Transcription factor combinations for inducing or reprogramming beta cells

Abstract

The present disclosure relates to the development of methods for making β-cells from differentiated, multipotent or pluripotent stem cells by introducing and expressing a combination of transcription factors. The present disclosure also relates to an isolated population comprising reprogrammed cells, compositions and their use in the treatment of diabetes. In various embodiments, the combination of transcription factors is selected from a group consisting of: (a) Pax6, Ngn3, MafA; (b) Rfx6, Ngn3, MafA; (c) Pax6, Ngn3, Rfx6, MafA; (d) Nkx6.1, Pdx1, MafA; and (e) Pdx1, Pax6, Rfx6, Neurodi.

Description

[0001] TRANSCRIPTION FACTOR COMBINATIONS FOR INDUCING OR REPROGRAMMING BETA CELLS

[0002] CROSS-REFERENCE TO RELATED APPLICATION

[0003] This application claims priority from Greece Patent Application No. 20240100187 filed on 13 March 2024, the contents of which are incorporated herein in their entirety by reference.

[0004] FIELD OF THE INVENTION

[0005] The present disclosure relates to the development of methods for reprogramming differentiated, multipotent, progenitor, or pluripotent stem cells into p-islet cells by introducing and expressing a novel combination of transcription factors or their encoding nucleotide sequences. The present disclosure also relates to an isolated population of reprogrammed cells, compositions and their use in the treatment of diabetes.

[0006] BACKGROUND OF THE INVENTION

[0007] Diabetes is a growing problem caused by the inability of the pancreas to regulate blood glucose levels (Kaul et al., 2012; Roder et al., 2016). Globally, about 140 million people have diabetes, and the number is predicted to rise to 300 million by 2025 (Hopkins, 2001). There are several strategies where people try to manage the disease such as administration of exogenous insulin, continuous glucose monitoring, use of insulin pumps, or closed loop systems. However, each come with their own set of limitations and does not give long term relief (Kaul et al., 2012; Roder et al., 2016; Kalra et al., 2013; Yun et al., 2015; Khursheed et al., 2019; Sena et al., 2010). A promising cure is islets transplantation. However, this procedure is limited by the shortage of donors, which impedes the widespread implementation of this therapy. Nonetheless, even though islets transplantation results have shown to be impractical, transplantation studies have been useful in demonstrating that cell replacement therapy is technically a feasible therapeutic option for diabetes fueled the search for an alternative [3-cells source.

[0008] In the past few decades, several regenerative approaches have emerged to replace p-cells from pluripotent stem cells (PSCs)-Embryonic stem cells (ESCs) or induced pluripotent stem cells (iPCs) through traditional differentiation approaches. Studies on pancreatic development in model organisms have identified important signals to produce -cells from PSCs in vitro (Huang et al., 2020). Based on these studies, many research groups have developed different differentiation protocols with the aim to recapitulate natural biological pathways to generate functional insulin-secreting p-cells (D’Amour et al., 2006; Rezania et al., 2012; Pagliuca et al., 2014; Russ et al., 2015; Nair et al., 2019; Liu et al. , 2021) as summarized in Table 1 . Table 1 . Overview of differentiation protocols for the generation of stem cells-derived p-cells.

[0009] Despite these efforts, existing protocols are very variable with efficiency (20-80%), time- consuming, expensive and difficult to reproduce. Moreover, derived p-cells often exhibit immaturity, non-functionality characterized by inadequate glucose-stimulated insulin secretion, and deficiencies in key transcription factors (TFs), leading to polyhormonal phenotypes. Recently, attention has shifted towards complementary approaches to progress the field, particularly, direct lineage reprogramming of developmental intermediates by forced expression of transcription factors. Direct cell reprogramming enables the conversion of one cell type (a source cell) directly into another cell type (a target cell) by forced expression of TFs that activate and stabilize the gene regulatory network (GRN) that defines target cell identity. Several computational methods have been established to analyze differential gene expression in source and target cell types, and to propose shortlists of candidate TFs for reprogramming (Chen et al., 2014; Taichai et al., 2012; Rackham et al., 2016; Weinreb et al., 2020). However, identifying TF combinations to efficiently convert source cells to target cells remains extremely challenging (Weinreb et al., 2020).

[0010] For p-cells, direct lineage reprogramming is increasingly proposed as an alternative to complex, lengthy and costly differentiation protocols. For example, TFs have been shown to reprogram certain cell types that are developmentally related to p-cells, e.g. pancreatic alpha and ductal cells, as well as certain gastrointestinal cells (Chen et al., 2014; Taichai et al., 2012; Rackham et al., 2016; Weinreb et al., 2020; Galivo et al., 2017; Akinci et al., 2012; Wang et al., 2018; Lee et al., 2013; Xiao et al., 2018). In 2008, Pdx1 , Ngn3 and MafA were shown to convert adult mouse pancreatic exocrine cells into p-cells in vivo, and since then the field has not progressed that well. This core set of TFs (sometimes supplemented by one or two additional TFs) have been shown to reprogram cells from other tissues derived from endoderm as shown in Figure 1 (Zhou et al., 2008; Tang et al., 2013; Sapir et al., 2005; Banga et al., 2014; Furuyama et al., 2019; McKimpson et al., 2019).

[0011] These reprogramming methods based on Pdx1 , Ngn3 and MafA expression have been shown to be sub-optimal and also result in immature [3-cell phenotype, emphasizing that there is a need to explore different starting cell sources with novel combinations of TFs for better reprogramming protocols.

[0012] Importantly, so far, cell culture protocols for reprogramming of stem cells into p-cells have been derived empirically and determined by trial and error, step by step elimination strategy and based on what has worked (or failed) before. There is a need to provide methods to enhance reprogramming of source cells to, for example, p-cells.

[0013] SUMMARY OF THE INVENTION

[0014] According to the present invention, novel combinations of TFs were identified and validated for reprogramming source cells (such as cultured pancreatic progenitor cells, cPPs) into p- cells in a more effective way compared to the known combination of Pdx1 , Ngn3 and MafA. Moreover, combinations of TFs were identified that can be delivered as a cocktail mix, or alternatively delivered sequentially, for reprogramming cPPs to p-cells. In addition, the p-cells reprogrammed using the novel combinations of the present disclosure are more competent and mature than those reprogrammed with the known combination of Pdx1 , Ngn3 and MafA.

[0015] In a first aspect, there is provided a composition comprising a combination of transcription factors or nucleotide sequences encoding the transcription factors for inducing or reprogramming a source cell into a [3-cell, wherein the source cell is a stem cell, a progenitor cell, a differentiated cell or mixtures thereof, and wherein the combination is selected from a group consisting of:

[0016] (a) Pax6, Ngn3, and MafA;

[0017] (b) Rfx6, Ngn3, and MafA;

[0018] (c) Pax6, Ngn3, Rfx6, and MafA;

[0019] (d) Nkx6.1 , Pdx1 , and MafA; and

[0020] (e) Pdx1 , Pax6, Rfx6, and Neurodi .

[0021] In some embodiments, Pax6 has 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%, at least 99%, or 100% amino acid sequence identity to SEQ ID NO: 1 or SEQ ID NO: 3; Ngn3 has 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%, at least 99%, or 100% amino acid sequence identity to SEQ ID NO: 5 or SEQ ID NO: 7; MafA has 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%, at least 99%, or 100% amino acid sequence identity to SEQ ID NO: 9 or SEQ ID NO: 11 ; Rfx6 has 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%, at least 99%, or 100% amino acid sequence identity to SEQ ID NO: 13 or SEQ ID NO: 15; Nkx6.1 has 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%, at least 99%, or 100% amino acid sequence identity to SEQ ID NO: 17 or SEQ ID NO: 19; Pdx1 has 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%, at least 99%, or 100% amino acid sequence identity to SEQ ID NO: 21 or SEQ ID NO: 23; and Neurodi has 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%, at least 99%, or 100% amino acid sequence identity to SEQ ID NO: 25 or SEQ ID NO: 27.

[0022] In some embodiments, Pax6 is encoded by a nucleotide sequence having at least 80%, at least 81%, at least 82%, 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%, at least 99%, or 100% identity to SEQ ID NO: 2 or SEQ ID NO: 4; Ngn3 is encoded by a nucleotide sequence having at least 80%, at least 81%, at least 82%, 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%, at least 99%, or 100% identity to SEQ ID NO: 6 or SEQ ID NO: 8; MafA is encoded by a nucleotide sequence having at least 80%, at least 81%, at least 82%, 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%, at least 99%, or 100% identity to SEQ ID NO: 10 or SEQ ID NO: 12; Rfx6 is encoded by a nucleotide sequence having at least 80%, at least 81%, at least 82%, 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%, at least 99%, or 100% identity to SEQ ID NO: 14 or SEQ ID NO: 16; Nkx6.1 is encoded by a nucleotide sequence having at least 80%, at least 81 %, at least 82%, 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%, at least 99%, or 100% to SEQ ID NO: 18 or SEQ ID NO: 20; Pdx1 is encoded by a nucleotide sequence having at least 80%, at least 81%, at least 82%, 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%, at least 99%, or 100% identity to SEQ ID NO: 22 or SEQ ID NO: 24; and Neurodi is encoded by a nucleotide sequence having at least 80%, at least 81 %, at least 82%, 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%, at least 99%, or 100% identity to SEQ ID NO: 26 or SEQ ID NO: 28.

[0023] In some embodiments, the stem cell is selected from a group consisting of pluripotent stem cell (PSC), multipotent stem cell, embryonic stem cell (ESC), and induced pluripotent stem cell (iPSC).

[0024] In some embodiments, the progenitor cell is a pancreatic progenitor cell. In some embodiments, the differentiated cell is an endoderm derived cell, such as a pancreatic cell (e.g. an exocrine pancreatic cell, a pancreatic duct cell, or an acinar pancreatic cell), a liver cell, an intestine cell, a stomach cell, a gall bladder cell.

[0025] In some embodiments, the source cell is mammalian cell, preferably a mouse cell or a human cell.

[0026] In a second aspect, there is provided a vector encoding at least the combination of transcription factors of the present disclosure.

[0027] In some embodiments, the vector is a viral vector; in particular a retrovirus, an adenovirus, a lentivirus, a herpes virus, a pox virus, or an adeno-associated virus vector.

[0028] In a third aspect, there is provided a method of inducing or reprogramming a source cell into a [3-cell, wherein the source cell is a stem cell, a progenitor cell a differentiated cell or mixtures thereof, comprising:

[0029] (i) contacting the source cell with the combination of the TFs or their encoding nucleotide sequences; and (ii) culturing the transduced cell in a cell media that supports growth of [3-cel Is.

[0030] In some embodiments, the nucleic acid sequences encoding the combination of transcription factors are located in one vector. In alternative embodiments, the nucleic acid sequences encoding the combination of transcription factors are located in multiple vectors. In such cases, the multiple vectors may be used simultaneously (i.e., as a mixture or delivered separately within one day) or sequentially (i.e., delivered in different days) to contact with the source cell.

[0031] In some embodiments, the method is in vivo, in vitro, or ex vivo.

[0032] In some embodiments, the nucleic acid sequences encoding the combination of transcription factors are operably linked to one or more inducible promoters. Advantageously, this allows for the production of a stable cell line of engineered source cells, whose fate can be switched upon activation of said inducible promoters. For example, inducible engineered source cells may be obtained in step (i) of the method of the third aspect.

[0033] In a fourth aspect, there is provided an isolated population of p-cells obtained by the method of the present disclosure. In some embodiments, the isolated population of p-cells of the present disclosure secrete more c-peptide in response to an increase in glucose compared to p-cells obtained using a combination of transcription factors Pdx1, Ngn3 and MafA.

[0034] In a fifth aspect, there is provided a pharmaceutical composition comprising a therapeutically effective amount of the isolated population of p-cells of the present disclosure and one or more pharmaceutically acceptable excipient.

[0035] In some embodiments the pharmaceutically acceptable excipient is selected from a group consisting of a buffer, a stabilizer, a preservative and combinations thereof.

[0036] In some embodiments, the pharmaceutical composition is an injectable formulation.

[0037] In a sixth aspect, there is provided for a method for treating a subject that is pre-diabetic or diabetic, comprising administering to the subject the isolated population of p-cells or the pharmaceutical composition of the present disclosure.

[0038] In a seventh aspect, there is provided use of the isolated population of p-cells of the present disclosure in the manufacture of a medicament for the treatment of diabetes in a subject in need thereof.

[0039] In an eighth aspect, there is provided a kit for use in a method of inducing or reprogramming a source cell into a p-cell, comprising i) a composition of any one of claims 1 to 4, or ii) one or more vectors comprising nucleic acid sequences encoding a combination of transcription factors selected from a group consisting of:

[0040] (a) Pax6, Ngn3, MafA;

[0041] (b) Rfx6, Ngn3, MafA;

[0042] (c) Pax6, Ngn3, Rfx6, MafA;

[0043] (d) Nkx6.1 , Pdx1 , MafA; and

[0044] (e) Pdx1 , Pax6, Rfx6, Neurodi ; or

[0045] (iii) transcription factors selected from a group consisting of:

[0046] (a) Pax6, Ngn3, MafA;

[0047] (b) Rfx6, Ngn3, MafA;

[0048] (c) Pax6, Ngn3, Rfx6, MafA;

[0049] (d) Nkx6.1 , Pdx1 , MafA; and

[0050] (e) Pdx1 , Pax6, Rfx6, Neurodi . BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Certain embodiments of the present disclosure are described more fully hereinafter with reference to the accompanying drawings.

[0052] Figure 1 shows a schematic of reprogramming endoderm cells to make insulin. Cells originating from endoderm can be converted to insulin positive cells by the induction and / or inhibition of TFs, with inhibitor TFs marked in dashed squares.

[0053] Figure 2 shows a schematic matrix for CombiCult® screening for transcription factor combinations that increase C-peptide expression.

[0054] Figure 3 shows an overview of negative and positive controls. In both the negative and positive control setups, cPPs are placed onto the beads using the same media. Media A consists of DMEM high glucose supplemented with Asc (50pg), 1% B27, EGF (50ng), FGF-7 (50ng), and RA (50nM). The purpose here is to guide the differentiation process towards pancreatic progenitors, particularly considering the early stage of cPPs. Following this, Media B is a combination of 5% KOSR, T3 (1 pM), and RA (25 nM), added to DMEM high glucose to foster endocrine progenitors. Finally, Media C comprises DMEM low glucose enriched with 10% FBS, T3 (1 pM), and Alk5i (10pM). In the negative control, no transfection occurs, while the positive control involves sequential transfection starting with Pdx1 , followed by Ngn3 and MafA.

[0055] Figure 4 shows scatter plots of reprogramming cPPs using new TF combinations. Groups with brackets indicate TF cocktails wherein TFs were provided as a mixture. Groups without brackets indicate TF cocktails wherein TFs were provided in sequence, -ve group has no TFs; +ve group has Pdx1 , Ngn3 and MafA.

[0056] Figure 5 shows FACS results of evaluation of reprogramming efficiency using identified TF combinations delivered in groups, (a) Quantification of reprogrammed cells (c-peptide+ cells) analyzed by FACS, (b) Histogram showing the fold change in reprogramming efficiency between controls (-ve group, and its efficiency was set as 1) and TF combinations.

[0057] Figure 6 shows histogram of glucose-dependent C-peptide secretion by spheroids containing cells reprogrammed using either a known TF combination or newly discovered TF combination 8226 in the presence of 2 mM glucose or 20 mM glucose. DETAILED DESCRIPTION OF THE INVENTION

[0058] Further details of the invention will now be described with reference to the following nonlimiting examples. Unless otherwise defined herein, technical and scientific terms used in the present description have the meanings that are commonly understood by those of ordinary skill in the art.

[0059] A. Definitions

[0060] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[0061] As used herein, the term “comprising” may include the embodiments “consisting of” and “consisting essentially of”. The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases that require the presence of the named features / steps and permit the presence of other features / steps. However, such description should be construed as also describing compositions, mixtures, or processes as “consisting of” and “consisting essentially of” the enumerated features / steps, which allows the presence of only the named features / steps, along with any impurities that might result therefrom, and excludes other features / steps.

[0062] As used herein, the term “therapeutically effective amount” or “efficacious amount” of a drug refers to an amount of the drug that is an amount sufficient to obtain a pharmacological response such as activating a biological target (e.g., increasing the expression or activity of Asns); or alternatively, is an amount of the drug that, when administered to a subject with a specified disorder or disease, is sufficient to have the intended effect, e g., treatment, alleviation, amelioration, palliation or elimination of one or more manifestations of the specified disorder or disease in the subject. A therapeutic effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses. Thus, a therapeutically effective amount may be administered in one or more administrations. The therapeutically effective amount will depend in part on the nature of the drug, the manner and route of administration, the stage and severity of the disease being treated, the weight and general state of health of the subject, and the judgment of the prescribing physician. Further, when the term “therapeutically effective amount” or “efficacious amount” is used to refer to a synergistic combination of antibiotic compounds, it means that each of said compounds is provided at an amount such that the combination, as a whole, would provide therapeutic effects. It would be understood that, for said synergistic combination of antibiotic compounds, the amount of one or both of said compounds in the combination, if used alone at the dose in the combination, may not provide a therapeutic effect.

[0063] As used herein, the term “subject” refers to animals, typically mammals. Any suitable mammal can be treated by a method described herein. Non-limiting examples of mammals include humans, non-human primates, domestic animals (e.g., dogs and cats), farm animals (e g., horses, cows, goats, sheep, pigs) and experimental animals (e.g., mouse, rat, rabbit, guinea pig). In some embodiments, a mammal is a human. A mammal can be any age or at any stage of development (e.g., an adult, teen, child, infant, or a mammal in utero). A mammal can be male or female. In some embodiments, a subject is a human. In some embodiments, a subject has or is diagnosed of having a particular disease, for example, heart failure.

[0064] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit, unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the present disclosure. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither, or both limits are included in the smaller ranges is also encompassed within the present disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the present disclosure.

[0065] As used herein, the term “transcription factor” or “TF” refers to a protein that binds to specific parts of DNA using DNA binding domains and is part of the system that controls the transcription of genetic information from DNA to RNA.

[0066] As used herein, the term “reprogramming” refers to the process that alters or reverses the differentiation state of a somatic cell. The cell can either be partially or terminally differentiated prior to the reprogramming. Reprogramming encompasses complete reversion of the differentiation state of a somatic cell to a pluripotent cell. Such complete reversal of differentiation produces an induced pluripotent (iPS) cell. A partial reversal of differentiation produces a partially induced pluripotent (PiPS) cell. Reprogramming also encompasses partial reversion of the differentiation state, for example to a multipotent state or to a somatic cell that is neither pluripotent or multipotent, but is a cell that has lost one or more specific characteristics of the differentiated cell from which it arises, e.g. direct reprogramming of a differentiated cell to a different somatic cell type. Reprogramming generally involves alteration, e.g., reversal, of at least some of the heritable patterns of nucleic acid modification (e g., methylation), chromatin condensation, epigenetic changes, genomic imprinting, etc., that occur during cellular differentiation as a zygote develops into an adult.

[0067] As used herein, the term “pluripotent” refers to a cell with the capacity, under different conditions, to differentiate to more than one differentiated cell type, and preferably to differentiate to cell types characteristic of all three germ cell layers. Pluripotent cells are characterized primarily by their ability to differentiate to more than one cell type, preferably to all three germ layers, using, for example, a nude mouse teratoma formation assay. Pluripotency is also evidenced by the expression of embryonic stem (ES) cell markers, although the preferred test for pluripotency is the demonstration of the capacity to differentiate into cells of each of the three germ layers.

[0068] As used herein, the term “differentiated cell” is meant any primary cell that is not, in its native form, pluripotent as that term is defined herein. It should be noted that placing many primary cells in culture can lead to some loss of fully differentiated characteristics. However, simply culturing such cells does not, on its own, render them pluripotent. The transition to pluripotency requires a reprogramming stimulus beyond the stimuli that lead to partial loss of differentiated character in culture. Reprogrammed pluripotent cells also have the characteristic of the capacity of extended passaging without loss of growth potential, relative to primary cell parents, which generally have capacity for only a limited number of divisions in culture. Stated another way, the term “differentiated cell” refers to a cell of a more specialized cell type derived from a cell of a less specialized cell type (e.g., a stem cell such as an induced pluripotent stem cell) in a cellular differentiation process.

[0069] As used herein, the term “an endoderm derived cell” refers to a cell of endoderm origin and includes any cell which has developed from an endoderm cell. Without wishing to be bound by theory, liver and pancreas progenitors develop from endoderm cells in the embryonic foregut. Shortly after their specification, liver and pancreas progenitors rapidly acquire markedly different cellular functions and regenerative capacities. These changes are elicited by inductive signals and genetic regulatory factors that are highly conserved among vertebrates. Interest in the development and regeneration of the organs has been fueled by the intense need for hepatocytes and pancreatic p cells in the therapeutic treatment of liver failure and type I diabetes. Studies in diverse model organisms and humans have revealed evolutionarily conserved inductive signals and transcription factor networks that elicit the differentiation of liver and pancreatic cells and provide guidance for how to promote hepatocyte and p cell differentiation from diverse stem and progenitor cell types. As used herein, a “vector” refers to a nucleic acid molecule, such as a dsDNA molecule that provides a useful biological or biochemical property to an inserted nucleotide sequence, such as the nucleic acid constructs or replacement cassettes described herein. Examples include plasmids, phages, autonomously replicating sequences (ARS), centromeres, and other sequences that are able to replicate or be replicated in vitro or in a host cell, or to convey a desired nucleic acid segment to a desired location within a host cell. A vector can have one or more restriction endonuclease recognition sites (whether type I, II or Ils) at which the sequences can be cut in a determinable fashion without loss of an essential biological function of the vector, and into which a nucleic acid fragment can be spliced or inserted in order to bring about its replication and cloning. Vectors can also comprise one or more recombination sites that permit exchange of nucleic acid sequences between two nucleic acid molecules. Vectors can further provide primer sites, e.g., for PCR, transcriptional and / or translational initiation and / or regulation sites, recombination signals, replicons, additional selectable markers, etc. A vector can further comprise one or more selectable markers suitable for use in the identification of cells transformed with the vector.

[0070] As used herein, the term “simultaneously” is used to mean that at least two agents (such as vectors) are delivered or administered concurrently or as a mixture. The term “sequentially” means that at least one agent is delivered or administered a period of time after other agents. The period may be any efficacious period, such as 30 min, 1 hr, 5 hr, 12 hr, 24 hr etc., and can be determined by the skilled person.

[0071] As used herein, the term “sequence identity” refers to sequence identity between two nucleic acid molecules or polypeptides. Identity can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. For example, when a position in the compared nucleotide sequence is occupied by the same base, then the molecules are identical at that position. A degree of sequence identity between nucleic acid or amino acid sequences is a function of the number of identical or matching nucleotides or amino acids at shared positions. For example, polypeptides having at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to specific polypeptides described herein and preferably exhibiting substantially the same functions, as well as polynucleotides encoding such polypeptides, are contemplated. Truncated versions of the specific polypeptides described herein preferably exhibiting substantially the same functions, as well as polynucleotides encoding such polypeptides, are also contemplated. Methods and computer programs for determining both sequence identity and similarity are publicly available, including, but not limited to, the GCG program package, BLASTP, BLASTN, FASTA, and the ALIGN program. The well-known Smith Waterman algorithm may also be used to determine similarity. The BLAST program is publicly available from NCBI and other sources.

[0072] B. Induction and reprogramming

[0073] As disclosed herein, the present disclosure relates to compositions, nucleic acid constructs, vectors, engineered source cells, and methods thereof for cell induction or reprogramming cell to p-cell, based, in part, on the surprisingly effect described herein of novel use and combinations of transcription factors that permit induction or reprogramming of differentiated or undifferentiated cells into p-cell. Such compositions, nucleic acid constructs, vectors, engineered source cells, and methods can be used for inducing or reprogramming p-cells in vitro, ex vivo, or in vivo, and these obtained p-cells can be used for treating diabetes.

[0074] An aspect of the present invention relates to compositions and methods for the induction or reprogramming of a source cell, such as a stem cell, a progenitor cell or a differentiated cell or mixtures thereof to a pancreatic p-cell using the following combinations of TFs identified in the present disclosure:

[0075] (a) Pax6, Ngn3, and MafA;

[0076] (b) Rfx6, Ngn3, and MafA;

[0077] (c) Pax6, Ngn3, Rfx6, and MafA;

[0078] (d) Nkx6.1 , Pdx1 , and MafA; and

[0079] (e) Pdx1 , Pax6, Rfx6, and Neurodi .

[0080] “Pax6” refers to the Pax6 protein having an amino acid sequence of SEQ ID NO: 1 (human) or SEQ ID NO: 3 (mouse). The term Pax6 also encompasses species variants, homologues, allelic forms, mutant forms, and equivalents thereof, including conservative substitutions, additions, deletions therein not adversely affecting the structure of function. Pax6 is referred in the art as aliases; paired box 6, AN, AN1, AN2, ASGD5, D11S812E, FVH1 , MGDA, WAGR. Human Pax6 is encoded by nucleic acid sequence of SEQ ID NO: 2. Mouse Pax6 is encoded by nucleic acid sequence of SEQ ID NO: 4. PAXAacts as a "master control" gene for the development of eyes and other sensory organs, certain neural and epidermal tissues as well as other homologous structures, usually derived from ectodermal tissues. Pax6 is expressed as a transcription factor when neural ectoderm receives a combination of weak Sonic hedgehog (SHH) and strong TGF-Beta signaling gradients. The term "Pax6” as used herein refers to a polypeptide having a naturally occurring amino acid sequence of a Pax6 protein or a fragment, variant, or derivative thereof that at least in part retains the biological activity of the naturally occurring protein. In addition to naturally-occurring allelic variants of the Pax6 sequences that may exist in the population, it will be appreciated that, as is the case for virtually all proteins, a variety of changes can be introduced into the sequences of SEQ ID NO: 1 or SEQ ID NO: 3 (referred to as "wild type" sequences) without substantially altering the functional (biological) activity of the polypeptides. Such variants are included within the scope of the terms "Pax6".

[0081] "Ngn3" refers to the Ngn3 protein having an amino acid sequence of SEQ ID NO: 5 (human) or SEQ ID NO: 7 (mouse). The term Ngn3 also encompasses species variants, homologues, allelic forms, mutant forms, and equivalents thereof, including conservative substitutions, additions, deletions therein not adversely affecting the structure of function. Ngn3 is referred in the art as aliases; neurogenin 3; Atoh5; Math4B; bHLHa7; NEUROG3. Human Ngn3 is encoded by nucleic acid sequence of SEQ ID NO: 6. Mouse Ngn3 is encoded by nucleic acid sequence of SEQ ID NO: 8. Ngn3 belongs to a family of basic helix- loop-helix transcription factors involved in the determination of neural precursor cells in the. The term "Ngn3” as used herein refers to a polypeptide having a naturally occurring amino acid sequence of a Ngn3 protein or a fragment, variant, or derivative thereof that at least in part retains the biological activity of the naturally occurring protein. In addition to naturally-occurring allelic variants of the Ngn3 sequences that may exist in the population, it will be appreciated that, as is the case for virtually all proteins, a variety of changes can be introduced into the sequences of SEQ ID NO: 5 or SEQ ID NO: 7 (referred to as "wild type" sequences) without substantially altering the functional (biological) activity of the polypeptides. Such variants are included within the scope of the terms "Ngn3".

[0082] "MafA" refers to the MafA protein having an amino acid sequence of SEQ ID NO: 9 (human) or SEQ ID NO: 11 (mouse). The term MafA also encompasses species variants, homologues, allelic forms, mutant forms, and equivalents thereof, including conservative substitutions, additions, deletions therein not adversely affecting the structure of function. MafA is referred in the art as aliases; v-maf musculoaponeurotic fibrosarcoma oncogene homolog A (avian), hMafA; RIPE3bl; MAFA. Human MafA is encoded by nucleic acid sequence of SEQ ID NO: 10. Mouse MafA is encoded by nucleic acid sequence of SEQ ID NO: 12. MAFA is a transcription factor that binds RIPE3b, a conserved enhancer element that regulates pancreatic beta cell-specific expression of the insulin gene. The term "MafA" as used herein refers to a polypeptide having a naturally occurring amino acid sequence of a MafA protein or a fragment, variant, or derivative thereof that at least in part retains the biological activity of the naturally occurring protein. In addition to naturally-occurring allelic variants of the MafA sequences that may exist in the population, it will be appreciated that, as is the case for virtually all proteins, a variety of changes can be introduced into the sequences of SEQ ID NO: 9 or SEQ ID NO: 11 (referred to as "wild type" sequences) without substantially altering the functional (biological) activity of the polypeptides. Such variants are included within the scope of the terms "MafA".

[0083] “Rfx6” refers to the Rfx6 protein having an amino acid sequence of SEQ ID NO: 13 (human) or SEQ ID NO: 15 (mouse). The term Rfx6 also encompasses species variants, homologues, allelic forms, mutant forms, and equivalents thereof, including conservative substitutions, additions, deletions therein not adversely affecting the structure of function. Rfx6 is referred in the art as aliases; regulatory factor X6, MTCHRS, MTFS, RFXDC1 , dJ955L16.1. Human Rfx6 is encoded by nucleic acid sequence of SEQ ID NO: 14. Mouse Rfx6 is encoded by nucleic acid sequence of SEQ ID NO: 16. Rfx6 is a member of the regulatory factor X (RFX) family of transcription factors, and specifically required for the differentiation of islet cells for the production of insulin. The term "Rfx6" as used herein refers to a polypeptide having a naturally occurring amino acid sequence of a Rfx6 protein or a fragment, variant, or derivative thereof that at least in part retains the biological activity of the naturally occurring protein. In addition to naturally-occurring allelic variants of the Rfx6 sequences that may exist in the population, it will be appreciated that, as is the case for virtually all proteins, a variety of changes can be introduced into the sequences of SEQ ID NO: 13 or SEQ ID NO: 15 (referred to as "wild type" sequences) without substantially altering the functional (biological) activity of the polypeptides, and such variants are included within the scope of the terms "Rfx6".

[0084] “Nkx6.1’’ refers to the Nkx6.1 protein having an amino acid sequence of SEQ ID NO: 17 (human) or SEQ ID NO: 19 (mouse). The term Nxk6.1 also encompasses species variants, homologues, allelic forms, mutant forms, and equivalents thereof, including conservative substitutions, additions, deletions therein not adversely affecting the structure of function. Nkx6.1 is referred in the art as aliases; NK6 homeobox 1 ; NKX6A; NKX6.1 and NKX6-1. Human Nkx6.1 is encoded by nucleic acid sequence of SEQ ID NO: 18. Mouse Nkx6.1 is encoded by nucleic acid sequence of SEQ ID NO: 20. In the pancreas, NKX6.1 is required for the development of beta cells and is a potent bifunctional transcription regulator that binds to AT -rich sequences within the promoter region of target genes. The term "Nkx6.1" as used herein refers to a polypeptide having a naturally occurring amino acid sequence of a Nkx6.1 protein or a fragment, variant, or derivative thereof that at least in part retains the biological activity of the naturally occurring protein. In addition to naturally-occurring allelic variants of the Nkx6.1 sequences that may exist in the population, it will be appreciated that, as is the case for virtually all proteins, a variety of changes can be introduced into the sequences of SEQ ID NO: 17 or SEQ ID NO: 19 (referred to as "wild type" sequences) without substantially altering the functional (biological) activity of the polypeptides, and such variants are included within the scope of the terms "Nkx6.1".

[0085] "Pdx1" refers to the Pdx1 protein having an amino acid sequence of SEQ ID NO: 21 (human) or SEQ ID NO: 23 (mouse). The term Pdx1 also encompasses species variants, homologues, allelic forms, mutant forms, and equivalents thereof, including conservative substitutions, additions, deletions therein not adversely affecting the structure of function. Pdx1 is referred in the art as aliases; pancreatic and duodenal homeobox 1 , IDX- 1, STF-I, PDX-I, M0DY4, Ipfl. Human Pdx1 is encoded by nucleic acid sequence of SEQ ID NO: 22. Mouse Pdx1 is encoded by nucleic acid sequence of SEQ ID NO: 24. Pdx1 protein is a transcriptional activator of several genes, including insulin, somatostatin, glucokinase, islet amyloid polypeptide, and glucose transporter type 2 (GLUT2). Pdx1 is a nuclear protein is involved in the early development of the pancreas and plays a major role in glucose- dependent regulation of insulin gene expression. Defects in the gene encoding the Pdx1 protein are a cause of pancreatic agenesis, which can lead to early-onset insulin-dependent diabetes mellitus (NIDDM), as well as maturity onset diabetes of the young type 4 (M0DY4). The term "Pdx1" as used herein refers to a polypeptide having a naturally occurring amino acid sequence of a Pdx1 protein or a fragment, variant, or derivative thereof that at least in part retains the biological activity of the naturally occurring protein. In addition to naturally-occurring allelic variants of the Pdx1 sequences that may exist in the population, it will be appreciated that, as is the case for virtually all proteins, a variety of changes can be introduced into the sequences of SEQ ID NO: 21 or SEQ ID NO: 23 (referred to as "wild type" sequences) without substantially altering the functional (biological) activity of the polypeptides. Such variants are included within the scope of the terms "Pdx1".

[0086] "Neurodi" refers to the Neurodi protein having an amino acid sequence of SEQ ID NO: 25 (human) or SEQ ID NO: 27 (mouse). The term Neurodi also encompasses species variants, homologues, allelic forms, mutant forms, and equivalents thereof, including conservative substitutions, additions, deletions therein not adversely affecting the structure of function. Neurodi is referred in the art as aliases; neuronal differentiation 1 , BETA2, BHF-1 , M0DY6, NEUROD, T2D, bHLHa3. Human Neurodi is encoded by nucleic acid sequence of SEQ ID NO: 26. Mouse Neurodi is encoded by nucleic acid sequence of SEQ ID NO: 28. Neurodi is a member of the Neurod family of basic helix-loop-helix (bHLH) transcription factors. It forms heterodimers with other bHLH proteins and activates transcription of genes that contain a specific DNA sequence known as the E-box, and regulates expression of the insulin gene. The term "Neurodi" as used herein refers to a polypeptide having a naturally occurring amino acid sequence of a Neurodi protein or a fragment, variant, or derivative thereof that at least in part retains the biological activity of the naturally occurring protein. In addition to naturally- occurring allelic variants of the Neurodi sequences that may exist in the population, it will be appreciated that, as is the case for virtually all proteins, a variety of changes can be introduced into the sequences of SEQ ID NO: 25 or SEQ ID NO: 27 (referred to as "wild type" sequences) without substantially altering the functional (biological) activity of the polypeptides. Such variants are included within the scope of the terms "Neurodi".

[0087] In some embodiments, the source cell is a stem cell selected from a group consisting of pluripotent stem cell (PSC), multipotent stem cell, embryonic stem cell (ESC), and induced pluripotent stem cell (iPSC). In some embodiments, the progenitor cell is a pancreatic progenitor cell, such as a cultured pancreatic progenitor cell.

[0088] In some embodiments, the source cell is a differentiated cell, preferably an endoderm derived cell. Examples of endoderm derived cell include but are not limited to a pancreatic cell (e.g. an exocrine pancreatic cell, a pancreatic duct cell, or an acinar pancreatic cell), a liver cell, an intestine cell, a stomach cell, a gall bladder cell.

[0089] In some embodiments, the transcription factors are delivered into the source cell by way of nucleic acid sequences having at least 80%, at least 81%, at least 82%, 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%, at least 99%, or 100% identity to the nucleotide sequence encoding the transcription factors, or byway of vectors comprising said nucleic acid sequences, due to gene code degeneracy. Said nucleic acid sequences may be DNA or RNA. Preferably, said nucleic acid sequences are mRNAs.

[0090] In some embodiments, the vector is a viral vector. Some viral-mediated expression methods employ retrovirus, adenovirus, lentivirus, herpes virus, pox virus, and adeno-associated virus (AAV) vectors, and such expression methods have been used in gene delivery and are well known in the art. Vectors used in the present invention can further comprise, in some embodiments, other nucleic acid elements for the regulation, expression, stabilization of the construct or of other vector genetic elements, for example, promoters, enhancers, TATA-box, ribosome binding sites, IRES, as known to one of ordinary skill in the art.

[0091] One vector may comprise one or more nucleotide sequences encoding the transcription factors. For example, nucleotide sequences encoding Pax6, Ngn3, and MafA respectively may be positioned under the control of the same or different promoters in one vector. Alternatively, two of Pax6, Ngn3, and MafA encoding sequences may locate in one vector, while the other encoding sequence is located in a different vector. Alternatively, each of Pax6, Ngn3, and MafA encoding sequences may locate in different vectors. This also applies to other combinations.

[0092] In embodiments wherein the nucleotide sequences encoding the combination of TFs locate in multiple (i.e., at least two) vectors, such vectors can be delivered to or contact with the source cell simultaneously or sequentially.

[0093] In some embodiments, the nucleic acid sequences encoding the combination of transcription factors are operably linked to one or more inducible promoters. Advantageously, this allows for the production of a stable cell line of engineered source cells, whose fate can be switched upon activation of said inducible promoters to express the transcription factors. Expression of the transcription factor combination will induce the engineered source cells to become, for example, p-cells. Inducible systems are known in the art, such as the Tet-on / off system which could be used to express one or more TFs, delivered via lentiviral vectors or integrated into one or more well-characterized safe harbor site(s) in the human genome. This system will allow precise temporal control over TF activation, enabling the circuits to function as molecular switches that can be turned on or off at any stage of differentiation.

[0094] In one preferred embodiment, the TF combinations may be delivered as mRNAs encoding the corresponding TFs. Preferably, these TF encoding mRNAs may be delivered by any suitable delivery vehicles known in the art, such as liposome nanoparticles (LNPs).

[0095] TF combinations of the present disclosure can be delivered to induce or reprogramming the source cell in vitro, ex vivo or in vivo.

[0096] The obtained p-cells (i.e., isolated population of p-cells) produced by the induction or reprogramming method of the present disclosure secrete more insulin in response to an increase in glucose compared to p-cells obtained using a known combination of transcription factors Pdx1 , Ngn3 and MafA. That is, the isolated population of p-cells of the present disclosure is more competent and mature than the reprogrammed p-cells obtained by known methods.

[0097] Thus, the isolated population of p-cells of the present disclosure may be used for treating diabetes (such as Type 1, Type 2, Type 3, and Type 4 diabetes mellitus) in a subject in need thereof. The isolated population of p-cells of the present disclosure may also be formulated as a pharmaceutical composition. Accordingly, an aspect of the present disclosure provides a pharmaceutical composition comprising a therapeutically effective amount of the isolated population of p-cells of the present disclosure and a pharmaceutically acceptable excipient.

[0098] Suitable pharmaceutically acceptable excipient known in the art may be used in the present disclosure. For example, the pharmaceutically acceptable excipient may be selected from a group consisting of a buffer, a stabilizer, a preservative, and combinations thereof. Choice of the cellular excipient and any further accompanying elements of the composition comprising a population of pancreatic p-cells as disclosed herein will be adapted in accordance with the dosage form of the formulation, as well as route and device used for administration. Preferably, the pharmaceutical composition is an injectable formulation. In some embodiments, a composition comprising a population of pancreatic p-cells can also comprise one or more other ingredients that facilitate the engraftment or functional mobilization of the pancreatic p- cells. Suitable ingredients include matrix proteins that support or promote adhesion of the pancreatic p-like cells, or complementary cell types, especially endothelial cells. In another embodiment, the composition may comprise absorbable or biodegradable matrix scaffolds.

[0099] Another aspect relates to a kit for use in a method of inducing or reprogramming a source cell into a p-cell, comprising i) a composition of any one of claims 1 to 4, or ii) one or more vectors comprising nucleic acid sequences encoding a combination of transcription factors selected from a group consisting of:

[0100] (a) Pax6, Ngn3, MafA;

[0101] (b) Rfx6, Ngn3, MafA;

[0102] (c) Pax6, Ngn3, Rfx6, MafA;

[0103] (d) Nkx6.1 , Pdx1 , MafA; and

[0104] (e) Pdx1 , Pax6, Rfx6, Neurodi ; or

[0105] (iii) transcription factors selected from a group consisting of:

[0106] (a) Pax6, Ngn3, MafA;

[0107] (b) Rfx6, Ngn3, MafA;

[0108] (c) Pax6, Ngn3, Rfx6, MafA;

[0109] (d) Nkx6.1, Pdx1 , MafA; and

[0110] (e) Pdx1 , Pax6, Rfx6, Neurodi .

[0111] In some embodiments, the kit comprises culture medium for culturing the source cell. The kit may be used in vivo or in vitro to induce or reprogram a source cell into a p-cell.

[0112] It should be understood that any and all embodiments of the present disclosure can be combined with technical features in any other embodiment or multiple other embodiments to obtain additional embodiments under the premise of no conflict. The invention includes such combinations resulting in further embodiments.

[0113] EXAMPLES

[0114] The following examples are intended to exemplify the present disclosures and are not limitations of the claimed invention. All molecules, compositions, methods, assays, and results disclosed in the examples and other sections of the specification, figures, and claims form part of the disclosure of the invention.

[0115] Example 1. Materials and methods mRNA production and transfection

[0116] DNA template preparation and mRNA synthesis were conducted according to HiScribeTM T7 ARCA mRNA Kit (with tailing) (NED, #E2060S) protocol. Human transcription factor sequences were used in the following examples.

[0117] For transfection with jetMESSENGER® (Axil Scientific, #150-07), 2 pg of mRNA were diluted in 200 pl of mRNA buffer (provided by the kit) followed by thorough mix for 10 seconds. Then 4 pl of jetMESSENGER® reagent was added and gently combined to the mix. After 10 minutes incubation at RT, transfection was performed by addition of the transfection mix to the cells in medium without antibiotics. For serial transfection, the transfection mix was sequentially added in the culture every 24 hours.

[0118] Immunofluorescence

[0119] For Immunostaining, cells were fixed with 4% Paraformaldehyde (PFA) (Sigma Aldrich, #78775) at room temperature (RT) for 30 minutes and washed three times with DPBS for 5 minutes each. For soluble proteins, blocking solution is 10% Bovine Serum Albumin (BSA) (Sigma Aldrich, #05470) or 10% Fetal Bovine Serum (FBS) (Thermo Scientific, #26140079) with 0.5% Triton X-100 (Sigma Aldrich, #T8787), whereas for membrane proteins, Triton X- 100 was excluded. Cells were incubated with blocking solution for an hour at RT. Primary antibody diluted in blocking solution, was added to cells and incubated at 4°C overnight. Next day, three washes were performed with DBPS for 5 min each. Secondary Antibody was diluted at 1 :500 in DPBS and incubated at RT for 2 hours in dark. Subsequently, three washes with DPBS were performed for 5 min each. For the visualization of cell nuclei, Hoechst 3342 dye (Thermo Fisher Scientific, #62249) was employed, with a dilution ration of 1 :10,000 (in DPBS). The dye was then incubated in dark for 5-10 minutes followed by three washes in DPBS. Lastly, the staining was detected using fluorescence microscopy (Inverted Live Cell Confocal Microscope-LSM800 Airy, Clinical Science Building (CSB)) (Inverted Fluorescence Live Cell Microscope-A07, CSB).

[0120] Flow cytometry immunostaininq

[0121] For the quantification of the efficiency of the serial transfection, FACS was performed. cPPs were dissociated from the beads and fixed with 10OpI of 4% PFA for 15 min at RT. Two washes were apply with staining buffer (BD, #554657) before permeabilized with blocking buffer for 30 min at RT. After centrifugation, conjugated antibodies were diluted at 1 :20 in blocking buffer for 30 min on ice in dark. Following incubation, two washes were performed with DPBS. At last, cells were suspended in DPBS and analyzed through FACS.

[0122] For the quantification of c-peptide+ cells using primary and secondary antibodies, a standardized protocol was implemented. Initially, cells were dissociated and fixed with 100 pl of 4% PFA for 30 minutes on ice. Subsequently, a single wash with 200 pl of DPBS was carried out to remove excess fixative and cellular debris before proceeding to blocking. Blocking was performed on ice for 30 minutes to minimize non-specific binding of antibodies. Following this, incubation with the primary antibody was conducted overnight at low temperature to facilitate specific binding of the primary antibody to its target antigen. The next day, the cells underwent two washes with blocking solution to remove unbound primary antibody, followed by blocking with the secondary antibody on ice for 2 hours to enable detection of the primary antibody. After two additional washes with DPBS to remove excess secondary antibody, the cells were carefully collected in FACS tubes for subsequent flow cytometry analysis to quantify the c- peptide+ cells accurately. The primary antibody used in this method is Mouse anti-pro-lnsulin c-peptide (Millipore, #05-1109) diluted at 1:1000. The secondary antibody used in this method is Donkey anti-Mouse, Alexa Fluor 488 (Thermo Fisher Scientific, #A-21202) diluted at 1 :500.

[0123] Fluorescence-activated cell sorting (FACS)

[0124] FACS (LSRFortessa X-20, CSB) was performed using an LSRFortessa X-20 (CSB) to assess and quantify transfected cells within the population. A dot plot of side scatter (SSC) versus forward scatter (FSC) was utilized to identify and separate live cells from doublets and dead cells. For the live cells, a dot plot of GFP / APC / PerCP versus FSC was created for additional analysis. All FACS data were evaluated using FlowJo software. CombiCult® workflow

[0125] Cultured Pancreatic Progenitors (cPPs) were selected as the initial material for the CombiCult® screening process due to their practicality, facilitating rapid and sustained expansion. Additionally, these cPPs exhibit developmental proximity to pancreatic p-cells. a) CombiCult® screen. i. Seeding of cells onto the beads. cPPs were seeded onto CombiCult® beads. For effective seeding, the beads were coated with matrigel (dilution 1 :100; BD, #354277) for at least 2 hours at 37°C. Single cells were generated from confluent cPPs with GCDR incubated for 10 min at 37°C. Cells were counted to achieve the desired seeding density, and following centrifugation, cPPs were reconstituted in their basal media plus supplements and Rock inhibitor Y27632 (15). Approximately 4.0 x 107 cPPs (250 cells / bead) were seeded onto 160,000 beads (40,000 / condition), and the mixture (cells and beads) were incubated at 37°C overnight. ii. Pool / Split process. Based on the designed matrix (Figure 2), the cell-seeded beads were distributed into a range of predetermined conditions (10 conditions, each expressing a specific TF ;(Pdx1 , Ngn3, Nkx2.2, Neurodi, Nkx6.1 , Maf (MafA, MafB), Isl 1 , Rfx6, Pax4, or Pax6) from in vitro transcribed mRNA) for four days using a splitpool process, allowing to test 10 thousand different combinations of TFs. iii. mRNA transfections and addition of fluorescence tags. Subsequent days, mRNAs-TFs transfections were performed according to the matrix followed by addition of CombiCult* specific fluorescent tags at each split (Figure 2). Simple medium with few growth factors and known molecules was used to push cPPs into p-cells via endocrine progenitors. First 2 days, DMEM high glucose was supplemented with Asc (50 pg), 1 % B27, EGF (50 ng), FGF-7 (50 ng) and RA (50 nM), while next two days 5% KOSR, T3 (1 pM) and RA (25 nM) were added to DMEM high glucose. From day 5 to day 11 , DMEM low glucose was enriched with 10% FBS, T3 (1 pM) and Alk5i (10 pM). iv. Screening assay. Following the pool / split process, the beads were stained at day 11 with antibody against c-peptide, functional pancreatic p-cells marker (Figure 2).

[0126] Concurrently with the CombiCult® screen, negative and positive controls were implemented to fine-tune the c-peptide staining protocol and establish criteria and parameters for isolating the positive beads. In both the cases, same media of CombiCult® experiment were adopted. However, positive reprogramming sample went through serial mRNA transfections of Pdx1 (day 1), Ngn3 (day 2) and MafA (day 3), known trio of factors that induce reprogramming to p- cells, while mRNA transfections were not performed in the negative control. The beads from the screen were transferred in Eppendorf tubes. Once the beads were settled, the media was removed and a wash with DPBS was performed to discard remaining media. Next, the beads were fixed with 4% PFA (in DBPS) at RT for 30 minutes and washed three times with DPBS for 5 minutes each. Subsequently, beads were incubated with blocking solution 10% BSA or FBS with 0.5% Triton X-100 for an hour. Primary antibody (Mouse anti-pro-lnsulin c-peptide (Millipore, #05-1109)) was diluted in blocking solution (1 :1000), added to beads and incubated at 4°C overnight. Next day, three washes were performed with DBPS for 5 min each. Secondary Antibody (Donkey anti-Mouse, Alexa Fluor 488 (Thermo Fisher Scientific, #A- 21202)) was diluted at 1 :500 in DPBS and incubated at RT for 2 hours in dark. Lastly, before detection, three washes with DPBS were performed for 5 min each. b) COPASTM sorting. Positive and negative controls (Figure 3), both unstained and stained, were utilized to define gating parameters, facilitating the discrimination between negative and positive bead populations. Subsequently, all stained beads from the CombiCult® experiment were subjected to screening using a particle flow sorter known as COPAS (Complex Object Parametric Analyzer and Sorter, Union Biometrica FP 2000 PRO, CSB). By utilizing the gating thresholds established by the positive and negative controls, the COPAS system analyzed the fluorescence intensity of the beads. Through this analysis, the COPAS system identified positive hits containing reprogrammed |3-cells, specifically c-peptide+beads. c) Bead digestion and isolation of tags. The c-peptide+beads were individually digested according to Plasticell’s proprietary protocol (Tarunina M, et al., 2014) to release the fluorescence tags accumulated by the bead during the experiment. d) Tag analysis. The released tags were analyzed using FACS and the data produced were submitted and deconvoluted by proprietary CombiCult* bioinformatics program Ariadne. e) Ariadne Bioinformatics. Ariadne has the capability to discern specific fluorescence signal intensities as distinct tags, thereby elucidating the pathways or combinations of four TFs responsible for reprogramming cPPs into |3-cells. Furthermore, through statistical analysis, it can rank the most effective reprogramming combinations (i.e., protocols) which are subsequently validated. Specifically, Ariadne ranks the protocols based on matching conditions, the number of beads within a cluster, cluster size, and the probability of the cluster occurring by chance. f) Validation.

[0127] The validation of the top-ranked protocols started with bead-based replication of the CombiCult® results. Approximately 4.0 x 107cPPs (250 cells / bead) were seeded onto matrigel-coated beads, followed by sequential delivery of 2 pg each TF mRNA over four consecutive days or simultaneous delivery of 2 pg total TF mRNA in a mixture in a single day, using the same media as the screening process. After an incubation period of 11 days, as described in (iii) of the CombiCult® workflow above, the beads were stained with antibody against c-peptide and screened using COPAS to identify those exhibiting c-peptide signals. The most effective combinations were determined by comparing the distribution of c-peptide fluorescence signals among negative and positive controls, as well as between sequential and simultaneous TF-delivery methods.

[0128] Subsequently, the ranked protocals underwent validation in spheroids, a common and natural method for obtaining p-cells. In this approach, cPPs were seeded as single cells in 10 cm dishes coated with matrigel (one dish containing 12 million cells, with two dishes per sample). TFs were then delivered sequentially over four days or simultaneously as a mixture in a single day, followed by the formation of spheroids using 24 million cells (200 cells per spheroid). Transfection was performed as described previously; for each 10 cm dish, 10 pg of mRNA was diluted in 1000 pl of mRNA buffer, and 20 pl of jetMESSENGER® was added to the mix. Media was changed regularly until day 11 , as for the reprogramming experiments. At this point, some spheroids were stained with antibody against c-peptide for immunofluorescence analysis, while others were dissociated into single cells and stained with c-peptide to quantify through FACS the reprogramming efficiency as a measure of c-peptide expression. Additionally, another subset of spheroids was used to measure the secretion of c-peptide upon stimulation with elevated glucose levels.

[0129] When delivered sequentially over four days, the combinations were delivered according to the following order:

[0130] Combination 2026: Ngn3 on day 1>Pax6 on day 2>Ngn3 on day 3>MafA on day 4; Combination 8226: Rfx6 on day 1 >Ngn3 on day 2>Ngn3 on day 3>MafA on day 4; Combination 0286: Pax6 on day 1 >Ngn3 on day 2>Rfx6 on day 3>MafA on day 4; Combination 5166: Nkx6.1 on day 1>Pdx1 on day 2>MafA on day 3>MafA on day 4; Combination 1084: Pdx1 on day 1 >Pax6 on day 2>Rfx6 on day 3>Neurod1 on day 4.

[0131] Glucose-stimulated insulin secretion Glucose-stimulated insulin secretion (GSIS) assays are frequently employed in research to evaluate the functional competence of differentiated and reprogrammed p-cells. These assays involve exposing the cells to different glucose concentrations, typically low glucose (2 mM) followed by high glucose (20 mM), to assess their insulin secretion response to glucose stimulation. C-peptide secreted in low and high glucose conditions was collected and quantified using an ELISA assay (Ultrasensitive C-peptide ELISA, Mercodia #10-1141-01).

[0132] Example 2. Identification and validation of novel TF combinations

[0133] Using cultured pancreatic progenitor cells (cPPs) as the source cell, 10,000 different combinations of 10 TFs (i.e., Pdx1 , Ngn3, MafA / B, Pax6, Pax4, Isl1 , Neurodi, Nkx6.1 , Nkx2.2 and Rfx6) important in pancreatic development were screened employing the Combicult® screening method. The TFs were delivered in the form of mRNA, thus leaving no genetic footprint.

[0134] These combinations were first validated with the bead-based replication CombiCult* method, wherein the TFs were added serially in order or as a mixture. It was found that for most combinations, there was no significant difference whether the TFs are added as a mixture or added sequentially in terms of reprogramming efficiency, except for combination 2026, which showed significantly better effects when added in sequence compared to the same TFs added as a mixture (Figure 4).

[0135] The five TF combinations were further subjected to validation in spheroids, which mimic natural environment for the cells to grow, according to the method described above, under f) Validation, wherein the TFs were added as a mixture. FACS results of the reprogramming efficiency show that about 2-4 times as many c-peptide+ cells were produced by the new TF combination groups than the comparator TF mixture (Fig. 5a). This is also reflected in the fold change in reprogramming efficiency between controls and TF combinations (Fig. 5b).

[0136] The combination 8226 was measured for the secretion of c-peptide upon stimulation. As shown in Figure 6, spheroids containing p-cells reprogrammed using the TF combination 8226 showed significant increase secretion of c-peptide upon glucose stimulation (8226_2mM glucose vs 8226_20mM glucose), indicating that p-cells reprogrammed by the combination 8226 is functional competent. It was further noted that even upon 20mM glucose stimulation, the combination 8226 induced more c-peptide secretion than the +ve control group (+ve_20mM glucose vs 8226_20mM glucose), indicating that p-cells reprogrammed by the combination 8226 are more competent and mature than those reprogrammed by the +ve control group.

[0137] Table 1 . Sequence listing of the present disclosure

[0138] While the subject matter of this disclosure has been described and shown in considerable detail with reference to certain illustrative aspects, including various combinations and sub- combinations of features, those skilled in the art will readily appreciate other aspects and variations and modifications thereof as encompassed within the scope of the present disclosure. Moreover, the descriptions of such aspects, combinations, and sub-combinations is not intended to convey that the claimed subject matter requires features or combinations of features other than those expressly recited in the claims. Accordingly, the scope of this disclosure is intended to include all modifications and variations encompassed within the spirit and scope of the following appended claims.

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Claims

Claims1. A composition comprising a combination of transcription factors or nucleic acid sequences encoding the transcription factors for inducing or reprogramming a source cell into a (3-cell, wherein the source cell is a stem cell, a progenitor cell, a differentiated cell or mixtures thereof, and wherein the combination is selected from a group consisting of:(a) Pax6, Ngn3, MafA;(b) Rfx6, Ngn3, MafA;(c) Pax6, Ngn3, Rfx6, MafA;(d) Nkx6.1, Pdx1 , MafA; and(e) Pdx1 , Pax6, Rfx6, Neurodi .

2. The composition of claim 1, wherein:Pax6 has at least 80% amino acid sequence identity to SEQ ID NO: 1 or SEQ ID NO: 3; Ngn3 has at least 80% amino acid sequence identity to SEQ ID NO: 5 or SEQ ID NO: 7; MafA has at least 80% amino acid sequence identity to SEQ ID NO: 9 or SEQ ID NO: 11 ; Rfx6 has at least 80% amino acid sequence identity to SEQ ID NO: 13 or SEQ ID NO: 15; Nkx6.1 has at least 80% amino acid sequence identity to SEQ ID NO: 17 or SEQ ID NO: 19;Pdx1 has at least 80% amino acid sequence identity to SEQ ID NO: 21 or SEQ ID NO: 23; andNeurodi has at least 80% amino acid sequence identity to SEQ ID NO: 25 or SEQ ID NO: 27.

3. The composition of claim 1 or 2, wherein: the nucleic acid sequence encoding Pax has at least 80% nucleic acid sequence identity to SEQ ID NO: 2 or SEQ ID NO: 4; the nucleic acid sequence encoding Ngn3 has at least 80% nucleic acid sequence identity to SEQ ID NO: 6 or SEQ ID NO: 8; the nucleic acid sequence encoding MafA has at least 80% nucleic acid sequence identity to SEQ ID NO: 10 or SEQ ID NO: 12; the nucleic acid sequence encoding Rfx6 has at least 80% nucleic acid sequence identity to SEQ ID NO: 14 or SEQ ID NO: 16; the nucleic acid sequence encoding Nkx6.1 has at least 80% nucleic acid sequence identity to SEQ ID NO: 18 or SEQ ID NO: 20; the nucleic acid sequence encoding Pdx1 has at least 80% nucleic acid sequence identity to SEQ ID NO: 22 or SEQ ID NO: 24; and the nucleic acid sequence encoding Neurodi has at least 80% nucleic acid sequence identity to SEQ ID NO: 24 or SEQ ID NO: 28.

4. The composition of any one of claims 1-3, wherein the nucleic acid sequences are mRNAs.

5. The composition of any one of claims 1 to 4, wherein the stem cell is selected from a group consisting of pluripotent stem cell (PSC), multipotent stem cell, embryonic stem cell (ESC), and induced pluripotent stem cell (iPSC), and the progenitor cell is a pancreatic progenitor cell such as a cultured progenitor cell.

6. The composition of any one of claims 1 to 4, wherein the differentiated cell is an endoderm derived cell.

7. The composition of any one of claims 1-6, wherein the source cell is mammalian cell, preferably a mouse cell or a human cell.

8. A vector comprising nucleic acid sequences encoding the combination of transcription factors described in any one of claims 1-4.

9. The vector of claim 8, wherein the vector is a viral vector; in particular a retrovirus, an adenovirus, a lentivirus, a herpes virus, a pox virus, or an adeno-associated virus vector.

10. A method of inducing or reprogramming a source cell into a |3-cell, wherein the source cell is a stem cell, a progenitor cell, a differentiated cell or mixtures thereof, comprising:(I) contacting the source cell with i) a composition of any one of claims 1 to 7, or ii) one or more vectors comprising nucleic acid sequences encoding a combination of transcription factors selected from a group consisting of:(a) Pax6, Ngn3, MafA;(b) Rfx6, Ngn3, MafA;(c) Pax6, Ngn3, Rfx6, MafA;(d) Nkx6.1 , Pdx1, MafA; and(e) Pdx1 , Pax6, Rfx6, Neurodi ; or(iii) transcription factors selected from a group consisting of:(a) Pax6, Ngn3, MafA;(b) Rfx6, Ngn3, MafA;(c) Pax6, Ngn3, Rfx6, MafA;(d) Nkx6.1 , Pdx1, MafA; and(e) Pdx1 , Pax6, Rfx6, Neurodi ; and(II) culturing the cell from step (I) in a cell media that supports growth of -cells.

11. The method of claim 10, wherein the nucleic acid sequences encoding the combination of transcription factors are located in one vector.

12. The method of claim 10, wherein the nucleic acid sequences encoding the combination of transcription factors are located in a plurality of vectors.

13. The method of claim 10 or 12, wherein the source cells are contacted with the transcription factors or vectors sequentially.

14. The method of any one of claims 10-13, wherein the stem cell is selected from a group consisting of pluripotent stem cell (PSC), multipotent stem cell, embryonic stem cell (ESC), induced pluripotent stem cell (iPSC); wherein the progenitor cell is a pancreatic progenitor cell; wherein the differentiated cell is an endoderm-derived cell.

15. The method of any one of claims 10-14, wherein the source cell is a mammalian cell, preferably a mouse cell or a human cell.

16. An isolated engineered source cell comprising nucleic acid sequences encoding a combination of transcription factors defined in any one of claims 1 to 4, operably linked to one or more inducible promoters.

17. An isolated population of |3-cells obtained by the method of any one of claims 10-15.

18. A pharmaceutical composition comprising a therapeutically effective amount of;(a) the isolated population of |3-cells of claim 17 and one or more pharmaceutically acceptable excipients, or(b) the composition of any one of claims 1 to 4 and one or more pharmaceutically acceptable excipients.

19. The pharmaceutical composition of claim 18, wherein the pharmaceutically acceptable excipient is selected from a group consisting of a buffer, a stabilizer, a preservative and combinations thereof.

20. The pharmaceutical composition of claim 18 or 19, wherein the composition is an injectable formulation.

21. The pharmaceutical composition of any one of claims 18 to 20, wherein the composition is formulated for simultaneous or sequential delivery of the transcription factors.

22. A method for treating a subject with diabetes or pre-diabetes, comprising administering to the subject an efficacious amount of the isolated population of p-cells of claim 16 or the pharmaceutical composition of any one of claims 18 to 21.

23. The method of claim 22, comprising simultaneous or sequential administration of transcription factors.

24. Use of the isolated population of p-cells of claim 17, or the pharmaceutical composition of any one of claims 18-to 21 in the manufacture of a medicament for the treatment of diabetes or pre-diabetes in a subject in need thereof.

25. A kit for use in a method of inducing or reprogramming a source cell into a p-cell, comprising i) a composition of any one of claims 1 to 4, or ii) one or more vectors comprising nucleic acid sequences encoding a combination of transcription factors selected from a group consisting of:(a) Pax6, Ngn3, MafA;(b) Rfx6, Ngn3, MafA;(c) Pax6, Ngn3, Rfx6, MafA;(d) Nkx6.1 , Pdx1, MafA; and(e) Pdx1 , Pax6, Rfx6, Neurodi ; or(iii) transcription factors selected from a group consisting of:(a) Pax6, Ngn3, MafA;(b) Rfx6, Ngn3, MafA;(c) Pax6, Ngn3, Rfx6, MafA;(d) Nkx6.1 , Pdx1, MafA; and(e) Pdx1 , Pax6, Rfx6, Neurodi .

26. The kit of claim 25, wherein the kit further comprises a culture medium for culturing the source cell.

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