Compositions and methods for reprogramming somatic cells

Abstract

Disclosed herein are compositions and methods for directly reprogramming somatic cells into insulin-producing and / or glucagon-producing cells both in vitro and in vivo. Also disclosed are viral and non-viral vectors containing the disclosed polynucleotides. Also disclosed is a method of reprogramming somatic cells into insulin-producing and / or glucagon-producing cells that involves delivering intracellularly into the somatic cells a polynucleotide encoding a reprogramming cocktail disclosed herein. Also disclosed herein is a method of treating a subject that involves delivering intracellularly into somatic cells of the subject a polynucleotide encoding a reprogramming cocktail disclosed herein.

Description

COMPOSITIONS AND METHODS FOR REPROGRAMMING SOMATIC CELLSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. Provisional Application No. 63 / 579,128, filed August 28, 2023, which is hereby incorporated herein by reference in its entirety.SEQUENCE LISTING

[0002] This application contains a sequence listing filed in ST.26 format entitled “321502-2130 Sequence Listing” created on August 28, 2024, and having 34,969 bytes. The content of the sequence listing is incorporated herein in its entirety.BACKGROUND OF THE INVENTION

[0003] Type I diabetes is driven by the autoimmune destruction of pancreatic beta cells and resulting decrease in insulin production. Allogenic islet transplantation is the current gold standard to obtain insulin-independence for patients with type I diabetes, but its implementation is limited due to a lack of donor tissue and required life-long immunosuppressants for patients after treatment. To address these limitations, several studies have focused on developing alternative cell sources for insulin production using cell reprogramming methods (e.g., iPSCs, viruses). However, to generate a functional cell-based therapy to treat type I diabetes, approximately 0.1 to 1 billion functional p-like cells need to be produced per patient.SUMMARY OF THE INVENTION

[0004] Disclosed herein are compositions and methods for reprogramming somatic cells into insulin-producing and / or glucagon-producing cells both in vitro and in vivo. In some embodiments, a polynucleotide is disclosed encoding at least 1 , 2, or 3 of the “plasticity-inducing" transcription factors Tcf3, Trp63, and Sox9, and at least 1 , 2, 3, 4, 5, 6, or 7 of the "fate- patterning" transcription factors Pdx1 , Ngn3, Mafa, Neurodi , Pax4, Nkx6.1 , and FoxA2. Combinations of these plasticity-inducing and fate-patterning proteins are referred to herein as a “reprogramming cocktail”.

[0005] Each and every combination of the “plasticity-inducing" transcription factors and "fate-patterning" transcription factors are disclosed. For example, in some embodiments, the reprogramming cocktail involves Tcf3, Trp63, Sox9, Pdx1 , Ngn3, Mafa, Neurodi , Pax4, Nkx6.1 , and FoxA2. For example, in some embodiments, the reprogramming cocktail involves Pdx1 and / or Ngn3.

[0006] In some embodiments, the reprogramming cocktail involves Trp63, Sox9, Pdx1 , Ngn3, Mafa, Neurodi , Pax4, Nkx6.1 , and FoxA2. In someembodiments, the reprogramming cocktail involves Tcf3, Sox9, Pdx1 , Ngn3, Mafa, Neurodi , Pax4, Nkx6.1 , and FoxA2. In some embodiments, the reprogramming cocktail involves Tcf3, Trp63, Pdx1 , Ngn3, Mafa, Neurodi , Pax4, Nkx6.1 , and FoxA2.

[0007] In some embodiments, the reprogramming cocktail involves Tcf3, Pdx1 , Ngn3, Mafa, Neurodi , Pax4, Nkx6.1 , and FoxA2. In some embodiments, the reprogramming cocktail involves Trp63, Pdx1 , Ngn3, Mafa, Neurodi , Pax4, Nkx6.1 , and FoxA2. In some embodiments, the reprogramming cocktail involves Sox9, Pdx1 , Ngn3, Mafa, Neurodi , Pax4, Nkx6.1 , and FoxA2.

[0008] In some embodiments, the reprogramming cocktail involves Tcf3, Trp63, Sox9, Ngn3, Mafa, Neurodi , Pax4, Nkx6.1 , and FoxA2. In some embodiments, the reprogramming cocktail involves Tcf3, Trp63, Sox9, Pdx1 , Mafa, Neurodi , Pax4, Nkx6.1 , and FoxA2. In some embodiments, the reprogramming cocktail involves Tcf3, Trp63, Sox9, Pdx1 , Ngn3, Neurodi , Pax4, Nkx6.1 , and FoxA2. In some embodiments, the reprogramming cocktail involves Tcf3, Trp63, Sox9, Pdx1 , Ngn3, Mafa, Pax4, Nkx6.1 , and FoxA2. In some embodiments, the reprogramming cocktail involves Tcf3, Trp63, Sox9, Pdx1 , Ngn3, Mafa, Neurodi , Nkx6.1 , and FoxA2. In some embodiments, the reprogramming cocktail involves Tcf3, Trp63, Sox9, Pdx1 , Ngn3, Mafa, Neurodi , Pax4, and FoxA2. In some embodiments, the reprogramming cocktail involves Tcf3, Trp63, Sox9, Pdx1 , Ngn3, Mafa, Neurodi , Pax4, and Nkx6.1.

[0009] In some embodiments, the reprogramming cocktail involves Trp63, Sox9, Ngn3, Mafa, Neurodi , Pax4, Nkx6.1 , and FoxA2. In some embodiments, the reprogramming cocktail involves Trp63, Sox9, Pdx1 , Mafa, Neurodi , Pax4, Nkx6.1 , and FoxA2. In some embodiments, the reprogramming cocktail involves Trp63, Sox9, Pdx1 , Ngn3, Neurodi , Pax4, Nkx6.1 , and FoxA2. In some embodiments, the reprogramming cocktail involves Trp63, Sox9, Pdx1 , Ngn3, Mafa, Pax4, Nkx6.1 , and FoxA2. In some embodiments, the reprogramming cocktail involves Trp63, Sox9, Pdx1 , Ngn3, Mafa, Neurodi , Nkx6.1, and FoxA2. In some embodiments, the reprogramming cocktail involves Trp63, Sox9, Pdx1 , Ngn3, Mafa, Neurodi , Pax4, and FoxA2. In some embodiments, the reprogramming cocktail involves Trp63, Sox9, Pdx1 , Ngn3, Mafa, Neurodi , Pax4, and Nkx6.1.

[0010] In some embodiments, the reprogramming cocktail involves Tcf3, Sox9, Ngn3, Mafa, Neurodi , Pax4, Nkx6.1 , and FoxA2. In some embodiments, the reprogramming cocktail involves Tcf3, Sox9, Pdx1 , Mafa, Neurodi , Pax4, Nkx6.1 , and FoxA2. In some embodiments, the reprogramming cocktail involves Tcf3, Sox9, Pdx1 , Ngn3, Neurodi , Pax4, Nkx6.1 , and FoxA2. In some embodiments, the reprogramming cocktail involves Tcf3, Sox9, Pdx1 , Ngn3, Mafa, Pax4, Nkx6.1 , andFoxA2. In some embodiments, the reprogramming cocktail involves Tcf3, Sox9, Pdx1 , Ngn3, Mafa, Neurodi , Nkx6.1, and FoxA2. In some embodiments, the reprogramming cocktail involves Tcf3, Sox9, Pdx1 , Ngn3, Mafa, Neurodi , Pax4, and FoxA2. In some embodiments, the reprogramming cocktail involves Tcf3, Sox9, Pdx1 , Ngn3, Mafa, Neurodi , Pax4, and Nkx6.1.

[0011] In some embodiments, the reprogramming cocktail involves Tcf3, Trp63, Ngn3, Mafa, Neurodi , Pax4, Nkx6.1 , and FoxA2. In some embodiments, the reprogramming cocktail involves Tcf3, Trp63, Pdx1 , Mafa, Neurodi , Pax4, Nkx6.1 , and FoxA2. In some embodiments, the reprogramming cocktail involves Tcf3, Trp63, Pdx1 , Ngn3, Neurodi , Pax4, Nkx6.1 , and FoxA2. In some embodiments, the reprogramming cocktail involves Tcf3, Trp63, Pdx1 , Ngn3, Mafa, Pax4, Nkx6.1 , and FoxA2. In some embodiments, the reprogramming cocktail involves Tcf3, Trp63, Pdx1 , Ngn3, Mafa, Neurodi , Nkx6.1, and FoxA2. In some embodiments, the reprogramming cocktail involves Tcf3, Trp63, Pdx1 , Ngn3, Mafa, Neurodi , Pax4, and FoxA2. In some embodiments, the reprogramming cocktail involves Tcf3, Trp63, Pdx1 , Ngn3, Mafa, Neurodi , Pax4, and Nkx6.1.

[0012] In some embodiments, the reprogramming cocktail involves Tcf3, Ngn3, Mafa, Neurodi , Pax4, Nkx6.1 , and FoxA2. In some embodiments, the reprogramming cocktail involves Tcf3, Pdx1 , Mafa, Neurodi , Pax4, Nkx6.1 , and FoxA2. In some embodiments, the reprogramming cocktail involves Tcf3, Pdx1 , Ngn3, Neurodi , Pax4, Nkx6.1 , and FoxA2. In some embodiments, the reprogramming cocktail involves Tcf3, Pdx1 , Ngn3, Mafa, Pax4, Nkx6.1 , and FoxA2. In some embodiments, the reprogramming cocktail involves Tcf3, Pdx1 , Ngn3, Mafa, Neurodi , Nkx6.1 , and FoxA2. In some embodiments, the reprogramming cocktail involves Tcf3, Pdx1 , Ngn3, Mafa, Neurodi , Pax4, and FoxA2. In some embodiments, the reprogramming cocktail involves Tcf3, Pdx1 , Ngn3, Mafa, Neurodi , Pax4, and Nkx6.1.

[0013] In some embodiments, the reprogramming cocktail involves Trp63, Ngn3, Mafa, Neurodi , Pax4, Nkx6.1 , and FoxA2. In some embodiments, the reprogramming cocktail involves Trp63, Pdx1 , Mafa, Neurodi , Pax4, Nkx6.1 , and FoxA2. In some embodiments, the reprogramming cocktail involves Trp63, Pdx1 , Ngn3, Neurodi , Pax4, Nkx6.1 , and FoxA2. In some embodiments, the reprogramming cocktail involves Trp63, Pdx1 , Ngn3, Mafa, Pax4, Nkx6.1 , and FoxA2. In some embodiments, the reprogramming cocktail involves Trp63, Pdx1 , Ngn3, Mafa, Neurodi , Nkx6.1 , and FoxA2. In some embodiments, the reprogramming cocktail involves Trp63, Pdx1 , Ngn3, Mafa, Neurodi , Pax4, andFoxA2. In some embodiments, the reprogramming cocktail involves Trp63, Pdx1 , Ngn3, Mafa, Neurodi , Pax4, and Nkx6.1.

[0014] In some embodiments, the reprogramming cocktail involves Sox9, Ngn3, Mafa, Neurodi , Pax4, Nkx6.1 , and FoxA2. In some embodiments, the reprogramming cocktail involves Sox9, Pdx1 , Mafa, Neurodi , Pax4, Nkx6.1 , and FoxA2. In some embodiments, the reprogramming cocktail involves Sox9, Pdx1 , Ngn3, Neurodi , Pax4, Nkx6.1 , and FoxA2. In some embodiments, the reprogramming cocktail involves Sox9, Pdx1 , Ngn3, Mafa, Pax4, Nkx6.1 , and FoxA2. In some embodiments, the reprogramming cocktail involves Sox9, Pdx1 , Ngn3, Mafa, Neurodi , Nkx6.1 , and FoxA2. In some embodiments, the reprogramming cocktail involves Sox9, Pdx1 , Ngn3, Mafa, Neurodi , Pax4, and FoxA2. In some embodiments, the reprogramming cocktail involves Sox9, Pdx1 , Ngn3, Mafa, Neurodi , Pax4, and Nkx6.1.

[0015] In some embodiments, the plasticity-inducing and fate-patterning proteins are mammalian proteins, such as human proteins.

[0016] Also disclosed are viral and non-viral vectors containing the disclosed polynucleotides. In particular embodiments, the vector is a recombinant bacterial plasmid. For example, in some embodiments, the non-viral vector has a pCDNA3 backbone. In some embodiments, the vector comprises an internal ribosome entry site (IRES).

[0017] Also disclosed is a method of reprogramming somatic cells into insulin-producing and / or glucagon-producing cells that involves delivering intracellularly into the somatic cells a polynucleotide encoding a reprogramming cocktail disclosed herein.

[0018] Also disclosed herein is a method of treating a subject that involves delivering intracellularly into somatic cells of the subject a polynucleotide encoding a reprogramming cocktail disclosed herein. In some embodiments, this method is used to treat diabetes in the subject. In some embodiments, this method is used to treat hypoglycemia in the subject. In some embodiments, this method is used to treat pancreatic injury or loss in the subject.

[0019] In some embodiments, after transfecting target cells with a polynucleotide encoding reprogramming cocktail disclosed herein, the cells can then pack the transfected genes (e.g. cDNA) into EVs, which can then induce endothelium in other somatic cells. Therefore, also disclosed is a method of reprogramming somatic cells into insulin-producing and / or glucagon-producing cells that involves exposing the somatic cell with an extracellular vesicle produced from a cell containing or expressing a reprogramming cocktail disclosed herein.

[0020] In these embodiments, the polynucleotides and compositions may be delivered to the somatic cell, or the donor cell, intracellularly via a gene gun, a microparticle or nanoparticle suitable for such delivery, transfection by electroporation, three-dimensional nanochannel electroporation, a tissue nanotransfection device, a liposome suitable for such delivery, or a deep-topical tissue nanoelectroinjection device. In some of these embodiments, the polynucleotides can be incorporated into a non-viral vector, such as a bacterial plasmid. In some embodiments, a viral vector can be used. For example, the polynucleotides can be incorporated into a viral vector, such as an adenoviral vector. However, in other embodiments, the polynucleotides are not delivered virally.

[0021] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.BRIEF DESCRIPTION OF FIGURES

[0022] FIG. 1 A shows Insulin 1 , Insulin 2, and Glucagon gene expression profiles of MEFs 7, 14 and 21 days after BEP with 3SP+7|3C factors or control. FIG. 1 B shows immunomicrographs depicting Insulin, nuclei (DAPI), and brightfield images MEFs 14 days after being transduced with 3SP+7|3C factors or control (scale bar = 20um, objective = 20x). Connecting letters represent data groups that are not significantly different from each other (p>0.05).

[0023] FIGs. 2A to 2F show the combined transfection of plasmid DNA encoding for 3 skin plasticity factors and 7 p cell patterning factors induces increased pancreatic islet gene expression in mouse embryonic fibroblasts in standard fibroblast media. Gene expression profiles of INS1-EGFP MEFs 1 and / or 4 days post-BEP with plasmid DNA cocktails encoding for control (pCMV6), 3 skin plasticity factors (3SP), Pdx1 , Ngn3 and Mafa (PNM), 7 beta cell patterning factors (7 C), or the combination of 3SP+7 C. FIG. 2A shows Pdx1 expression 1- and 4-days post- BEP. B) Pdx1 expression 1-day post-BEP. FIGs. 2C-2E show insulin 1 , insulin 2, and glucagon expression 1- and 4 days post-BEP. FIG. 2F shows EGFP expression 4 post-BEP. Connecting letters represent data groups that are not significantly different from each other (p>0.05).

[0024] FIGs. 3 shows insulin gene expression is maximum in fibroblasts 14 post-BEP with 3SP+7PC. Insulin 1 , insulin 2, glucagon, somatostatin, and Pdx1 gene expression profiles of INS1-EGFP MEFs 7-, 14-, and 21-days post-BEP with plasmid DNA cocktails encoding for control (pCMV6) or the combination of3SP+7pC. Connecting letters represent data groups that are not significantly different from each other (p>0.05).

[0025] FIG. 4 shows insulin protein expression observed in smaller populations of fibroblasts transfected with control (pCMV6) or 3SP+7 C. FIG. 4 contains immunomicrographs depicting nuclei (DAPI), insulin (a-insulin), and merged images (with brightfield) of INS1-EGFP MEFs 14 days post-BEP with control or 3SP+7PC (scale bar = 20um, objective = 20x).

[0026] FIGs. 5A and 5B show insulin and co-localized EGFP protein expression observed in larger populations of fibroblasts transfected with control (pCMV6) or 3SP+7PC. FIG. 5A contains immunomicrographs depicting nuclei (DAPI), insulin (a-insulin), and merged images of INS1-EGFP MEFs 14 days postNEP with control or 3SP+7PC (scale bar = 20um, objective = 20x). FIG. 5B contains immunomicrographs depicting nuclei (DAPI), EGFP (a-GFP), insulin (a-insulin), and merged images of INS1-EGFP MEFs 14 days post-NEP with control or 3SP+7PC (scale bar = 20um, objective = 20x).

[0027] FIGs. 6A and 6B show three skin plasticity factors (3SP) greatly decrease heterochromatin markers and significantly open the chromatin landscape for potential reprogramming. FIG. 6A shows cytological fluorescent analysis of heterochromatin markers (H3K9me3) for MEFs transfected with all combinations of skin plasticity factors or controls (NT, iBET, pCMV6) 1- and 3-days post-BEP. FIG. 6B is a histological example depicting nuclei (DAPI), H3K9me3, and merged images of MEFs used to create corresponding H3K9me3 cytological fluorescent analysis of 3SP factor or control (NT, iBET, pCMV6) combinations 7 days post-BEP. (scale bar = 100um, objective = 20x).

[0028] FIG. 7 shows INS1-EGFP protein expression and islet-like MEF clustering 14 days post-BEP following short-term trypsin treatment 3 days post-BEP. Immunomicrographs depicting brightfield, nuclei (DAPI), EGFP (a-GFP), insulin (a- insulin), c-peptide (a-c-peptide), and merged images of INS1-EGFP MEFs 14 days post-BEP with control or 3SP+7PC (scale bar = 32.5um, objective = 20x).DETAILED DESCRIPTION

[0029] Disclosed herein are compositions and methods for reprogramming somatic cells into insulin-producing and / or glucagon-producing both in vitro and in vivo.Compositions

[0030] Disclosed are polynucleotides encoding at least 1 , 2, or 3 of the “plasticity-inducing" transcription factors Tcf3, Trp63, and Sox9, and at least 1 , 2, 3,4, 5, 6, or 7 of the "fate-patterning" transcription factors Pdx1 , Ngn3, Mafa, Neurodi , Pax4, Nkx6.1 , and FoxA2 (“reprogramming cocktail”).

[0031] The amino acid and nucleic acid sequences encoding Tcf3, Trp63, Sox9, Pdx1 , Ngn3, Mafa, Neurodi , Pax4, Nkx6.1 , and FoxA2 are known in the art.

[0032] In some embodiments, human Tcf3 has the amino acid sequence: MNQPQRMAPVGTDKELSDLLDFSMMFPLPVTNGKGRPASLAGAQFGGSGLEDRP SSGSWGSGDQSSSSFDPSRTFSEGTHFTESHSSLSSSTFLGPGLGGKSGERGAYA SFGRDAGVGGLTQAGFLSGELALNSPGPLSPSGMKGTSQYYPSYSGSSRRRAAD GSLDTQPKKVRKVPPGLPSSVYPPSSGEDYGRDATAYPSAKTPSSTYPAPFYVAD GSLHPSAELWSPPGQAGFGPMLGGGSSPLPLPPGSGPVGSSGSSSTFGGLHQHE RMGYQLHGAEVNGGLPSASSFSSAPGATYGGVSSHTPPVSGADSLLGSRGTTAG SSGDALGKALASIYSPDHSSNNFSSSPSTPVGSPQGLAGTSQWPRAGAPGALSPS YDGGLHGLQSKIEDHLDEAIHVLRSHAVGTAGDMHTLLPGHGALASGFTSPMSLGG RHAGLVGGSHPEDGLAGSTSLMHNHAALPSQPGTLPDLSRPPDSYSGLGRAGATA AASEIKREEKEDEENTSAADHSEEEKKELKAPRARTSPDEDEDDLLPPEQKAEREK ERRVANNARERLRVRDINEAFKELGRMCQLHLNSEKPQTKLLILHQAVSVILNLEQQ VRERNLNPKAACLKRREEEKVSGWGDPQMVLSAPHPGLSEAHNPAGHM (SEQ ID NO:1).

[0033] In some embodiments, human Tcf3 is encoded by the nucleic acid sequence: ATGAACCAGCCGCAGAGGATGGCGCCTGTGGGCACAGACAAGGAGCTCAGTG ACCTCCTGGACTTCAGCATGATGTTCCCGCTGCCTGTCACCAACGGGAAGGGC CGGCCCGCCTCCCTGGCCGGGGCGCAGTTCGGAGGTTCAGGTCTTGAGGACC GGCCCAGCTCAGGCTCCTGGGGCAGCGGCGACCAGAGCAGCTCCTCCTTTGA CCCCAGCCGGACCTTCAGCGAGGGCACCCACTTCACTGAGTCGCACAGCAGCC TCTCTTCATCCACATTCCTGGGACCGGGACTCGGAGGCAAGAGCGGTGAGCGG GGCGCCTATGCCTCCTTCGGGAGAGACGCAGGCGTGGGCGGCCTGACTCAGG CTGGCTTCCTGTCAGGCGAGCTGGCCCTCAACAGCCCCGGGCCCCTGTCCCCT TCGGGCATGAAGGGGACCTCCCAGTACTACCCCTCCTACTCCGGCAGCTCCCG GCGGAGAGCGGCAGACGGCAGCCTAGACACGCAGCCCAAGAAGGTCCGGAAG GTCCCGCCGGGTCTTCCATCCTCGGTGTACCCACCCAGCTCAGGTGAGGACTA CGGCAGGGATGCCACCGCCTACCCGTCCGCCAAGACCCCCAGCAGCACCTAT CCCGCCCCCTTCTACGTGGCAGATGGCAGCCTGCACCCCTCAGCCGAGCTCTG GAGTCCCCCGGGCCAGGCGGGCTTCGGGCCCATGCTGGGTGGGGGCTCATCC CCGCTGCCCCTCCCGCCCGGTAGCGGCCCGGTGGGCAGCAGTGGAAGCAGCA GCACGTTTGGTGGCCTGCACCAGCACGAGCGTATGGGCTACCAGCTGCATGGA GCAGAGGTGAACGGTGGGCTCCCATCTGCATCCTCCTTCTCCTCAGCCCCCGGAGCCACGTACGGCGGCGTCTCCAGCCACACGCCGCCTGTCAGCGGGGCCGAC AGCCTCCTGGGCTCCCGAGGGACCACAGCTGGCAGCTCCGGGGATGCCCTCG GCAAAGCACTGGCCTCGATCTACTCCCCGGATCACTCAAGCAATAACTTCTCGT CCAGCCCTTCTACCCCCGTGGGCTCCCCCCAGGGCCTGGCAGGAACGTCACA GTGGCCTCGAGCAGGAGCCCCCGGTGCCTTATCGCCCAGCTACGACGGGGGT CTCCACGGCCTGCAGAGTAAGATAGAAGACCACCTGGACGAGGCCATCCACGT GCTCCGCAGCCACGCCGTGGGCACAGCCGGCGACATGCACACGCTGCTGCCT GGCCACGGGGCGCTGGCCTCAGGTTTCACCAGTCCCATGTCGCTGGGTGGGC GGCACGCAGGCCTGGTTGGAGGCAGCCACCCCGAGGACGGCCTCGCAGGCA GCACCAGCCTCATGCACAACCACGCGGCCCTCCCCAGCCAGCCAGGCACCCT CCCTGACCTGTCTCGGCCTCCCGACTCCTACAGTGGGCTAGGGCGAGCAGGTG CCACGGCGGCCGCCAGCGAGATCAAGCGGGAGGAGAAGGAGGACGAGGAGA ACACGTCAGCGGCTGACCACTCGGAGGAGGAGAAGAAGGAGCTGAAGGCCCC CCGGGCCCGGACCAGCCCAGACGAGGACGAGGACGACCTTCTCCCCCCAGAG CAGAAGGCCGAGCGGGAGAAGGAGCGCCGGGTGGCCAATAACGCCCGGGAG CGGCTGCGGGTCCGTGACATCAACGAGGCCTTTAAGGAGCTGGGGCGCATGT GCCAACTGCACCTCAACAGCGAGAAGCCCCAGACCAAACTGCTCATCCTGCAC CAGGCTGTCTCGGTCATCCTGAACTTGGAGCAGCAAGTGCGAGAGCGGAACCT GAATCCCAAAGCAGCCTGTTTGAAACGGCGAGAAGAGGAAAAGGTGTCAGGTG TGGTTGGAGACCCCCAGATGGTGCTTTCAGCTCCCCACCCAGGCCTGAGCGAA GCCCACAACCCCGCCGGGCACATG (SEQ ID N0:1 1).

[0034] In some embodiments, human Trp63 has the amino acid sequence: MNFETSRCATLQYCPDPYIQRFVETPAHFSWKESYYRSTMSQSTQTNEFLSPEVF QHIWDFLEQPICSVQPIDLNFVDEPSEDGATNKIEISMDCIRMQDSDLSDPMWPQYT NLGLLNSMDQQIQNGSSSTSPYNTDHAQNSVTAPSPYAQPSSTFDALSPSPAIPSN TDYPGPHSFDVSFQQSSTAKSATWTYSTELKKLYCQIAKTCPIQIKVMTPPPQGAVI RAMPVYKKAEHVTEWKRCPNHELSREFNEGQIAPPSHLIRVEGNSHAQYVEDPIT GRQSVLVPYEPPQVGTEFTTVLYN FMCNSSCVGG M NRRPI LI I VTLETRDGQVLG R RCFEARICACPGRDRKADEDSIRKQQVSDSTKNGDGTKRPFRQNTHGIQMTSIKKR RSPDDELLYLPVRGRETYEMLLKIKESLELMQYLPQHTIETYRQQQQQQHQHLLQK QTSIQSPSSYGNSSPPLNKMNSMNKLPSVSQLINPQQRNALTPTTIPDGMGANIPM MGTHMPMAGDMNGLSPTQALPPPLSMPSTSHCTPPPPYPTDCSIVSFLARLGCSS CLDYFTTQGLTTIYQIEHYSMDDLASLKIPEQFRHAIWKGILDHRQLHEFSSPSHLLR TPSSASTVSVGSSETRGERVIDAVRFTLRQTISFPPRDEWNDFNFDMDARRNKQQ RIKEEGE (SEQ ID NO:2).

[0035] In some embodiments, human Trp63 is encoded by the nucleic acid sequence:ATGAATTTTGAAACTTCACGGTGTGCCACCCTACAGTACTGCCCTGACCCTTAC ATCCAGCGTTTCGTAGAAACCCCAGCTCATTTCTCTTGGAAAGAAAGTTATTACC GATCCACCATGTCCCAGAGCACACAGACAAATGAATTCCTCAGTCCAGAGGTTT TCCAGCATATCTGGGATTTTCTGGAACAGCCTATATGTTCAGTTCAGCCCATTGA CTTGAACTTTGTGGATGAACCATCAGAAGATGGTGCGACAAACAAGATTGAGAT TAGCATGGACTGTATCCGCATGCAGGACTCGGACCTGAGTGACCCCATGTGGC CACAGTACACGAACCTGGGGCTCCTGAACAGCATGGACCAGCAGATTCAGAAC GGCTCCTCGTCCACCAGTCCCTATAACACAGACCACGCGCAGAACAGCGTCAC GGCGCCCTCGCCCTACGCACAGCCCAGCTCCACCTTCGATGCTCTCTCTCCAT CACCCGCCATCCCCTCCAACACCGACTACCCAGGCCCGCACAGTTTCGACGTG TCCTTCCAGCAGTCGAGCACCGCCAAGTCGGCCACCTGGACGTATTCCACTGA ACTGAAGAAACTCTACTGCCAAATTGCAAAGACATGCCCCATCCAGATCAAGGT GATGACCCCACCTCCTCAGGGAGCTGTTATCCGCGCCATGCCTGTCTACAAAAA AGCTGAGCACGTCACGGAGGTGGTGAAGCGGTGCCCCAACCATGAGCTGAGC CGTGAATTCAACGAGGGACAGATTGCCCCTCCTAGTCATTTGATTCGAGTAGAG GGGAACAGCCATGCCCAGTATGTAGAAGATCCCATCACAGGAAGACAGAGTGT GCTGGTACCTTATGAGCCACCCCAGGTTGGCACTGAATTCACGACAGTCTTGTA CAATTTCATGTGTAACAGCAGTTGTGTTGGAGGGATGAACCGCCGTCCAATTTTAATCATTGTTACTCTGGAAACCAGAGATGGGCAAGTCCTGGGCCGACGCTGCTT TGAGGCCCGGATCTGTGCTTGCCCAGGAAGAGACAGGAAGGCGGATGAAGATA GO ATCAG AAAGCAG C AAGTTTCG GAG AGTAC AAAGAACG GTG ATG GTACG AAG CGCCCGTTTCGTCAGAACACACATGGTATCCAGATGACATCCATCAAGAAACGA AGATCCCCAGATGATGAACTGTTATACTTACCAGTGAGGGGCCGTGAGACTTAT GAAATGCTGTTGAAGATCAAAGAGTCCCTGGAACTCATGCAGTACCTTCCTCAG CACACAATTG AAACGTAC AG G CAACAG CAAC AG CAGC AG CACC AGCACTTACTT CAGAAACAGACCTCAATACAGTCTCCATCTTCATATGGTAACAGCTCCCCACCT CTG AAC AAAATG AACAGC ATG AACAAG CTG CCTTCTGTG AG CCAG CTTATCAAC CCTCAGCAGCGCAACGCCCTCACTCCTACAACCATTCCTGATGGCATGGGAGC CAACATTCCCATGATGGGCACCCACATGCCAATGGCTGGAGACATGAATGGACT CAGCCCCACCCAGGCACTCCCTCCCCCACTCTCCATGCCATCCACCTCCCACT GCACACCCCCACCTCCGTATCCCACAGATTGCAGCATTGTCAGTTTCTTAGCGA GGTTGGGCTGTTCATCATGTCTGGACTATTTCACGACCCAGGGGCTGACCACCA TCTATCAGATTGAGCATTACTCCATGGATGATCTGGCAAGTCTGAAAATCCCTGA GCAATTTCGACATGCGATCTGGAAGGGCATCCTGGACCACCGGCAGCTCCACG AATTCTCCTCCCCTTCTCATCTCCTGCGGACCCCAAGCAGTGCCTCTACAGTCA GTGTGGGCTCCAGTGAGACCCGGGGTGAGCGTGTTATTGATGCTGTGCGATTCACCCTCCGCCAGACCATCTCTTTCCCACCCCGAGATGAGTGGAATGACTTCAACTTTG AC ATG G ATG CTCGCCGC AATAAG CAACAG CGC ATCAAAG AG G AG GG G GA G (SEQ ID NO: 12).

[0036] In some embodiments, human Sox9 has the amino acid sequence: MNLLDPFMKMTDEQEKGLSGAPSPTMSEDSAGSPCPSGSGSDTENTRPQENTFP KGEPDLKKESEEDKFPVCIREAVSQVLKGYDWTLVPMPVRVNGSSKNKPHVKRPM NAFMVWAQAARRKLADQYPHLHNAELSKTLGKLWRLLNESEKRPFVEEAERLRVQ HKKDHPDYKYQPRRRKSVKNGQAEAEEATEQTHISPNAIFKALQADSPHSSSGMS EVHSPGEHSGQSQGPPTPPTTPKTDVQPGKADLKREGRPLPEGGRQPPIDFRDVD IGELSSDVISNIETFDVNEFDQYLPPNGHPGVPATHGQVTYTGSYGISSTAATPASA GHVWMSKQQAPPPPPQQPPQAPPAPQAPPQPQAAPPQQPAAPPQQPQAHTLTTL SSEPGQSQRTHIKTEQLSPSHYSEQQQHSPQQIAYSPFNLPHYSPSYPPITRSQYD YTDHQNSSSYYSHAAGQGTGLYSTFTYMNPAQRPMYTPIADTSGVPSIPQTHSPQ HWEQPVYTQLTRP (SEQ ID NO:3).

[0037] In some embodiments, human Sox9 is encoded by the nucleic acid sequence: ATGAATCTCCTGGACCCCTTCATGAAGATGACCGACGAGCAGGAGAAGGGCCT GTCCGGCGCCCCCAGCCCCACCATGTCCGAGGACTCCGCGGGCTCGCCCTGC CCGTCGGGCTCCGGCTCGGACACCGAGAACACGCGGCCCCAGGAGAACACGT TCCCCAAGGGCGAGCCCGATCTGAAGAAGGAGAGCGAGGAGGACAAGTTCCC CGTGTGCATCCGCGAGGCGGTCAGCCAGGTGCTCAAAGGCTACGACTGGACG CTGGTGCCCATGCCGGTGCGCGTCAACGGCTCCAGCAAGAACAAGCCGCACG TCAAGCGGCCCATGAACGCCTTCATGGTGTGGGCGCAGGCGGCGCGCAGGAA GCTCGCGGACCAGTACCCGCACTTGCACAACGCCGAGCTCAGCAAGACGCTG GGCAAGCTCTGGAGACTTCTGAACGAGAGCGAGAAGCGGCCCTTCGTGGAGG AGGCGGAGCGGCTGCGCGTGCAGCACAAGAAGGACCACCCGGATTACAAGTA CCAGCCGCGGCGGAGGAAGTCGGTGAAGAACGGGCAGGCGGAGGCAGAGGA GGCCACGGAGCAGACGCACATCTCCCCCAACGCCATCTTCAAGGCGCTGCAGG CCGACTCGCCACACTCCTCCTCCGGCATGAGCGAGGTGCACTCCCCCGGCGA GCACTCGGGGCAATCCCAGGGCCCACCGACCCCACCCACCACCCCCAAAACC GACGTGCAGCCGGGCAAGGCTGACCTGAAGCGAGAGGGGCGCCCCTTGCCAG AGGGGGGCAGACAGCCCCCTATCGACTTCCGCGACGTGGACATCGGCGAGCT GAGCAGCGACGTCATCTCCAACATCGAGACCTTCGATGTCAACGAGTTTGACCA GTACCTGCCGCCCAACGGCCACCCGGGGGTGCCGGCCACGCACGGCCAGGTC ACCTACACGGGCAGCTACGGCATCAGCAGCACCGCGGCCACCCCGGCGAGCG CGGGCCACGTGTGGATGTCCAAGCAGCAGGCGCCGCCGCCACCCCCGCAGCA GCCCCCACAGGCCCCGCCGGCCCCGCAGGCGCCCCCGCAGCCGCAGGCGGC GCCCCCACAGCAGCCGGCGGCACCCCCGCAGCAGCCACAGGCGCACACGCTGACCACGCTGAGCAGCGAGCCGGGCCAGTCCCAGCGAACGCACATCAAGACGG AGCAGCTGAGCCCCAGCCACTACAGCGAGCAGCAGCAGCACTCGCCCCAACA GATCGCCTACAGCCCCTTCAACCTCCCACACTACAGCCCCTCCTACCCGCCCAT CACCCGCTCACAGTACGACTACACCGACCACCAGAACTCCAGCTCCTACTACAG CCACGCGGCAGGCCAGGGCACCGGCCTCTACTCCACCTTCACCTACATGAACC CCGCTCAGCGCCCCATGTACACCCCCATCGCCGACACCTCTGGGGTCCCTTCC ATCCCGCAGACCCACAGCCCCCAGCACTGGGAACAACCCGTCTACACACAGCT ACTCGACCT (SEQ ID N0:13).

[0038] In some embodiments, human Pdx1 has the amino acid sequence: MNGEEQYYAATQLYKDPCAFQRGPAPEFSASPPACLYMGRQPPPPPPHPFPGAL GALEQGSPPDISPYEVPPLADDPAVAHLHHHLPAQLALPHPPAGPFPEGAEPGVLE EPNRVQLPFPWMKSTKAHAWKGQWAGGAYAAEPEENKRTRTAYTRAQLLELEKE FLFNKYISRPRRVELAVMLNLTERHIKIWFQNRRMKWKKEEDKKRGGGTAVGGGG VAEPEQDCAVTSGEELLALPPPPPPGGAVPPAAPVAAREGRLPPGLSASPQPSSVA PRRPQEPR (SEQ ID NO:4).

[0039] In some embodiments, human Pdx1 is encoded by the nucleic acid sequence: ATGAACGGCGAGGAGCAGTACTACGCGGCCACGCAGCTTTACAAGGACCCATG CGCGTTCCAGCGAGGCCCGGCGCCGGAGTTCAGCGCCAGCCCCCCTGCGTGC CTGTACATGGGCCGCCAGCCCCCGCCGCCGCCGCCGCACCCGTTCCCTGGCG CCCTGGGCGCGCTGGAGCAGGGCAGCCCCCCTGACATCTCCCCGTACGAGGT GCCCCCCCTCGCCGACGACCCCGCGGTGGCGCACCTTCACCACCACCTCCCG GCTCAGCTCGCGCTCCCCCACCCGCCCGCCGGGCCCTTCCCGGAGGGAGCCG AACCGGGCGTCCTGGAGGAGCCCAACCGCGTCCAGCTGCCTTTCCCATGGATG AAGTCTACCAAAGCTCACGCGTGGAAAGGCCAGTGGGCAGGCGGCGCCTACG CTGCGGAGCCGGAGGAGAACAAGCGGACGCGCACGGCCTACACGCGCGCACA GCTGCTAGAGCTGGAGAAGGAGTTCCTATTCAACAAGTACATCTCACGGCCGC GCCGGGTGGAGCTGGCTGTCATGTTGAACTTGACCGAGAGACACATCAAGATC TGGTTCCAAAACCGCCGCATGAAGTGGAAAAAGGAGGAGGACAAGAAGCGCGG CGGCGGGACAGCTGTCGGGGGTGGCGGGGTCGCGGAGCCTGAGCAGGACTG CGCCGTGACCTCCGGCGAGGAGCTTCTGGCGCTGCCGCCGCCGCCGCCCCCC GGAGGTGCTGTGCCGCCCGCTGCCCCCGTTGCCGCCCGAGAGGGCCGCCTGC CGCCTGGCCTTAGCGCGTCGCCACAGCCCTCCAGCGTCGCGCCTCGGCGGCC GCAGGAACCACGA (SEQ ID NO: 14).

[0040] In some embodiments, human Ngn3 has the amino acid sequence: MTPQPSGAPTVQVTRETERSFPRASEDEVTCPTSAPPSPTRTRGNCAEAEEGGCR GAPRKLRARRGGRSRPKSELALSKQRRSRRKKANDRERNRMHNLNSALDALRGVLPTFPDDAKLTKIETLRFAHNYIWALTQTLRIADHSLYALEPPAPHCGELGSPGGSPG DWGSLYSPVSQAGSLSPAASLEERPGLLGATSSACLSPGSLAFSDFL (SEQ ID NO:5).

[0041] In some embodiments, human Ngn3 is encoded by the nucleic acid sequence: ATGACGCCTCAACCCTCGGGTGCGCCCACTGTCCAAGTGACCCGTGAGACGGA GCGGTCCTTCCCCAGAGCCTCGGAAGACGAAGTGACCTGCCCCACGTCCGCC CCGCCCAGCCCCACTCGCACACGGGGGAACTGCGCAGAGGCGGAAGAGGGA GGCTGCCGAGGGGCCCCGAGGAAGCTCCGGGCACGGCGCGGGGGACGCAGC CGGCCTAAGAGCGAGTTGGCACTGAGCAAGCAGCGACGGAGTCGGCGAAAGA AGGCCAACGACCGCGAGCGCAATCGAATGCACAACCTCAACTCGGCACTGGAC GCCCTGCGCGGTGTCCTGCCCACCTTCCCAGACGACGCGAAGCTCACCAAGAT CGAGACGCTGCGCTTCGCCCACAACTACATCTGGGCGCTGACTCAAACGCTGC GCATAGCGGACCACAGCTTGTACGCGCTGGAGCCGCCGGCGCCGCACTGCGG GGAGCTGGGCAGCCCAGGCGGTTCCCCCGGGGACTGGGGGTCCCTCTACTCC CCAGTCTCCCAGGCTGGCAGCCTGAGTCCCGCCGCGTCGCTGGAGGAGCGAC CCGGGCTGCTGGGGGCCACCTCTTCCGCCTGCTTGAGCCCAGGCAGTCTGGC TTTCTCAGATTTTCTG (SEQ ID NO:15).

[0042] In some embodiments, human Mafa has the amino acid sequence: MAAELAMGAELPSSPLAIEYVNDFDLMKFEVKKEPPEAERFCHRLPPGSLSSTPLST PCSSVPSSPSFCAPSPGTGGGGGAGGGGGSSQAGGAPGPPSGGPGAVGGTSGK PALEDLYWMSGYQHHLNPEALNLTPEDAVEALIGSGHHGAHHGAHHPAAAAAYEA FRGPGFAGGGGADDMGAGHHHGAHHAAHHHHAAHHHHHHHHHHGGAGHGGGA GHHVRLEERFSDDQLVSMSVRELNRQLRGFSKEEVIRLKQKRRTLKNRGYAQSCR FKRVQQRHILESEKCQLQSQVEQLKLEVGRLAKERDLYKEKYEKLAGRGGPGSAG GAGFPREPSPPQAGPGGAKGTADFFL (SEQ ID NO:6).

[0043] In some embodiments, human Mafa is encoded by the nucleic acid sequence: ATGGCCGCGGAGCTGGCGATGGGCGCCGAGCTGCCCAGCAGCCCGCTGGCC ATCGAGTACGTCAACGACTTCGACCTGATGAAGTTCGAGGTGAAGAAGGAGCC TCCCGAGGCCGAGCGCTTCTGCCACCGCCTGCCGCCAGGCTCGCTGTCCTCG ACGCCGCTCAGCACGCCCTGCTCCTCCGTGCCCTCCTCGCCCAGCTTCTGCGC GCCCAGCCCGGGCACCGGCGGCGGCGGCGGCGCGGGGGGCGGCGGCGGCT CGTCTCAGGCCGGGGGCGCCCCCGGGCCGCCGAGCGGGGGCCCCGGCGCC GTCGGGGGCACCTCGGGGAAGCCGGCGCTGGAGGATCTGTACTGGATGAGCG GCTACCAGCATCACCTCAACCCCGAGGCGCTCAACCTGACGCCCGAGGACGC GGTGGAGGCGCTCATCGGCAGCGGCCACCACGGCGCGCACCACGGCGCGCACCACCCGGCGGCCGCCGCAGCCTACGAGGCTTTCCGCGGCCCGGGCTTCGCG GGCGGCGGCGGAGCGGACGACATGGGCGCCGGCCACCACCACGGCGCGCAC CACGCCGCCCACCACCACCACGCCGCCCACCACCACCACCACCACCACCACCA CCATGGCGGCGCGGGACACGGCGGTGGCGCGGGCCACCACGTGCGCCTGGA GGAGCGCTTCTCCGACGACCAGCTGGTGTCCATGTCGGTGCGCGAGCTGAACC GGCAGCTCCGCGGCTTCAGCAAGGAGGAGGTCATCCGGCTCAAGCAGAAGCG GCGCACGCTCAAGAACCGCGGCTACGCGCAGTCCTGCCGCTTCAAGCGGGTG CAGCAGCGGCACATTCTGGAGAGCGAGAAGTGCCAACTCCAGAGCCAGGTGG AGCAGCTGAAGCTGGAGGTGGGGCGCCTGGCCAAAGAGCGGGACCTGTACAA GGAGAAATACGAGAAGCTGGCGGGCCGGGGCGGCCCCGGGAGCGCGGGCGG GGCCGGTTTCCCGCGGGAGCCTTCGCCGCCGCAGGCCGGTCCCGGCGGGGCCAAGGGCACGGCCGACTTCTTCCTG (SEQ ID NO: 16).

[0044] In some embodiments, human Neurodi has the amino acid sequence: MTKSYSESGLMGEPQPQGPPSWTDECLSSQDEEHEADKKEDDLEAMNAEEDSLR NGGEEEDEDEDLEEEEEEEEEDDDQKPKRRGPKKKKMTKARLERFKLRRMKANA RERNRMHGLNAALDNLRKWPCYSKTQKLSKIETLRLAKNYIWALSEILRSGKSPDL VSFVQTLCKGLSQPTTNLVAGCLQLNPRTFLPEQNQDMPPHLPTASASFPVHPYSY QSPGLPSPPYGTMDSSHVFHVKPPPHAYSAALEPFFESPLTDCTSPSFDGPLSPPL SINGNFSFKHEPSAEFEKNYAFTMHYPAATLAGAQSHGSIFSGTAAPRCEIPIDNIMS FDSHSHHERVMSAQLNAIFHD (SEQ ID NO:7).

[0045] In some embodiments, human Neurodi is encoded by the nucleic acid sequence:ATGACCAAATCGTACAGCGAGAGTGGGCTGATGGGCGAGCCTCAGCCCCAAGG TCCTCCAAGCTGGACAGACGAGTGTCTCAGTTCTCAGGACGAGGAGCACGAGG CAGACAAGAAGGAGGACGACCTCGAAGCCATGAACGCAGAGGAGGACTCACTG AGGAACGGGGGAGAGGAGGAGGACGAAGATGAGGACCTGGAAGAGGAGGAA GAAGAGGAAGAGGAGGATGACGATCAAAAGCCCAAGAGACGCGGCCCCAAAAA G AAG AAG ATG ACTAAG G CTCG CCTGG AGCGTTTTAAATTG AG ACG CATG AAGG CTAACGCCCGGGAGCGGAACCGCATGCACGGACTGAACGCGGCGCTAGACAA CCTGCGCAAGGTGGTGCCTTGCTATTCTAAGACGCAGAAGCTGTCCAAAATCGA GACTCTGCGCTTGGCCAAGAACTACATCTGGGCTCTGTCGGAGATCCTGCGCT CAGGCAAAAGCCCAGACCTGGTCTCCTTCGTTCAGACGCTTTGCAAGGGCTTAT CCCAACCCACC ACCAACCTGGTTGCG GG CTG CCTG CAACTCAATCCTCG G ACTTTTCTGCCTGAGCAGAACCAGGACATGCCCCCCCACCTGCCGACGGCCAGCGC TTCCTTCCCTGTACACCCCTACTCCTACCAGTCGCCTGGGCTGCCCAGTCCGCC TTACGGTACCATGGACAGCTCCCATGTCTTCCACGTTAAGCCTCCGCCGCACGC CTACAGCGCAGCGCTGGAGCCCTTCTTTGAAAGCCCTCTGACTGATTGCACCAGCCCTTCCTTTGATGGACCCCTCAGCCCGCCGCTCAGCATCAATGGCAACTTCT CTTTCAAACACGAACCGTCCGCCGAGTTTGAGAAAAATTATGCCTTTACCATGCA CTATCCTGCAGCGACACTGGCAGGGGCCCAAAGCCACGGATCAATCTTCTCAG GCACCGCTGCCCCTCGCTGCGAGATCCCCATAGACAATATTATGTCCTTCGATA GCCATTCACATCATGAGCGAGTCATGAGTGCCCAGCTCAATGCCATATTTCATG AT (SEQ ID NO: 17).

[0046] In some embodiments, human Pax4 has the amino acid sequence: MNQLGGLFVNGRPLPLDTRQQIVRLAVSGMRPCDISRILKVSNGCVSKILGRYYRTG VLEPKGIGGSKPRLATPPWARIAQLKGECPALFAWEIQRQLCAEGLCTQDKTPSVS SINRVLRALQEDQGLPCTRLRSPAVLAPAVLTPHSGSETPRGTHPGTGHRNRTIFSP SQAEALEKEFQRGQYPDSVARGKLATATSLPEDTVRVWFSNRRAKWRRQEKLKW EMQLPGASQGLTVPRVAPGIISAQQSPGSVPTAALPALEPLGPSCYQLCWATAPER CLSDTPPKACLKPCWGHLPPQPNSLDSGLLCLPCPSSHCPLASLSGSQALLWPGC PLLYGLE (SEQ ID NO:8).

[0047] In some embodiments, human Pax4 is encoded by the nucleic acid sequence: ATGAACCAGCTTGGGGGGCTCTTTGTGAATGGCCGGCCCCTGCCTCTGGATAC CCGGCAGCAGATTGTGCGGCTAGCAGTCAGTGGAATGCGGCCCTGTGACATCT CACGGATCCTTAAGGTATCTAATGGCTGTGTGAGCAAGATCCTAGGGCGTTACT ACCGCACAGGTGTCTTGGAGCCAAAGGGCATTGGGGGAAGCAAGCCACGGCT GGCTACACCCCCTGTGGTGGCTCGAATTGCCCAGCTGAAGGGTGAGTGTCCAG CCCTCTTTGCCTGGGAAATCCAACGCCAGCTTTGTGCTGAAGGGCTTTGCACCC AGGACAAGACTCCCAGTGTCTCCTCCATCAACCGAGTCCTGCGGGCATTACAG GAGGACCAGGGACTACCGTGCACACGGCTCAGGTCACCAGCTGTTTTGGCTCC AGCTGTCCTCACTCCCCATAGTGGCTCTGAGACTCCCCGGGGTACCCACCCAG GGACCGGCCACCGGAATCGGACTATCTTCTCCCCAAGCCAGGCAGAGGCACTG GAGAAAGAGTTCCAGCGTGGGCAGTATCCTGATTCAGTGGCCCGTGGAAAGCT GGCTACTGCCACCTCTCTGCCTGAGGACACGGTGAGGGTCTGGTTTTCCAACA GAAGAGCCAAATGGCGTCGGCAAGAGAAGCTCAAGTGGGAAATGCAGCTGCCA GGTGCTTCCCAGGGGCTGACTGTACCAAGGGTTGCCCCAGGAATCATCTCTGC ACAGCAGTCCCCTGGCAGTGTGCCCACAGCAGCCCTGCCTGCCCTGGAACCAC TGGGTCCCTCCTGCTATCAGCTGTGCTGGGCAACAGCACCAGAAAGGTGTCTG AGTGACACCCCACCTAAAGCCTGTCTCAAGCCCTGCTGGGGCCACTTGCCCCC ACAGCCGAATTCCCTGGACTCAGGACTGCTTTGCCTTCCTTGCCCTTCCTCCCA CTGTCCCCTGGCCAGTCTTAGTGGCTCTCAGGCCCTGCTCTGGCCTGGCTGCC CACTACTGTATGGCTTGGAA (SEQ ID NO:18).

[0048] In some embodiments, human Nkx6.1 has the amino acid sequence: MLAVGAMEGTRQSAFLLSSPPLAALHSMAEMKTPLYPAAYPPLPAGPPSSSSSSSS SSSPSPPLGTHNPGGLKPPATGGLSSLGSPPQQLSAATPHGINDILSRPSMPVASG AALPSASPSGSSSSSSSSASASSASAAAAAAAAAAAAASSPAGLLAGLPRFSSLSP PPPPPGLYFSPSAAAVAAVGRYPKPLAELPGRTPIFWPGVMQSPPWRDARLACTP HQGSILLDKDGKRKHTRPTFSGQQIFALEKTFEQTKYLAGPERARLAYSLGMTESQ VKVWFQNRRTKWRKKHAAEMATAKKKQDSETERLKGASENEEEDDDYNKPLDPN SDDEKITQLLKKHKSSSGGGGGLLLHASEPESSS (SEQ ID NO:9).

[0049] In some embodiments, human Nkx6.1 is encoded by the nucleic acid sequence: ATGTTAGCGGTGGGGGCAATGGAGGGCACCCGGCAGAGCGCATTCCTGCTCA GCAGCCCTCCCCTGGCCGCCCTGCACAGCATGGCCGAGATGAAGACCCCGCT GTACCCTGCCGCGTATCCCCCGCTGCCTGCCGGCCCCCCCTCCTCCTCGTCCT CGTCGTCGTCCTCCTCGTCGCCCTCCCCGCCTCTGGGCACCCACAACCCAGGC GGCCTGAAGCCCCCGGCCACGGGGGGGCTCTCATCCCTCGGCAGCCCCCCGC AGCAGCTCTCGGCCGCCACCCCACACGGCATCAACGATATCCTGAGCCGGCCC TCCATGCCCGTGGCCTCGGGGGCCGCCCTGCCCTCCGCCTCGCCCTCCGGTT CCTCCTCCTCCTCTTCCTCGTCCGCCTCTGCCTCCTCCGCCTCTGCCGCCGCC GCGGCTGCTGCCGCGGCCGCAGCCGCCGCCTCATCCCCGGCGGGGCTGCTG GCCGGACTGCCACGCTTTAGCAGCCTGAGCCCGCCGCCGCCGCCGCCCGGGC TCTACTTCAGCCCCAGCGCCGCGGCCGTGGCCGCCGTGGGCCGGTACCCCAA GCCGCTGGCTGAGCTGCCTGGCCGGACGCCCATCTTCTGGCCCGGAGTGATG CAGAGCCCGCCCTGGAGGGACGCACGCCTGGCCTGTACCCCTCATCAAGGAT CCATTTTGTTGGACAAAGACGGGAAGAGAAAACACACGAGACCCACTTTTTCCG GACAGCAGATCTTCGCCCTGGAGAAGACTTTCGAACAAACAAAATACTTGGCGG GGCCCGAGAGGGCTCGTTTGGCCTATTCGTTGGGGATGACAGAGAGTCAGGTC AAGGTCTGGTTCCAGAACCGCCGGACCAAGTGGAGGAAGAAGCACGCTGCCG AGATGGCCACGGCCAAGAAGAAGCAGGACTCGGAGACAGAGCGCCTCAAGGG GGCCTCGGAGAACGAGGAAGAGGACGACGACTACAATAAGCCTCTGGATCCCA ACTCGGACGACGAGAAAATCACGCAGCTGTTGAAGAAGCACAAGTCCAGCAGC GGCGGCGGCGGCGGCCTCCTACTGCACGCGTCCGAGCCGGAGCTCATCC (SEQ ID NO:19).

[0050] In some embodiments, human FoxA2 has the amino acid sequence: MLGAVKMEGHEPSDWSSYYAEPEGYSSVSNMNAGLGMNGMNTYMSMSAAAMG SGSGNMSAGSMNMSSYVGAGMSPSLAGMSPGAGAMAGMGGSAGAAGVAGMGP HLSPSLSPLGGQAAGAMGGLAPYANMNSMSPMYGQAGLSRARDPKTYRRSYTHA KPPYSYISLITMAIQQSPNKMLTLSEIYQWIMDLFPFYRQNQQRWQNSIRHSLSFNDCFLKVPRSPDKPGKGSFWTLHPDSGNMFENGCYLRRQKRFKCEKQLALKEAAGAA GSGKKAAAGAQASQAQLGEAAGPASETPAGTESPHSSASPCQEHKRGGLGELKG TPAAALSPPEPAPSPGQQQQAAAHLLGPPHHPGLPPEAHLKPEHHYAFNHPFSINN LMSSEQQHHHSHHHHQPHKMDLKAYEQVMHYPGYGSPMPGSLAMGPVTNKTGL DASPLAADTSYYQGVYSRPIMNSS (SEQ ID NO: 10).

[0051] In some embodiments, human FoxA2 is encoded by the nucleic acid sequence: ATGCTGGGAGCGGTGAAGATGGAAGGGCACGAGCCGTCCGACTGGAGCAGCT ACTATGCAGAGCCCGAGGGCTACTCCTCCGTGAGCAACATGAACGCCGGCCTG GGGATGAACGGCATGAACACGTACATGAGCATGTCGGCGGCCGCCATGGGCA GCGGCTCGGGCAACATGAGCGCGGGCTCCATGAACATGTCGTCGTACGTGGG CGCTGGCATGAGCCCGTCCCTGGCGGGGATGTCCCCCGGCGCGGGCGCCATG GCGGGCATGGGCGGCTCGGCCGGGGCGGCTGGCGTGGCGGGCATGGGGCC GCACTTGAGTCCCAGCCTGAGCCCGCTCGGGGGGCAGGCGGCCGGGGCCAT GGGCGGCCTGGCCCCCTACGCCAACATGAACTCCATGAGCCCCATGTACGGG CAGGCGGGCCTGAGCCGCGCCCGCGACCCCAAGACCTACAGGCGCAGCTACA CGCACGCAAAGCCGCCCTACTCGTACATCTCGCTCATCACCATGGCCATCCAG CAGAGCCCCAACAAGATGCTGACGCTGAGCGAGATCTACCAGTGGATCATGGA CCTCTTCCCCTTCTACCGGCAGAACCAGCAGCGCTGGCAGAACTCCATCCGCC ACTCGCTCTCCTTCAACGACTGTTTCCTGAAGGTGCCCCGCTCGCCCGACAAG CCCGGCAAGGGCTCCTTCTGGACCCTGCACCCTGACTCGGGCAACATGTTCGA GAACGGCTGCTACCTGCGCCGCCAGAAGCGCTTCAAGTGCGAGAAGCAGCTG GCGCTGAAGGAGGCCGCAGGCGCCGCCGGCAGCGGCAAGAAGGCGGCCGCC GGGGCCCAGGCCTCACAGGCTCAACTCGGGGAGGCCGCCGGGCCGGCCTCC GAGACTCCGGCGGGCACCGAGTCGCCTCACTCGAGCGCCTCCCCGTGCCAGG AGCACAAGCGAGGGGGCCTGGGAGAGCTGAAGGGGACGCCGGCTGCGGCGC TGAGCCCCCCAGAGCCGGCGCCCTCTCCCGGGCAGCAGCAGCAGGCCGCGG CCCACCTGCTGGGCCCGCCCCACCACCCGGGCCTGCCGCCTGAGGCCCACCT GAAGCCGGAACACCACTACGCCTTCAACCACCCGTTCTCCATCAACAACCTCAT GTCCTCGGAGCAGCAGCACCACCACAGCCACCACCACCACCAGCCCCACAAAA TGGACCTCAAGGCCTACGAACAGGTGATGCACTACCCCGGCTACGGTTCCCCC ATGCCTGGCAGCTTGGCCATGGGCCCGGTCACGAACAAAACGGGCCTGGACG CCTCGCCCCTGGCCGCAGATACCTCCTACTACCAGGGGGTGTACTCCCGGCCC ATTATGAACTCCTCT (SEQ ID NO:20).

[0052] Therefore, disclosed herein is a viral or non-viral vector comprising the nucleic acid sequence SEQ ID NO:11 , SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ IDNO: 19, SEQ ID NO:20, or any combination thereof, or a variant thereof 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 SEQ ID NO:11 , SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and / or SEQ ID NQ:20.

[0053] In order to express a polypeptide or functional nucleic acid, the nucleotide coding sequence may be inserted into appropriate expression vector. Therefore, also disclosed is a non-viral vector comprising a polynucleotide comprising two or more nucleic acid sequences encoding the reprogramming cocktail, wherein the two or more nucleic acid sequences are operably linked to an expression control sequence. In some embodiments, the nucleic acid sequences are operably linked to a single expression control sequence. In other embodiments, the nucleic acid sequences are operably linked to two or more separate expression control sequences. In some embodiments, the non-viral vector comprises a plasmid selected from the group plRES-hrGFP-21 , pAd-IRES-GFP, and pCDNA3.0.

[0054] Methods to construct expression vectors containing genetic sequences and appropriate transcriptional and translational control elements are well known in the art. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described in Sambrook et al., Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Press, Plainview, N.Y., 1989), and Ausubel et al., Current Protocols in Molecular Biology (John Wiley & Sons, New York, N.Y., 1989).

[0055] Expression vectors generally contain regulatory sequences necessary elements for the translation and / or transcription of the inserted coding sequence. For example, the coding sequence is preferably operably linked to a promoter and / or enhancer to help control the expression of the desired gene product.

[0056] The “control elements” or “regulatory sequences” are those nontranslated regions of the vector — enhancers, promoters, 5' and 3' untranslated regions — which interact with host cellular proteins to carry out transcription and translation. Such elements may vary in their strength and specificity.

[0057] A “promoter” is generally a sequence or sequences of DNA that function when in a relatively fixed location in regard to the transcription start site. A “promoter” contains core elements required for basic interaction of RNA polymerase and transcription factors and can contain upstream elements and response elements.

[0058] “Enhancer” generally refers to a sequence of DNA that functions at no fixed distance from the transcription start site and can be either 5' or 3' to the transcription unit. Furthermore, enhancers can be within an intron as well as withinthe coding sequence itself. They are usually between 10 and 300 bp in length, and they function in cis. Enhancers function to increase transcription from nearby promoters. Enhancers, like promoters, also often contain response elements that mediate the regulation of transcription. Enhancers often determine the regulation of expression.

[0059] An “endogenous” enhancer / promoter is one which is naturally linked with a given gene in the genome. An “exogenous” or “heterologous” enhancer / promoter is one which is placed in juxtaposition to a gene by means of genetic manipulation (i.e., molecular biological techniques) such that transcription of that gene is directed by the linked enhancer / promoter.

[0060] Promoters used in biotechnology are of different types according to the intended type of control of gene expression. They can be generally divided into constitutive promoters, tissue-specific or development-stage-specific promoters, inducible promoters, and synthetic promoters.

[0061] Constitutive promoters direct expression in virtually all tissues and are largely, if not entirely, independent of environmental and developmental factors. As their expression is normally not conditioned by endogenous factors, constitutive promoters are usually active across species and even across kingdoms. Examples of constitutive promoters include CMV, EF1a, SV40, PGK1 , Ubc, Human beta actin, and CAG.

[0062] Tissue-specific or development-stage-specific promoters direct the expression of a gene in specific tissue(s) or at certain stages of development. For plants, promoter elements that are expressed or affect the expression of genes in the vascular system, photosynthetic tissues, tubers, roots and other vegetative organs, or seeds and other reproductive organs can be found in heterologous systems (e.g. distantly related species or even other kingdoms) but the most specificity is generally achieved with homologous promoters (i.e. from the same species, genus or family). This is probably because the coordinate expression of transcription factors is necessary for regulation of the promoter's activity.

[0063] The performance of inducible promoters is not conditioned to endogenous factors but to environmental conditions and external stimuli that can be artificially controlled. Within this group, there are promoters modulated by abiotic factors such as light, oxygen levels, heat, cold and wounding. Since some of these factors are difficult to control outside an experimental setting, promoters that respond to chemical compounds, not found naturally in the organism of interest, are of particular interest. Along those lines, promoters that respond to antibiotics, copper, alcohol, steroids, and herbicides, among other compounds, have been adapted andrefined to allow the induction of gene activity at will and independently of other biotic or abiotic factors.

[0064] The two most commonly used inducible expression systems for research of eukaryote cell biology are named Tet-Off and Tet-On. The Tet-Off system makes use of the tetracycline transactivator (tTA) protein, which is created by fusing one protein, TetR (tetracycline repressor), found in Escherichia coli bacteria, with the activation domain of another protein, VP16, found in the Herpes Simplex Virus. The resulting tTA protein is able to bind to DNA at specific TetO operator sequences. In most Tet-Off systems, several repeats of such TetO sequences are placed upstream of a minimal promoter such as the CMV promoter. The entirety of several TetO sequences with a minimal promoter is called a tetracycline response element (TRE), because it responds to binding of the tetracycline transactivator protein tTA by increased expression of the gene or genes downstream of its promoter. In a Tet-Off system, expression of TRE-controlled genes can be repressed by tetracycline and its derivatives. They bind tTA and render it incapable of binding to TRE sequences, thereby preventing transactivation of TRE-controlled genes. A Tet- On system works similarly, but in the opposite fashion. While in a Tet-Off system, tTA is capable of binding the operator only if not bound to tetracycline or one of its derivatives, such as doxycycline, in a Tet-On system, the rtTA protein is capable of binding the operator only if bound by a tetracycline. Thus the introduction of doxycycline to the system initiates the transcription of the genetic product. The Tet- On system is sometimes preferred over Tet-Off for its faster responsiveness.

[0065] In some embodiments, the nucleic acid sequences encoding ETV2, FOXC2, and / or FLI1 are operably linked to the same expression control sequence. Alternatively, internal ribosome entry sites (IRES) elements can be used to create multigene, or polycistronic, messages. IRES elements are able to bypass the ribosome scanning model of 5' methylated Cap dependent translation and begin translation at internal sites. IRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, creating polycistronic messages. By virtue of the IRES element, each open reading frame is accessible to ribosomes for efficient translation. Multiple genes can be efficiently expressed using a single promoter / enhancer to transcribe a single message.

[0066] Disclosed are non-viral vectors containing one or more polynucleotides disclosed herein operably linked to an expression control sequence. Examples of such non-viral vectors include the oligonucleotide alone or in combination with a suitable protein, polysaccharide or lipid formulation. Non-viralmethods present certain advantages over viral methods, with simple large scale production and low host immunogenicity being just two. Previously, low levels of transfection and expression of the gene held non-viral methods at a disadvantage; however, recent advances in vector technology have yielded molecules and techniques with transfection efficiencies similar to those of viruses.

[0067] Examples of suitable non-viral vectors include, but are not limited to plRES-hrGFP-2a, pAd-IRES-GFP, and pCDNA3.0.

[0068] The compositions disclosed can be used therapeutically in combination with a pharmaceutically acceptable carrier. By “pharmaceutically acceptable” is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject, along with the nucleic acid or vector, without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. The carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art.

[0069] The materials may be in solution, suspension (for example, incorporated into microparticles, liposomes, or cells). These may be targeted to a particular cell type via antibodies, receptors, or receptor ligands. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Senter, et al., Bioconjugate Chem., 2:447-451 , (1991); Bagshawe, K.D., Br. J. Cancer, 60:275-281 , (1989); Bagshawe, et al., Br. J. Cancer, 58:700-703, (1988); Senter, et al., Bioconjugate Chem., 4:3-9, (1993); Battelli, et al., Cancer Immunol. Immunother., 35:421-425, (1992); Pietersz and McKenzie, Immunolog. Reviews, 129:57-80, (1992); and Roffler, et al., Biochem. Pharmacol, 42:2062-2065, (1991)). Vehicles such as “stealth” and other antibody conjugated liposomes (including lipid mediated drug targeting to colonic carcinoma), receptor mediated targeting of DNA through cell specific ligands, lymphocyte directed tumor targeting, and highly specific therapeutic retroviral targeting of murine glioma cells in vivo. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Hughes et al., Cancer Research, 49:6214-6220, (1989); and Litzinger and Huang, Biochimica et Biophysica Acta, 1104:179-187, (1992)). In general, receptors are involved in pathways of endocytosis, either constitutive or ligand induced. These receptors cluster in clathrin-coated pits, enter the cell via clathrin-coated vesicles, pass through an acidified endosome in which the receptors are sorted, and then either recycle to the cell surface, become stored intracellularly, or are degraded in lysosomes. The internalization pathways serve a variety offunctions, such as nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, dissociation and degradation of ligand, and receptor-level regulation. Many receptors follow more than one intracellular pathway, depending on the cell type, receptor concentration, type of ligand, ligand valency, and ligand concentration. Molecular and cellular mechanisms of receptor-mediated endocytosis has been reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399-409 (1991)).

[0070] Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. A.R. Gennaro, Mack Publishing Company, Easton, PA 1995. Typically, an appropriate amount of a pharmaceutically- acceptable salt is used in the formulation to render the formulation isotonic.Examples of the pharmaceutically-acceptable carrier include, but are not limited to, saline, Ringer's solution and dextrose solution. The pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5. Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of composition being administered.

[0071] Pharmaceutical carriers are known to those skilled in the art. These most typically would be standard carriers for administration of drugs to humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH. The compositions can be administered intramuscularly or subcutaneously. Other compounds will be administered according to standard procedures used by those skilled in the art.

[0072] Pharmaceutical compositions may include carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the molecule of choice. Pharmaceutical compositions may also include one or more active ingredients such as antimicrobial agents, antiinflammatory agents, anesthetics, and the like.

[0073] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose,dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.

[0074] Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.

[0075] Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders may be desirable..

[0076] Some of the compositions may potentially be administered as a pharmaceutically acceptable acid- or base- addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, trialkyl and aryl amines and substituted ethanolamines.

[0077] The herein disclosed compositions, including pharmaceutical composition, may be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated. For example, the disclosed compositions can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally. The compositions may be administered orally, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, transdermally, extracorporeally, ophthalmically, vaginally, rectally, intranasally, topically or the like, including topical intranasal administration or administration by inhalant.Methods

[0078] Also disclosed are methods of reprogramming somatic cells into insulin-producing and / or glucagon-producing that involve delivering intracellularly into the somatic cells a polynucleotide encoding reprogramming cocktail disclosed herein. In some embodiments, the nucleic acid sequences are present in non-viral vectors. In some embodiments, the nucleic acid sequences are operably linked to anexpression control sequence. In other embodiments the nucleic acids are operably linked to two or more expression control sequences.

[0079] A variety of methods are known in the art and suitable for introduction of nucleic acid into a cell, including viral and non-viral mediated techniques. Examples of typical non-viral mediated techniques include, but are not limited to, electroporation, calcium phosphate mediated transfer, nucleofection, sonoporation, heat shock, magnetofection, liposome mediated transfer, microinjection, microprojectile mediated transfer (nanoparticles), cationic polymer mediated transfer (DEAE-dextran, polyethylenimine, polyethylene glycol (PEG) and the like) or cell fusion.

[0080] In some embodiments, after transfecting target cells with a polynucleotide encoding reprogramming cocktail disclosed herein, the cells can then pack the transfected genes (e.g. cDNA) into EVs, which can then induce endothelium in other somatic cells. Similarly, cells transfected with a miR-200b inhibitor will tend to exocytose part of that inhibitor in EVs, which could subsequently be used to induce endothelium in other / remote somatic cells. Therefore, also disclosed is a method of reprogramming somatic cells into insulin-producing and / or glucagon-producing that involves exposing the somatic cell with an extracellular vesicle produced from a cell containing or expressing a reprogramming cocktail disclosed herein.

[0081] Therefore, disclosed are methods of reprogramming somatic cells into insulin-producing and / or glucagon-producing that involve exposing the somatic cells to extracellular vesicles (EVs) isolated from cells expressing or containing exogenous polynucleotides encoding reprogramming cocktail disclosed herein. EVs secreted by the donor cells can then collected from the culture medium. These EVs can then be administered to the somatic cells to reprogram them into vasculogenic cells and / or endothelial cells. In some embodiments, the donor cells can be any stromal / support cell from connective or epithelial tissues, including (but not limited to) skin fibroblasts, muscle fibroblast, skin epithelium, gut epithelium, and ductal epithelium.

[0082] Exosomes and microvesicles are EVs that differ based on their process of biogenesis and biophysical properties, including size and surface protein markers. Exosomes are homogenous small particles ranging from 40 to 150 nm in size and they are normally derived from the endocytic recycling pathway. In endocytosis, endocytic vesicles form at the plasma membrane and fuse to form early endosomes. These mature and become late endosomes where intraluminal vesicles bud off into an intra-vesicular lumen. Instead of fusing with the lysosome, these multivesicular bodies directly fuse with the plasma membrane and release exosomes into the extracellular space. Exosome biogenesis, protein cargo sorting, and releaseinvolve the endosomal sorting complex required for transport (ESCRT complex) and other associated proteins such as Alix and Tsg101. In contrast, microvesicles, are produced directly through the outward budding and fission of membrane vesicles from the plasma membrane, and hence, their surface markers are largely dependent on the composition of the membrane of origin. Further, they tend to constitute a larger and more heterogeneous population of extracellular vesicles, ranging from 150 to 1000 nm in diameter. However, both types of vesicles have been shown to deliver functional mRNA, miRNA and proteins to recipient cells.

[0083] In some embodiments, the polynucleotides are delivered to the somatic cells, or the donor cells for EVs, intracellularly via a gene gun, a microparticle or nanoparticle suitable for such delivery, transfection by electroporation, three-dimensional nanochannel electroporation, a tissue nanotransfection device, a liposome suitable for such delivery, or a deep-topical tissue nanoelectroinjection device. In some embodiments, a viral vector can be used. However, in other embodiments, the polynucleotides are not delivered virally.

[0084] Electroporation is a technique in which an electrical field is applied to cells in order to increase permeability of the cell membrane, allowing cargo (e.g., reprogramming factors) to be introduced into cells. Electroporation is a common technique for introducing foreign DNA into cells.

[0085] Tissue nanotransfection allows for direct cytosolic delivery of cargo (e.g., reprogramming factors) into cells by applying a highly intense and focused electric field through arrayed nanochannels, which benignly nanoporates the juxtaposing tissue cell members, and electrophoretically drives cargo into the cells.

[0086] In one embodiment, the disclosed compositions are administered in a dose equivalent to parenteral administration of about 0.1 ng to about 100 g per kg of body weight, about 10 ng to about 50 g per kg of body weight, about 100 ng to about 1 g per kg of body weight, from about 1 pg to about 100 mg per kg of body weight, from about 1 pg to about 50 mg per kg of body weight, from about 1 mg to about 500 mg per kg of body weight; and from about 1 mg to about 50 mg per kg of body weight. Alternatively, the amount of the disclosed compositions administered to achieve a therapeutic effective dose is about 0.1 ng, 1 ng, 10 ng, 100 ng, 1 pg, 10 pg, 100 pg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 500 mg per kg of body weight or greater.

[0087] In some embodiments, the disclosed compositions and methods are used to create a vasculature that can serve as a scaffolding structure. Thisscaffolding structure can then be used, for example, to aid in the repair of nerve tissue. Applications of this include peripheral nerve injuries, and pathological / injurious insults to the central nervous system such as traumatic brain injury or stroke. In some embodiments, the created vasculature can be used to nourish composite tissue transplants, or any tissue graft.

[0088] In some embodiments, the disclosed compositions and methods are used to convert “unwanted” tissue (e.g., fat, scar tissue) into vasculature. Such newly formed vasculature is expected to “resorb” under non-ischemic conditions.Definitions

[0089] The term “subject” refers to any individual who is the target of administration or treatment. The subject can be a vertebrate, for example, a mammal. Thus, the subject can be a human or veterinary patient. The term “patient” refers to a subject under the treatment of a clinician, e.g., physician.

[0090] The term “therapeutically effective" refers to the amount of the composition used is of sufficient quantity to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration only requires a reduction or alteration, not necessarily elimination.

[0091] The term “pharmaceutically acceptable” refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit / risk ratio.

[0092] The term “carrier” means a compound, composition, substance, or structure that, when in combination with a compound or composition, aids or facilitates preparation, storage, administration, delivery, effectiveness, selectivity, or any other feature of the compound or composition for its intended use or purpose. For example, a carrier can be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject.

[0093] The term “treatment” refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially orcompletely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.

[0094] The term “inhibit” refers to a decrease in an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels.

[0095] The term “polypeptide” refers to amino acids joined to each other by peptide bonds or modified peptide bonds, e.g., peptide isosteres, etc. and may contain modified amino acids other than the 20 gene-encoded amino acids. The polypeptides can be modified by either natural processes, such as post-translational processing, or by chemical modification techniques which are well known in the art. Modifications can occur anywhere in the polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. The same type of modification can be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide can have many types of modifications. Modifications include, without limitation, acetylation, acylation, ADP- ribosylation, amidation, covalent cross-linking or cyclization, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of a phosphytidylinositol, disulfide bond formation, demethylation, formation of cysteine or pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristolyation, oxidation, pergylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, and transfer-RNA mediated addition of amino acids to protein such as arginylation. (See Proteins - Structure and Molecular Properties 2nd Ed., T.E. Creighton, W.H. Freeman and Company, New York (1993); Posttranslational Covalent Modification of Proteins, B.C. Johnson, Ed., Academic Press, New York, pp. 1-12 (1983)).

[0096] As used herein, the term “amino acid sequence” refers to a list of abbreviations, letters, characters or words representing amino acid residues. The amino acid abbreviations used herein are conventional one letter codes for the amino acids and are expressed as follows: A, alanine; B, asparagine or aspartic acid; C,cysteine; D aspartic acid; E, glutamate, glutamic acid; F, phenylalanine; G, glycine; H histidine; I isoleucine; K, lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q, glutamine; R, arginine; S, serine; T, threonine; V, valine; W, tryptophan; Y, tyrosine; Z, glutamine or glutamic acid.

[0097] The phrase “nucleic acid” as used herein refers to a naturally occurring or synthetic oligonucleotide or polynucleotide, whether DNA or RNA or DNA-RNA hybrid, single-stranded or double-stranded, sense or antisense, which is capable of hybridization to a complementary nucleic acid by Watson-Crick basepairing. Nucleic acids can also include nucleotide analogs (e.g., BrdU), and non- phosphodiester internucleoside linkages (e.g., peptide nucleic acid (PNA) or thiodiester linkages). In particular, nucleic acids can include, without limitation, DNA, RNA, cDNA, gDNA, ssDNA, dsDNA or any combination thereof.

[0098] A “nucleotide" as used herein is a molecule that contains a base moiety, a sugar moiety, and a phosphate moiety. Nucleotides can be linked together through their phosphate moieties and sugar moieties creating an internucleoside linkage. The term “oligonucleotide” is sometimes used to refer to a molecule that contains two or more nucleotides linked together. The base moiety of a nucleotide can be adenine-9-yl (A), cytosine-1-yl (C), guanine-9-yl (G), uracil-1 -yl (U), and thymin-1-yl (T). The sugar moiety of a nucleotide is a ribose or a deoxyribose. The phosphate moiety of a nucleotide is pentavalent phosphate. A non-limiting example of a nucleotide would be 3 -AMP (3’-adenosine monophosphate) or 5’-GMP (5’- guanosine monophosphate).

[0099] A nucleotide analog is a nucleotide that contains some type of modification to the base, sugar, and / or phosphate moieties. Modifications to nucleotides are well known in the art and would include, for example, 5- methylcytosine (5-me-C), 5 hydroxymethyl cytosine, xanthine, hypoxanthine, and 2- aminoadenine as well as modifications at the sugar or phosphate moieties.

[0100] Nucleotide substitutes are molecules having similar functional properties to nucleotides, but which do not contain a phosphate moiety, such as peptide nucleic acid (PNA). Nucleotide substitutes are molecules that will recognize nucleic acids in a Watson-Crick or Hoogsteen manner, but are linked together through a moiety other than a phosphate moiety. Nucleotide substitutes are able to conform to a double helix type structure when interacting with the appropriate target nucleic acid.

[0101] The term “vector” or “construct” refers to a nucleic acid sequence capable of transporting into a cell another nucleic acid to which the vector sequence has been linked. The term “expression vector” includes any vector, (e.g., a plasmid,cosmid or phage chromosome) containing a gene construct in a form suitable for expression by a cell (e.g., linked to a transcriptional control element). “Plasmid” and “vector” are used interchangeably, as a plasmid is a commonly used form of vector. Moreover, the invention is intended to include other vectors which serve equivalent functions.

[0102] The term “operably linked to” refers to the functional relationship of a nucleic acid with another nucleic acid sequence. Promoters, enhancers, transcriptional and translational stop sites, and other signal sequences are examples of nucleic acid sequences operably linked to other sequences. For example, operable linkage of DNAto a transcriptional control element refers to the physical and functional relationship between the DNA and promoter such that the transcription of such DNA is initiated from the promoter by an RNA polymerase that specifically recognizes, binds to and transcribes the DNA.

[0103] For purposes herein, the % sequence identity of a given nucleotides or amino acids sequence C to, with, or against a given nucleic acid sequence D (which can alternatively be phrased as a given sequence C that has or comprises a certain % sequence identity to, with, or against a given sequence D) is calculated as follows:

[0104] 100 times the fraction W / Z,

[0105] where W is the number of nucleotides or amino acids scored as identical matches by the sequence alignment program in that program’s alignment of C and D, and where Z is the total number of nucleotides or amino acids in D. It will be appreciated that where the length of sequence C is not equal to the length of sequence D, the % sequence identity of C to D will not equal the % sequence identity of D to C. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software.

[0106] By “specifically hybridizes” is meant that a probe, primer, or oligonucleotide recognizes and physically interacts (that is, base-pairs) with a substantially complementary nucleic acid (for example, a c-met nucleic acid) under high stringency conditions, and does not substantially base pair with other nucleic acids.

[0107] The term “stringent hybridization conditions” as used herein mean that hybridization will generally occur if there is at least 95% and preferably at least 97% sequence identity between the probe and the target sequence. Examples of stringent hybridization conditions are overnight incubation in a solution comprising 50%formamide, 5X SSC (150 mM NaCI, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5X Denhardt’s solution, 10% dextran sulfate, and 20 .g / ml denatured, sheared carrier DNA such as salmon sperm DNA, followed by washing the hybridization support in 0.1 X SSC at approximately 65°C. Other hybridization and wash conditions are well known and are exemplified in Sambrook et al, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y. (1989), particularly chapter 11.Embodiments

[0108] Embodiment 1 . A polynucleotide comprising at least one nucleic acid sequences encoding Tcf3, Trp63, Sox9, or a combination thereof and at least one nucleic acid sequence encoding Pdx1 , Ngn3, Mafa, Neurodi , Pax4, Nkx6.1 , FoxA2, or any combination thereof.

[0109] Embodiment 2. A non-viral vector comprising the polynucleotide of embodiment 1 , wherein the two or more nucleic acid sequences are operably linked to an expression control sequence.

[0110] Embodiment 3. The non-viral vector of embodiment 2, wherein each of the nucleic acid sequences are operably linked to a single expression control sequence.

[0111] Embodiment 4. The non-viral vector of embodiment 2 or 3, wherein the non-viral vector comprises a plasmid selected from the group plRES-hrGFP-2a, pAd-IRES-GFP, and pCDNA3.0.

[0112] Embodiment 5. The non-viral vector of any one of embodiments 2 to 4, wherein the polynucleotide is encapsulated in a liposome, microparticle or nanoparticle suitable for intracellular delivery.

[0113] Embodiment 6. A method of reprogramming a somatic cell into insulinproducing and / or glucagon-producing, comprising a) delivering intracellularly into the somatic cell one or more proteins selected from the group consisting of Tcf3, Trp63, and Sox9, along with one or more proteins selected from the group consisting of Pdx1 , Ngn3, Mafa, Neurodi , Pax4, Nkx6.1 , and FoxA2, b) delivering intracellularly into the somatic cell one polynucleotides encoding one or more proteins selected from the group consisting of Tcf3, Trp63, and Sox9, along with one or more proteins selected from the group consisting of Pdx1 , Ngn3, Mafa, Neurodi , Pax4, Nkx6.1 , and FoxA2; c) exposing the somatic cell to an extracellular vesicle produced from a cell containing or expressing one or more proteins selected from the group consistingof Tcf3, Trp63, and Sox9, along with one or more proteins selected from the group consisting of Pdx1 , Ngn3, Mafa, Neurodi , Pax4, Nkx6.1 , and FoxA2; or d) exposing the somatic cell to an extracellular vesicle produced from a cell containing or expressing polynucleotides encoding one or more proteins selected from the group consisting of Tcf3, Trp63, and Sox9, along with one or more proteins selected from the group consisting of Pdx1 , Ngn3, Mafa, Neurodi , Pax4, Nkx6.1 , and FoxA2.

[0114] Embodiment 7. The method of embodiment 7, wherein the proteins or polynucleotides are administered sequentially.

[0115] Embodiment 8. The method of embodiment 7, comprising delivering intracellularly into the somatic cell the polynucleotide of embodiment 1 , the non-viral vector of any one of embodiments 2 or 5, or the composition of embodiment 6.

[0116] Embodiment 9. The method of any one of claims 7 to 13, wherein the somatic cell is a skin cell.

[0117] Embodiment 10. The method of any one of embodiments 7 to 14, wherein intracellular delivery comprises three-dimensional nanochannel electroporation.

[0118] Embodiment 11. The method of any one of embodiments 7 to 14, wherein intracellular delivery comprises delivery by a tissue nanotransfection device.

[0119] Embodiment 12. The method of any one of embodiments 7 to 14, wherein intracellular delivery comprises delivery by a deep-topical tissue nanoelectroinjection device.

[0120] A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.EXAMPLESExample 1: Pancreatic Cells Derived from Fibroblasts using Non-viral Cell Reprogramming Methods.Introduction

[0121] Type I diabetes is driven by the autoimmune destruction of pancreatic beta cells and resulting decrease in insulin production. Allogenic islet transplantation is the current gold standard to obtain insulin-independence for patients with type I diabetes, but its implementation is limited due to a lack of donor tissue and required life-long immunosuppressants for patients after treatment. To address these limitations, several studies have focused on developing alternative cell sources for insulin production using cell reprogramming methods (e.g., iPSCs, viruses).However, to generate a functional cell-based therapy to treat type I diabetes, approximately 0.1 to 1 billion functional p-like cells need to be produced per patient. Thus, to meet this cell demand, the ideal cell source used for reprogramming should be highly accessible, easily isolated, and easily expanded from a patient biopsy. One cell source that meets these criteria is skin dermal fibroblasts (DFs). However, current approaches to p-cell derivation from fibroblasts face several hurdles, including tedious pre-processing steps and heavy reliance on viral vectors. Here we report on the development of a non-viral approach to p-cell derivation from fibroblasts via electroporation-based gene delivery.Materials and Methods

[0122] The objective of this study was to investigate whether the delivery of plasmid DNA encoding for a unique combination of transcription factors for skin plasticity (e.g., Tcf3, Sox9) and beta cell patterning (e.g., Pdx1 , Ngn3) could drive the direct reprogramming of fibroblasts into i Cs. Three transcription factors (3SP) were identified as potentiall important for the plasticity observed in adult stem cells in the skin and seven transcription factors (7pC) that have been identified as important for the biological development of pancreatic beta cells or the reprogramming of other cell types (e.g., hepatocytes) into beta pancreatic beta cells. Plasmid DNA encoding for the identified transcription factors or pCMV6 (sham / control plasmid DNA) was delivered to fibroblasts in vitro using bulk electroporation (BEP). All studies used mouse embryonic fibroblasts (MEFs). Cell gene and protein expression were analyzed at predetermined time points using qRT-PCR or immunohistology as appropriate. Data was collected, processed, and quantified using automated tools and / or completed by blinded investigators. Post hoc exclusion criteria included statistical outliers (i.e., Studentized residual > 1 .5 interquartile range for data set).Results and Discussion

[0123] Results demonstrated that fibroblasts transduced with all ten of the 3SP+7PC factors led to an increase in mRNA expression for both insulin 1 and insulin 2 by day 14 after BEP (Figure 1A). Moreover, this increased insulin 1 / 2 expression correlated with a small population (<1%) of transduced cells expressing the insulin protein by day 14 after BEP as well (Figure 1 B). Glucagon expression was also increased on day 7 after BEP in MEFs transduced with the 3SP+7PC factors compared to control. However, glucagon expression was observed to steadily decrease over time Figure 1A.Conclusion

[0124] These findings suggest that fibroblasts transduced with plasmid DNA encoding for 3SP+7PC factors have the potential to reprogram into induced p cells.Current experiments are working to further characterize the insulin production (e.g., ELISAs) from these transduced cells as well as their sensitivity to glucose (e.g., glucose stimulated insulin response assays). The development of an alternative cell source to treat (and potentially cure) type I diabetes could reduce the healthcare and financial burden associated with this condition for not only the patients but the global healthcare system.Example 2:

[0125] FIGs. 2A to 2F show the combined transfection of plasmid DNA encoding for 3 skin plasticity factors and 7 p cell patterning factors induces increased pancreatic islet gene expression in mouse embryonic fibroblasts in standard fibroblast media. Gene expression profiles of INS1-EGFP MEFs 1 and / or 4 days post-BEP with plasmid DNA cocktails encoding for control (pCMV6), 3 skin plasticity factors (3SP), Pdx1 , Ngn3 and Mafa (PNM), 7 beta cell patterning factors (7pC), or the combination of 3SP+7pC. FIG. 2A shows Pdx1 expression 1- and 4-days post- BEP. B) Pdx1 expression 1-day post-BEP. FIGs. 2C-2E show insulin 1 , insulin 2, and glucagon expression 1- and 4 days post-BEP. FIG. 2F shows EGFP expression 4 post-BEP. Connecting letters represent data groups that are not significantly different from each other (p>0.05).

[0126] FIGs. 3 shows insulin gene expression is maximum in fibroblasts 14 post-BEP with 3SP+7pC. Insulin 1 , insulin 2, glucagon, somatostatin, and Pdx1 gene expression profiles of INS1-EGFP MEFs 7-, 14-, and 21-days post-BEP with plasmid DNA cocktails encoding for control (pCMV6) or the combination of 3SP+7PC. Connecting letters represent data groups that are not significantly different from each other (p>0.05).

[0127] FIG. 4 shows insulin protein expression observed in smaller populations of fibroblasts transfected with control (pCMV6) or 3SP+7PC. FIG. 4 contains immunomicrographs depicting nuclei (DAPI), insulin (a-insulin), and merged images (with brightfield) of INS1-EGFP MEFs 14 days post-BEP with control or 3SP+7PC (scale bar - 20um, objective = 20x).

[0128] FIGs. 5A and 5B show insulin and co-localized EGFP protein expression observed in larger populations of fibroblasts transfected with control (pCMV6) or 3SP+7PC. FIG. 5A contains immunomicrographs depicting nuclei (DAPI), insulin (a-insulin), and merged images of INS1-EGFP MEFs 14 days postNEP with control or 3SP+7PC (scale bar = 20um, objective = 20x). FIG. 5B contains immunomicrographs depicting nuclei (DAPI), EGFP (a-GFP), insulin (a-insulin), and merged images of INS1-EGFP MEFs 14 days post-NEP with control or 3SP+7PC (scale bar = 20um, objective = 20x).

[0129] FIGs. 6A and 6B show three skin plasticity factors (3SP) greatly decrease heterochromatin markers and significantly open the chromatin landscape for potential reprogramming. FIG. 6A shows cytological fluorescent analysis of heterochromatin markers (H3K9me3) for MEFs transfected with all combinations of skin plasticity factors or controls (NT, iBET, pCMV6) 1- and 3-days post-BEP. FIG. 6B is a histological example depicting nuclei (DAPI), H3K9me3, and merged images of MEFs used to create corresponding H3K9me3 cytological fluorescent analysis of 3SP factor or control (NT, iBET, pCMV6) combinations 7 days post-BEP. (scale bar = 100um, objective = 20x).

[0130] FIG. 7 shows INS1-EGFP protein expression and islet-like MEF clustering 14 days post-BEP following short-term trypsin treatment 3 days post-BEP. Immunomicrographs depicting brightfield, nuclei (DAPI), EGFP (a-GFP), insulin (a- insulin), c-peptide (a-c-peptide), and merged images of INS1-EGFP MEFs 14 days post-BEP with control or 3SP+7pC (scale bar = 32.5um, objective = 20x).

[0131] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.

[0132] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

WHAT IS CLAIMED IS:

1. A polynucleotide comprising at least one nucleic acid sequences encoding Tcf3, Trp63, Sox9, or a combination thereof and at least one nucleic acid sequence encoding Pdx1 , Ngn3, Mafa, Neurodi , Pax4, Nkx6.1 , FoxA2, or any combination thereof.

2. A non-viral vector comprising the polynucleotide of claim 1 , wherein the two or more nucleic acid sequences are operably linked to an expression control sequence.

3. The non-viral vector of claim 2, wherein each of the nucleic acid sequences are operably linked to a single expression control sequence.

4. The non-viral vector of claim 2, wherein the non-viral vector comprises a plasmid selected from the group plRES-hrGFP-2a, pAd-IRES-GFP, and pCDNA3.0.

5. The non-viral vector of claim 2, wherein the polynucleotide is encapsulated in a liposome, microparticle or nanoparticle suitable for intracellular delivery.

6. A method of reprogramming a somatic cell into insulin-producing and / or glucagon-producing, comprising(a) delivering intracellularly into the somatic cell one or more proteins selected from the group consisting of Tcf3, Trp63, and Sox9, along with one or more proteins selected from the group consisting of Pdx1 , Ngn3, Mafa, Neurodi , Pax4, Nkx6.1 , and FoxA2,(b) delivering intracellularly into the somatic cell one polynucleotides encoding one or more proteins selected from the group consisting of Tcf3, Trp63, and Sox9, along with one or more proteins selected from the group consisting of Pdx1 , Ngn3, Mafa, Neurodi, Pax4, Nkx6.1 , and FoxA2;(c) exposing the somatic cell to an extracellular vesicle produced from a cell containing or expressing one or more proteins selected from the group consisting of Tcf3, Trp63, and Sox9, along with one or more proteins selected from the group consisting of Pdx1 , Ngn3, Mafa, Neurodi , Pax4, Nkx6.1 , and FoxA2; or(d) exposing the somatic cell to an extracellular vesicle produced from a cell containing or expressing polynucleotides encoding one or more proteins selected from the group consisting of Tcf3, Trp63, and Sox9, along with one or more proteins selected from the group consisting of Pdx1 , Ngn3, Mafa, Neurodi , Pax4, Nkx6.1 , and FoxA2.

7. The method of claim 7, wherein the proteins or polynucleotides are administered sequentially.

8. The method of claim 7, comprising delivering intracellularly into the somatic cell the polynucleotide of claim 1 , the non-viral vector of claim 2, or the composition of claim 6.

9. The method of claim 7, wherein the somatic cell is a skin cell.

10. The method of claim 7, wherein intracellular delivery comprises three- dimensional nanochannel electroporation.

11. The method of claim 7, wherein intracellular delivery comprises delivery by a tissue nanotransfection device.

12. The method of claim 7, wherein intracellular delivery comprises delivery by a deep-topical tissue nanoelectroinjection device.

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