both a faster rate of full-term development of all iPSC embryos and survival to maturity of all iPSC mice (Fig. 4g-i, Table 1). A cocktail of OSKM and OSK can not only induce complete cell fate reset in a dish but also reverse aging in various animal tissues (Browder et al., 2022; Chen et al., 2021; Chondronasiou et al., 2022; Lu et al., 2020; Ocampo et al., 2016; Sarkar et al., 2020). However, the much-anticipated lifespan extension for wild-type mice remains undiscovered (Ocampo et al., 2016).It is reasonable to speculate that a cocktail that can induce higher quality iPSCs (Figures 4g-i, 5f) and support normal adult development of tissues despite possible background leakage of tet-inducible promoters (Meyer-Ficca et al., 2004) could also outperform wild-type factors in reversing animal aging.

[0418] Notably, all reprogramming cocktails that improved the developmental potential of mouse iPSCs, including OSKM vs. OKSM (Carey et al., 2011), OSK (Buganim et al., 2014), and SKM vs. OSKM (Velychko et al., 2019a), as well as the highly cooperative Sox2 introduced in this study, also reduced the rate of cell division (Figure 1i). Some cell culture interventions that improve iPSC quality also inhibit cell division (Liu et al., 2014). Therefore, it is tempting to theorize that the secret to complete resetting lies in limiting cell proliferation (Figure 6k). This suggests that: 1) OSKM vs. OKSM may be a key factor in determining whether or not the reprogramming cocktail is effective. AV1) by increasing Oct / Sox cooperativity, as in KM reprogramming; 2) by increasing the ratio of Sox2 to Oct4, as in SKM reprogramming but also in Oct4 heterozygous knockout ESCs (Karwacki-Neisius et al., 2013); 3) by depleting or omitting Myc (or other proliferation inducers), as in OKS (Figure 5f, Figure S10k) (Buganim et al., 2014), but also in OSKM versus OKSM cassettes (Carey et al., 2011) and Myc-depleted ESCs (Scognamiglio et al., 2016); 4) by reducing the expression level of reprogramming factors, as in episomal versus viral vectors (Figure 5f) (Velychko et al., 2019a). Following the episomal reprogramming protocol, we generated human iPSC lines for this study using a construct carrying shRNA against TP53 (Okita et al., 2011) to knock down a key tumor suppressor and accelerate cell proliferation. This likely caused a loss of genetic imprinting in the human iPSC lines (Figure 5r). Given the mouse developmental performance results shown here (Figure 5f), knocking down our genome guardian is likely detrimental to iPSC quality and should be avoided.

[0419] Mouse naive pluripotent cells are the most developmentally potent culture currently available. Mice have a high potential to generate (or maintain) an unusual stability of their naive pluripotent fate, allowing embryonic quiescence at the blastocyst stage, known as diapause (Boroviak et al., 2015). The unusual developmental potential of mouse naive pluripotent cells and their increased capacity for homologous recombination repair have enabled unprecedented genetic engineering of this species (Thomas and Capecchi, 1987). Mice remain the only species in which the generation of all-iPSC animals has been reported (Boland et al., 2009; Kang et al., 2009; Zhao et al., 2009); germline competence has only been reported for mice and rats (Hamanaka et al., 2011), highlighting the limitations of current technology. Naive pluripotent cells have reduced levels of Myc compared with induced ones (Bernemann et al., 2011; Ying et al., 2008), which could be a direct effect of high levels of Sox2 in naive cells (Metz et al., 2022) (Figure 6e). Here, we showed that Sox2 / Oct4 heterodimerization is reduced during naive-to-induced differentiation (Figure 6b-c) and restored during induced-to-naive conversion (Figure 6d). For the first time, a common mechanism for the reduced developmental potential in induced vs. naive and 4N-off vs. 4N-on pluripotent cells (Figure 6d-h) was found. Although the Sox / Oct model of developmental resetting presented here (Figure 6k) is consistent with other studies placing Sox2 at the top of the pluripotency network hierarchy (Buganim et al., 2012; Chronis et al., 2017; Liu et al., 2015; Luo et al., 2021; Malik et al., 2019; Tremble et al., 2021; White et al., 2016b), the critical role of Sox2 and Sox2 / Oct4 heterodimerization in highly developmentally competent (naive) pluripotency has not previously been elucidated.We found that even transient episomal expression of S*K was sufficient to generate naive-like hiPSCs in both naive and induced human cultures (Fig. 11e-h, Fig. 12). Episomal S*K resetting also enabled us to generate teratoma-competent biPSCs (Fig. 10z-ab), providing the first in vivo evidence of enhanced developmental potential for non-mouse cells. Finally, S*K resetting could increase the developmental potential of already naive mouse ESCs and accelerate their ability to generate whole ESC mice (Fig. 10z-ab, 12d). Such unprecedented results across species have not been achieved by any other naive reset technology. The in vivo evidence for naive resetting in three species is the ultimate proof for the "heterodimer model" of the naive-to-induced pluripotency continuum. The model explains the action of each Yamanaka factor in pluripotency: high levels of Sox2 and Klf4 expression and Sox2 / Oct4 dimerization promote a preimplantation naive state characterized by the highest developmental potential, whereas reduced Sox2 levels result in reduced Sox2 / Oct4 dimerization, and Oct4 alone and Myc binding promote cell proliferation and guidance.

[0420] Long-term culture in naive medium results in epigenetic abnormalities and loss of germ cell potential in both humans and mice (Keshet and Benvenisty, 2021; Alves-Lopez et al., 2023; Choi et al., 2017). Our work offers an alternative: here we propose a protocol for inducing high-grade pluripotency across species: 1) generation of induced iPSCs using OS*KM; 2) naive resetting of induced cells using S*K (Figure 11i). Furthermore, existing induced cultures of different species could be efficiently reset using simple exposure of episomes or mRNA encoding S*K (Figure 12). We hypothesize that genetically engineered factors designed to induce higher-quality iPSCs with improved efficiency could exceed the performance of wild-type factors in reversing aging. The A61V mutant discovered in this study may allow us to elucidate the role of Sox / Oct dimerization in partial reprogramming-mediated rejuvenation, enhancing our understanding of this process. It will certainly be interesting to investigate how super-SOX-containing cocktails affect animal t...

Claims

1. a) an HMG (high mobility group) domain of any of the amino acid sequences of SEQ ID NOs: 1 to 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 that shares at least 82% sequence identity with the HMG domain defined in (a), assuming that the substitutions defined in (a) are retained; SoxB1 factor variants, including

2. moreover, a) the amino acids at positions 43 to 47 in the HMG domain of any of SEQ ID NOs: 1 to 3 are substituted with the amino acid sequence of SEQ ID NO: 7; and / or b) the amino acids at positions 65 to 86 in the HMG domain of any of SEQ ID NOs: 1 to 3 are substituted with the amino acid sequence of SEQ ID NO: 8; and / or c) the SoxB1 factor variant comprises an amino acid sequence that shares at least 82% sequence identity with the SoxB1 factor variant of a) and / or b), assuming that the substitutions defined in a) and / or b) are retained; A SoxB1 factor variant according to claim 1a).

3. The SoxB1 factor variant according to claim 1 or 2, wherein the SoxB1 factor variant is a Sox2 factor variant.

4. A SoxB1 factor variant described in any one of claims 1 to 3, wherein the SoxB1 factor variant further comprises the amino acid sequence of SEQ ID NO: 9, preferably wherein the amino acid sequence of SEQ ID NO: 9 is linked to the C-terminus of the HMG domain defined in claim 1 or 2.

5. The SoxB1 factor variant of claim 4, comprising or consisting of the amino acid sequence of SEQ ID NO: 13 or 14 or a SoxB1 factor variant sharing at least 96% sequence identity with the amino acid sequence of SEQ ID NO: 13 or 14.

6. (a) an HMG domain of the amino acid sequence of SEQ ID NO:15, wherein amino acids 24 to 28 of SEQ ID NO:15 are replaced with the amino acid sequence of SEQ ID NO:16; or (b) an amino acid sequence that shares at least 82% sequence identity with the HMG domain defined in (a), assuming that the substitutions defined in (a) are retained; Sox17 factor variants, including

7. a) Sox factors and POU factors; or b) Sox factors and POU domains; or c) an HMG domain and a POU factor; or d) HMG domain and POU domain A fusion protein comprising or consisting of: Sox factors i. a SoxB1 factor or a variant of the SoxB1 factor according to any one of claims 1 to 5; or ii. Sox17 or a variant of the Sox17 factor according to claim 6. Selected from: The HMG domain iii. An HMG domain according to claim 1a) or b). Selected from: The POU factor is 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 Selected from: The POU domain is x. The POU domain of SEQ ID NO: 17 or 18 or a variant thereof sharing at least 82% sequence identity with SEQ ID NO: 17 or 18 Selected from: Preferably, the fusion protein retains amino acids 1 to 50, 78 and / or 82 of SEQ ID NO: 17 or 18, respectively.

8. a) Sox factors and POU factors; or b) Sox factors and POU domains; or c) an HMG domain and a POU factor; or d) HMG domain and POU domain A complex or composition comprising or consisting of A complex or composition, wherein the Sox factor, HMG domain, POU factor and POU domain are selected from the Sox factor, HMG domain, POU factor and POU domain defined in claim 7.

9. - a SoxB1 factor variant according to any one of claims 1 to 5, a Sox17 factor variant according to claim 6 or a fusion protein according to claim 7; and / or - a complex according to claim 8; and / or - one or more additional reprogramming factor(s) A nucleic acid molecule or combination of nucleic acid molecules encoding

10. A vector or a combination of vectors comprising the nucleic acid molecule or combination of nucleic acid molecules according to claim 9.

11. (a) a cell or (b) a cell derived from said cell, The cell (a) comprises: (i) a SoxB1 factor variant according to any one of claims 1 to 5, a Sox17 factor variant according to claim 6, or a fusion protein according to claim 7, and / or a complex or composition according to claim 8; and / or (ii) a nucleic acid molecule or a combination of nucleic acid molecules according to claim 9; and / or (iii) A vector or a combination of vectors according to claim 10. or the cell (b) is modified by the presence thereof, The cell of (b) preferably retains the phenotype of the cell of (a).

12. A method for increasing cooperativity between a Sox factor and a POU factor, 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.

13. 13. The method of claim 12, wherein the average number and / or strength of interactions is increased by substituting the amino acid at position 61 of the HMG domain with a hydrophobic amino acid selected from valine, leucine, isoleucine, phenylalanine, methionine, tryptophan or proline, preferably substituting the amino acid at position 61 of the HMG domain with valine.

14. 1. A method for producing induced pluripotent stem cell(s) (iPSC(s)) from non-pluripotent cell(s), comprising culturing the non-pluripotent cell(s) under conditions suitable for reprogramming the non-pluripotent cell(s) into induced pluripotent stem cell(s) (iPSC(s)), wherein the conditions result in the non-pluripotent cell(s): a) a SoxB1 factor variant according to any one of claims 1 to 5 or a Sox17 factor variant and a POU factor according to claim 6; and / or b) a fusion protein according to claim 7 or a complex or composition according to claim 8; and / or c) a variant of the SoxB1 factor and a Klf family member according to claim 5; and optionally: - inhibitors of p53 function; and / or - one or more additional reprogramming factor(s) and preferably (co)expressing them in said non-pluripotent cell(s).

15. 1. A reprogramming method for rejuvenating aged cell(s), tissue(s), organ(s) or organism(s), comprising: a) a SoxB1 factor variant according to any one of claims 1 to 5 or a Sox17 factor variant and a POU factor according to claim 6; and / or b) a fusion protein according to claim 7 or a complex or composition according to claim 8; and / or c) a variant of the SoxB1 factor and a Klf family member according to claim 5; and optionally: - inhibitors of p53 function; and / or - one or more additional reprogramming factor(s) , preferably by (co)expressing them in said aged cell(s), tissue(s), organ(s) or organism(s).

16. 1. A method for the conversion of pluripotent cell(s) into high-grade naive pluripotent cell(s), comprising culturing pluripotent cell(s) under conditions suitable for converting said pluripotent cell(s) into high-grade naive pluripotent cell(s), wherein said conditions result in said pluripotent cell(s): a) a SoxB1 factor or a SoxB1 factor variant according to any one of claims 1 to 5 and a Klf family member; and / or b) a variant SoxB1 factor according to claim 5; and optionally: - inhibitors of p53 function; and / or - one or more additional reprogramming factor(s) , preferably by (co)expressing them in said pluripotent cell(s).

17. a) a variant of the SoxB1 factor according to any one of claims 1 to 5; and / or b) a variant of the Sox17 factor according to claim 6; and / or c) a fusion protein according to claim 7; and / or d) a complex or composition according to claim 8; and / or e) a nucleic acid molecule or a combination of nucleic acid molecules according to claim 9; and / or f) a vector or vector combination according to claim 10; and / or g) a cell according to claim 11; and optionally instructions for use of the kit. A kit of parts comprising or consisting of:

18. 1. A method for producing rejuvenated cell(s), tissue(s), organ(s) or organism(s), comprising: in aged cell(s), tissue(s), organ(s) or organism(s): a) a SoxB1 factor variant according to any one of claims 1 to 5 or a Sox17 factor variant and a POU factor according to claim 6; and / or b) a fusion protein according to claim 7 or a complex or composition according to claim 8; and / or c) a variant of the SoxB1 factor and a Klf family member according to claim 5; and optionally one or more additional reprogramming factor(s). increasing the levels of, preferably by (co)expressing them in said aged cell(s), tissue(s), organ(s) or organism(s); thereby producing rejuvenated cell(s), tissue(s), organ(s) or organism(s).

19. In low-grade pluripotent cell(s), a) a SoxB1 factor or a SoxB1 factor variant according to any one of claims 1 to 5 or a Sox17 factor variant according to claim 6 and a Klf family member; and / or b) a fusion protein according to claim 7 or a complex or composition according to claim 8 and a Klf family member; and optionally one or more additional reprogramming factor(s). increasing the levels of, preferably by (co)expressing them in said low-grade pluripotent cell(s); A method for producing high-grade naive pluripotent cell(s), comprising the step of thereby producing naive pluripotent cell(s).

20. the low-grade pluripotent cell(s) are iPSC(s); Optionally: - the iPSC(s) are iPSC(s) obtained or obtainable by the method of claim 14; or 20. The method of claim 19, further comprising, prior to step a), providing iPSCs by carrying out the method of claim 14.

21. The (co)expression is integration-free (co)expression, preferably integration-free (co)expression: - a nucleic acid molecule according to claim 9; or - A vector according to claim 10, wherein the vector is preferably an episomal vector. The method according to any one of claims 14 to 16 and 18 to 20, wherein the method is from

22. An induced pluripotent stem cell (iPSC), a high-grade naive pluripotent cell, or a rejuvenated cell, tissue, or organ, which is produced or can be produced by the method according to any one of claims 14 to 16 and 18 to 21, respectively.

23. (i) expression of at least one, preferably at least two, more preferably at least three, pluripotency-specific marker(s) selected from Oct4, Sox2, Nanog, and Klf4; and / or (ii) the ability to differentiate into each of the three primitive germ layers 23. The induced pluripotent stem cell (iPSC) of claim 22, characterized in that:

24. compared with corresponding non-naive pluripotent cells or low-grade naive pluripotent cells (i) Enhanced generative capacity; (ii) higher expression levels 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 six naive pluripotency-specific marker(s) selected from Klf17, Klf4, Sox2, Susd2, Argfx, and Dnmt3l; (iii) higher expression levels of primitive endoderm-specific marker(s) selected from at least one of Gata6 and Sox17; (iv) activated POU5f1 distal enhancer; (v) a reactivated X chromosome in the female lineage; (vi) reduced DNA methylation; (vii) enhanced ability to differentiate, preferably into germ cells; (viii) an enhanced ability to contribute to the development of embryo(s) and / or animal(s); and / or (ix) A combination of two or more of (i) to (viii) The high-grade naive pluripotent cell according to claim 22, characterized in that:

25. - as medicine; - in regenerative medicine; and / or - In the treatment or prevention of age-related diseases 25. The induced pluripotent stem cells (iPSCs), high-grade naive pluripotent cells or rejuvenated cells, tissues or organs according to any one of claims 22 to 24 for use.

26. 25. Use of the induced pluripotent stem cells (iPSCs), high-grade naive pluripotent cells or rejuvenated cells, tissues or organs according to any of claims 22 to 24 for generating differentiated cell(s), tissue(s), organ(s) or organism(s).

27. The differentiated cell(s), tissue(s) or organ(s) are (i) Graft; (ii) an organoid; or (iii) Cultured meat 27. The use according to claim 26, wherein

28. 22. Use of high grade naive pluripotent cell(s) obtained by the method of any of claims 19 to 21 for generating embryo(s) or animal(s), preferably chimeric embryo(s) or chimeric animal(s).

29. 1. A method for inducing high-grade naive pluripotency in embryo(s) thereby enhancing the viability and / or developmental potential of said embryo(s), comprising: a) a variant of SoxB1 factor according to any one of claims 1 to 5 or a variant of Sox17 factor according to claim 6 and optionally a Klf family member and / or a POU factor; and / or b) a fusion protein according to claim 7 or a complex according to claim 8 and optionally a Klf family member. The method comprises increasing the level(s) of

30. For the development of culture media for the cultivation or induction of naive pluripotent stem cells a) a variant of the SoxB1 factor according to any one of claims 1 to 5; and / or b) a variant of the Sox17 factor according to claim 6; and / or c) a fusion protein according to claim 7; and / or d) a complex or composition according to claim 8; and / or e) a nucleic acid molecule or a combination of nucleic acid molecules according to claim 9; and / or f) a vector or vector combination according to claim 10, and / or g) Cells according to claims 11 and / or 22 Use of.

31. For the development of differentiation media for the induction of germline and other cell types and tissues, a) a variant of the SoxB1 factor according to any one of claims 1 to 5; and / or b) a variant of the Sox17 factor according to claim 6; and / or c) a fusion protein according to claim 7; and / or d) a complex or composition according to claim 8; and / or e) a nucleic acid molecule or a combination of nucleic acid molecules according to claim 9; and / or f) a vector or vector combination according to claim 10, and / or g) Cells according to claims 11 and / or 22 Use of.

32. A germline differentiation medium comprising the cells of claim 11 and a molecule that blocks DNA binding or expression of an Oct family member.

33. A method for developing or optimizing a culture medium, comprising testing whether the cells of claims 11 and / or 22 retain at least one phenotypic or genotypic marker over at least one cell cycle or passage.

34. 23. A method for developing or optimizing a differentiation medium, preferably a germline differentiation medium, comprising the step of testing whether the cells of claim 11 or 22 exhibit at least one marker representative of a germline or a cell type further differentiated therefrom.

35. 34. The method of claim 33, wherein said at least one cell cycle or passage is at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 cell cycles or passages.

36. At least one marker representative of the induced pluripotent stem cells of claim 22, (i) expression of at least one, preferably at least two, more preferably at least three, pluripotency-specific marker(s) selected from Oct4, Sox2, Nanog, and Klf4; and / or (ii) the ability to differentiate into each of the three primitive germ layers 36. The method of claim 33, 34 or 35, wherein the

37. At least one marker representative of the high-grade naive pluripotent cells of claim 22 compared to corresponding non-naive pluripotent cells or low-grade naive pluripotent cells: (i) Enhanced generative capacity; (ii) higher expression levels 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 six naive pluripotency-specific marker(s) selected from Klf17, Klf4, Sox2, Susd2, Argfx, and Dnmt3l; (iii) higher expression levels of primitive endoderm-specific marker(s) selected from at least one of Gata6 and Sox17; (iv) activated POU5f1 distal enhancer; (v) a reactivated X chromosome in the female lineage; (vi) reduced DNA methylation; (vii) enhanced ability to differentiate, preferably into germ cells; (viii) an enhanced ability to contribute to the development of embryo(s) and / or animal(s); and / or (ix) A combination of two or more of (i) to (viii) 36. The method of claim 33, 34 or 35, wherein the

38. 36. The method of any one of claims 33 to 35, wherein retaining at least one marker of the phenotype of the cell of claim 11 comprises retaining at least one of the following phenotypes of the original cell bearing the SoxB1 factor variant of any one of claims 1 to 5 or the Sox17 factor variant of claim 6: an increased 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 naive 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 the female lineage; reduced DNA methylation; an increased ability to differentiate, preferably into germ cells; and / or an increased ability to contribute to the development of embryo(s) and / or animal(s).

39. 39. The method of any one of claims 33 to 38, further comprising the step of producing a developed or optimized medium.

40. 40. A cell culture medium produced or preparable by the method of any one of claims 33 or 35 to 39.

41. A germline differentiation medium produced or producible by the method of any one of claims 34 to 39.

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