WO2011130618A2 - Compositions and methods for producing glycosylated human pdx-1 protein and methods of use therefore - Google Patents

Abstract

The invention generally features a yeast culture system for producing large quantities of biologically active recombinant glycosylated human Pdxl polypeptides, and therapeutic and prophylactic methods featuring such polypeptides.

Description

COMPOSITIONS AND METHODS FOR PRODUCING GLYCOSYLATED HUMAN PDX-1 PROTEIN AND METHODS OF USE THEREFORE

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.

61/325,183, filed on April 16, 2010, the entire contents of each of which are incorporated herein by reference.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY

SPONSORED RESEARCH

This work was supported by the following grants from the National Institutes of Health, Grant Nos: NIDDK DK071831_, The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

The incidence of both Type 1 and especially Type 2 diabetes is increasing dramatically and diabetes now affects roughly 8% of the U.S. population.

Therefore, more than ever, there is an urgent need for new treatment modalities for diabetes. Although the two forms of diabetes have different pathophysiologic mechanisms, a shared feature is insulin insufficiency due to β-cell deficiency. To cure diabetes, researchers are pursuing strategies for restoring β-cell mass by searching for factors that either promote endogenous pancreatic-cell regeneration or reprogram non-pancreatic cells into insulin-producing cells (IPCs). Until recently, over-expression of key pancreatic transcription factor (PTF) genes by means of viral vectors was the most effective way to stimulate pancreatic -cell differentiation into β-cells and to reprogram liver cells/liver stem cells into IPCs.

Several key PTF proteins including PDX1, Ngn3, NeuroD, and Pax4 are critical for pancreatic β-cell differentiation and maturation and they all contain a special amino acid sequence called a protein transduction domain (PTD). The most effective and well characterized PTDs are positively charged cationic sequences that allow proteins to rapidly enter living cells or whole organs. It has been suggested that molecules containing these types of PTDs transduce cells by lipid raft-mediated macropinocytosis and activate the transcription of their target genes. This strategy opens a new avenue for reactivating β-cell development or for directing stem-cell differentiation with protein therapy. PDXl contains a highly basic sequence of 16 amino acids, which constitutes an antennapedia-like PTD that facilitates penetration of plasma membranes. This PTD allows PDXl to cross the cell and nuclear membranes, eliciting biological effects independent of endocytosis.

The PDXl gene consists of two exons coding for a protein of 283 amino acids with a predicted molecular mass of 31 kDa. PDXl is now widely regarded as a master transcriptional regulator in the pancreas and is critical for the development, regeneration, and maintenance of β-cell function. During embryogenesis, the PDXl gene is expressed in all progenitor cells differentiating toward the exocrine, pancreatic ducts, and endotrine pancreas. In adults, PDXl expression is restricted mainly to β-cells and plays a key role in insulin gene expression. A recent publication by the present inventors offers a proof-of-principle demonstration that treatment of streptozotocin-induced diabetic mice with recombinant PDXl protein (rPDXl) promotes, β-cell regerneration and transient liver-cell reprogramming, leading to restoration of normoglycemia. Although rPDXl treatment of diabetic mice is a promising avenue, the in vivo biologic activity of bacterially- expressed rPDXl protein is much lower than transgene-expressed rPDXl. This is at least partially due to the limitations of the prokaryotic bacterial expression system since the rPDXl lacks post-translational modifications. In addition, an additional tag is needed for protein affinity purification, which may affect a protein's biological function, creating immunogenic epitopes that can stimulate antibody production.

The use of Escherichia coil as an expression system has draw- backs when used to manufacture rPDXl. PDXl is a large protein that is glycosylated post-translationally. There is evidence that this modification plays a specific role in the binding of PDXl to DNA and it directly correlates with glucose-stimu lated insulin secretion in β-cells. Since the E. coli expression system lacks the ability to glycosylate proteins post- translationally, improved methods for producing biologically active PDXl are required.

SUMMARY OF THE INVENTION

As described below, the present invention features a culture system for producing large quantities of biologically active PDX- 1 protein, recombinant glycosylated human Pdxl polypeptides, and therapeutic and prophylactic methods featuring such polypeptides.

Compositions and articles defined by the invention were isolated or otherwise manufactured in connection with the examples provided below. Other features and advantages of the invention will be apparent from the detailed description, and from the claims.

Definitions

By "Pancreatic and Duodenal Homeobox - 1 (Pdx-1) polypeptide" is meant a protein or fragment thereof having at least 85% homology to the sequence provided at GenBank Accession No. NP_000200, NP_032840, AAI11593, or a sequence encoded by NM008814, and having DNA binding or transcriptional regulation activity.

An exemplary human PDX-1 amino acid sequence is provided below:

1 mngeeqyyaa tqlykdpcaf qrgpapefsa sppaclymgr qpppppphpf pgalgaleqg

61 sppdispyev ppladdpava hlhhhlpaql alphppagpf pegaepgvle epnrvqlpfp

121 wmkstkahaw kgqwaggaya aepeenkrtr taytraqlle lekeflfnky isrprrvela

181 vmlnlterhi kiwfqnrrmk wkkeedkkrg ggtavggggv aepeqdcavt sgeellalpp

241 ppppggavpp aapvaaregr lppglsaspq pssvaprrpq epr

By "Pancreatic and Duodenal Homeobox - 1 (Pdx-1)" nucleic acid sequence is meant a nucleic acid sequence encoding PDX-1. Exemplary pdx-1 nucleic acid sequences include BC111592 and NM_008814.

By "adult cell" is meant a somatic cell not derived from an embryo or germ cell.

By "inducing regeneration" is meant inducing the generation of a cell, tissue or organ. Methods of regeneration include, but are not limited to, neogenesis, replication, cell proliferation, transdifferentiation, or any other method that involves the production of additional cells that resemble a cell of interest.

By "protein transduction domain" is meant an amino acid sequence that facilitates protein entry into a cell or cell organelle. Exemplary protein transduction domains include but are not limited to a minimal unidecapeptide protein transduction domain (corresponding to residues 47-57 of HIV-1 TAT comprising

YGRKKRRQRRR), a polyarginine sequence comprising a number of arginines sufficient to direct entry into a cell (e.g., 3, 4, 5, 6, 7, 8 or 9 arginines), a VP22 domain (Zender et al., Cancer Gene Ther. 2002 Jun;9(6):489-96), and an

antennapedia protein transduction domain (Noguchi et al., Diabetes 2003; 52(7):1732- 1737). See, also, Nat Biotechnol. 2001 Dec;19(12):1173-6.

By "reprogramming" is meant altering a cell such that at least one protein product is produced in the reprogrammed cell that is not produced in the cell prior to reprogramming (or in a corresponding control cell). Typically, the reprogrammed cell has an altered transcriptional or translational profile, such that the reprogrammed cell expresses a set of proteins not expressed in the cell prior to reprogramming (or in a corresponding control cell).

By "liver-derived cell" is meant any cell derived from the liver. Such cells include hepatocytes, liver stem cells, primary or immortalized cultures of liver cells, or any other cell obtained from the liver.

By "pancreatic transcription factor" is meant any transcription factor expressed in a pancreatic tissue. Exemplary pancreatic transcription factors include, but are not limited to, Pdx-1, Pdx-l/VP16, Ngn3, Pax4, NeuroDl, Nkx2.2 (mouse NM_010919, NP_035049; human C075092, AAH75092), Nkx6.1 (mouse

NP_659204, AF357883; human P78426, NM_006168) Isll (mouse NM_021459, NP_067434; human NM_002202, NP_002193), Pax6 (mouse BC036957,

AAH36957; human NP_000271, BC011953), and MafA (v-maf musculoaponeurotic fibrosarcoma oncogene homolog A), human NP_963883, NM_201589; mouse NP_919331, NM_194350).

By "agent" is meant any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or fragments thereof.

By "ameliorate" is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease.

By "alteration" is meant a change (increase or decrease) in the expression levels or activity of a gene or polypeptide as detected by standard art known methods such as those described herein. As used herein, an alteration includes a 10% change in expression levels, preferably a 25% change, more preferably a 40% change, and most preferably a 50% or greater change in expression levels. "

By "analog" is meant a molecule that is not identical, but has analogous functional or structural features. For example, a polypeptide analog retains the biological activity of a corresponding naturally-occurring polypeptide, while having certain biochemical modifications that enhance the analog's function relative to a naturally occurring polypeptide. Such biochemical modifications could increase the analog's protease resistance, membrane permeability, or half-life, without altering, for example, ligand binding. An analog may include an unnatural amino acid.

In this disclosure, "comprises," "comprising," "containing" and "having" and the like can have the meaning ascribed to them in U.S. Patent law and can mean " includes," "including," and the like; "consisting essentially of" or "consists essentially" likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.

"Detect" refers to identifying the presence, absence or amount of the analyte to be detected.

By "detectable label" is meant a composition that when linked to a molecule of interest renders the latter detectable, via spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include radioactive isotopes, magnetic beads, metallic beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (for example, as commonly used in an ELISA), biotin, digoxigenin, or haptens.

By "disease" is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ. Examples of diseases include diabetes.

By "effective amount" is meant the amount of a required to ameliorate the symptoms of a disease relative to an untreated patient. The effective amount of active compound(s) used to practice the present invention for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an "effective" amount.

The invention provides a number of targets that are useful for the development of highly specific drugs to treat or a disorder characterized by the methods delineated herein. In addition, the methods of the invention provide a facile means to identify therapies that are safe for use in subjects. In addition, the methods of the invention provide a route for analyzing virtually any number of compounds for effects on a disease described herein with high- volume throughput, high sensitivity, and low complexity.

By "fragment" is meant a portion of a polypeptide or nucleic acid molecule. This portion contains, preferably, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.

By "isolated polynucleotide" is meant a nucleic acid (e.g., a DNA) that is free of the genes which, in the naturally-occurring genome of the organism from which the nucleic acid molecule of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuc lease digestion) independent of other sequences. In addition, the term includes an RNA molecule that is transcribed from a DNA molecule, as well as a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence.

By an "isolated polypeptide" is meant a polypeptide of the invention that has been separated from components that naturally accompany it. Typically, the polypeptide is isolated when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated.

Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, a polypeptide of the invention. An isolated polypeptide of the invention may be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.

By "marker" is meant any protein or polynucleotide having an alteration in expression level or activity that is associated with a disease or disorder.

As used herein, "obtaining" as in "obtaining an agent" includes synthesizing, purchasing, or otherwise acquiring the agent.

As used herein, the terms "prevent," "preventing," "prevention," "prophylactic treatment" and the like refer to reducing the probability of developing a disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disorder or condition.

"Primer set" means a set of oligonucleotides that may be used, for example, for PCR. A primer set would consist of at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 30, 40, 50, 60, 80, 100, 200, 250, 300, 400, 500, 600, or more primers.

By "reduces" is meant a negative alteration of at least 10%, 25%, 50%, 75%, or 100%.

By "reference" is meant a standard or control condition.

A "reference sequence" is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset of or the entirety of a specified sequence; for example, a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence. For polypeptides, the length of the reference polypeptide sequence will generally be at least about 16 amino acids, preferably at least about 20 amino acids, more preferably at least about 25 amino acids, and even more preferably about 35 amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the length of the reference nucleic acid sequence will generally be at least about 50 nucleotides, preferably at least about 60 nucleotides, more preferably at least about 75 nucleotides, and even more preferably about 100 nucleotides or about 300 nucleotides or any integer thereabout or therebetween.

By "substantially identical" is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). Preferably, such a sequence is at least 60%, more preferably 80% or 85%, and more preferably 90%, 95% or even 99% identical at the amino acid level or nucleic acid to the sequence used for comparison.

Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e^"3 and e^"100 indicating a closely related sequence.

By "subject" is meant a mammal, including, but not limited to, a human or non-human mammal, such as a bovine, equine, canine, ovine, or feline.

As used herein, the terms "treat," treating," "treatment," and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.

Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

As used herein, the terms "treat," treating," "treatment," and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.

Unless specifically stated or obvious from context, as used herein, the term "or" is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms "a", "an", and "the" are understood to be singular or plural.

Unless specifically stated or obvious from context, as used herein, the term "about" is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.

The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a Western blot showing the detection of secreted rPDXl protein from supernatants (S 1-S8). Eight yeast cones were randomly selected from a 500 μg/ml Zeocin YPD plate, seeded into 5 ml of BMGY medium, and incubated for 24 hour at 28 C. Cells were collected by centrifugation and resuspended to BMMY medium for continuing incubation for an additional 72 hours at 28 C. Methanol was added to a final concentration of 0.5% every 24 hours to maintain induction of rPDXl expression. The expressed rPDXl in the culture media was detected by Western blotting with anti-PDXI antibodies (1 : 1000) following SDS- PAGE separation and protein transferring to the PVDF membrane. Figure legend: M - molecular weight markers: C - positive control for PIM protein from INS- 1 cell lysate; SI -S8 represent the rPDXI-producing yeast clones

Figures 2A and 2B are Western blots showing the Time-dependent rPDXl expression. Figure 2 A is a time-course of rPDXl expression. Culture supernatant (20μ1) from the SI clone fermentation was collected at 12, 24, 48. 72 and 96 hours, respectively. The supernatants were subjected to SDS-PAGE and blots were probed with anti-PDXI antibodies (1 : 1000). Figure 2B shows the detection of rPDXl protein expression in two compartments. The 96 hour-culture supernatants and yeast cell lysates were subjected to protein SDS-PAGE, and the rPDXl proteins were probed by Western blotting using anti-PDXI antibodies. Lane 1 is supernatant (30 μΐ), lane 2 cell lysate for yeast cells (100 μg), and lane 3 is rat insulinoma INS-1 cell lysate as positive control (100 μg).

Figure 3 is a Western blot that confirms rPDXl glycosylation. The purified rPDXl from the yeast culture supernatants was treated without ( -) or with (+) PNGase F for 5 hours and the proteins were fractioned by SDS-PAGE and blotted with anti-PDXI antibodies. Arrows indicate the sizes of the rPDXl protein before or after the enzymatic digestion

Figures 4A and B depict the purification (Figure 4A) and identification (Figure 4B) of rPDXl protein. The secreted rPDXl protein from culture supernatant, concentrated by ammonium sulfate precipitation, and purified by heparin-chromatography was separated by SDS-PAGE and either stained with Coomassie blue (Figure 4A) or probed by Western blotting (Figure 4B) using anti- PDX1 antibodies (1 : 1000) following transfer to the PVDF membrane. Lane 1 - supernatants from the 96 h yeast cultures; lane 2 - the precipitate from 60% saturated ammonium sulfate precipitation; lane 3 - a fraction of the eluate from the heparinaga rose column

Figure 5 is a graph showing the bological function of rPDXl. Huh-7 cells transfected with the insulin promoter-luciferase reporter gene construct were treated with or without the rPDXl protein for 24 hours. Cell lysates were collected for determination of luciferase activity. Renilla luciferase was used as an internal control for transfection efficiency. Bars represent means plus SD and this result is one of the three independent experiments. The difference between control and treated groups was significant (p < 0.001. t-test).

DETAILED DESCRIPTION OF THE INVENTION

The invention features a culture sytstem and related compositions and methods for producing biologically active glycosylated PDX1 proteins that are useful for the treatment or prevention of diabetes.

Pichia pastoris is widely used as an expression system that has the capacity to generate post-translationally modified proteins. Moreover, P.

pastoris can express proteins extracellularly, eliminating the need to use a His- tag for protein purification. Also, P. pastoris does not secrete many intrinsic proteins, simplifying the process of purification of the recombinant protein from the culture medium. Finally, P. pastoris is a methylotrophic organism that can be cultured to high cell densities at relatively low cost. In the present study, the aim was to construct an efficient system for the expression and purification of rPDXl in P. pastoris.

Pancreatic duodenal homeobox 1 protein functions in the development of the embryonic pancreas and plays a key role in pancreatic β-cell differentiation, maturation, generation, and maintenance of normal pancreatic β-cell insulin- producing function. Purified recombinant PDX1 ( rPDXl ) may be a useful tool for many research and clinical applications, however, using the Escherichia coli expression system has several drawbacks for producing quality PDX1 protein. To explore the yeast expression system for generating rPDXl protein, the cDNA coding for the full-length human PDX1 gene was cloned into the secreting expression organism Pichia pastoris, anti-Western blotting analysis of culture medium from methanol-induced expression yeast clones demonstrated that the reDXl was secreted into the culture medium, had a molecular weight by SDS-PAGE of 50 kDa, and was glycosylated. The predicted size of the mature unmodified PDX1 polypeptide is 31 kDa, suggesting that eukaryotic post-translational modifications are the result of the increased molecular weight. The recombinant protein was purified to greater than 95% purity using a combined ammonium sulfate

precipitation with heparin- agarose chromatography. Finally, 120 μg of the protein was obtained in high purity from 1 L of the culture supernatant. Bioactivity of the rPDXl was confirmed by the ability to penetrate cell membranes and activation of an insulin-luciferase reporter gene. These results indicate that the P. pastoris expression system can be used to produce a fully functional human rPDXl for both research and clinical application

The invention features compositions and methods that are useful for generating biologically active glycosylated recombinant human PDX1.

The present invention provides methods of treating disease and/or disorders or symptoms thereof which comprise administering a therapeutically effective amount of a pharmaceutical composition comprising a compound of the formulae herein to a subject (e.g., a mammal such as a human). Thus, one embodiment is a method of treating a subject suffering from or susceptible to diabetes type I or II, or having a disease or disorder characterized by a deficiency in β-cell number or biological activity or symptom thereof. The method includes the step of administering to the mammal a therapeutic amount of an amount of a compound herein sufficient to treat the disease or disorder or symptom thereof, under conditions such that the disease or disorder is treated.

The methods herein include administering to the subject (including a subject identified as in need of such treatment) an effective amount of a compound described herein, or a composition described herein to produce such effect. Identifying a subject in need of such treatment can be in the judgment of a subject or a health care professional and can be subjective (e.g. opinion) or objective (e.g. measurable by a test or diagnostic method). The therapeutic methods of the invention (which include prophylactic treatment) in general comprise administration of a therapeutically effective amount of the compounds herein, such as a compound of the formulae herein to a subject (e.g., animal, human) in need thereof, including a mammal, particularly a human. Such treatment will be suitably administered to subjects, particularly humans, suffering from, having, susceptible to, or at risk for a disease, disorder, or symptom thereof. Determination of those subjects "at risk" can be made by any objective or subjective determination by a diagnostic test or opinion of a subject or health care provider (e.g., genetic test, enzyme or protein marker, Marker (as defined herein), family history, and the like). The compounds herein may be also used in the treatment of any other disorders in which insulin insufficiency may be implicated.

In one embodiment, the invention provides a method of monitoring treatment progress. The method includes the step of determining a level of diagnostic marker (Marker) (e.g., any target delineated herein modulated by a compound herein, a protein or indicator thereof, etc.) or diagnostic measurement (e.g., screen, assay) in a subject suffering from or susceptible to a disorder or symptoms thereof associated with a reduction in β-cell number or biological activity (e.g., insulin production), in which the subject has been administered a therapeutic amount of a compound herein sufficient to treat the disease or symptoms thereof. The level of Marker determined in the method can be compared to known levels of Marker in either healthy normal controls or in other afflicted patients to establish the subject's disease status. In preferred embodiments, a second level of Marker in the subject is determined at a time point later than the determination of the first level, and the two levels are compared to monitor the course of disease or the efficacy of the therapy. In certain preferred embodiments, a pre-treatment level of Marker in the subject is determined prior to beginning treatment according to this invention; this pre-treatment level of Marker can then be compared to the level of Marker in the subject after the treatment commences, to determine the efficacy of the treatment.

Pancreatic Transcription Factors

The differentiation and maturation of endocrine islet cells during embryonic development is a complex process that is controlled by a unique pattern of gene regulation. Numerous pancreatic transcription factors (PTFs) are known to play important roles in specifying the different cell types found within the pancreas. Of these transcription factors, Pancreatic and Duodenal Homeobox gene- 1 (Pdx-1) has the greatest likelihood for encoding key proteins that distinguish liver and pancreas. During embryogenesis, Pdx- 1, which is expressed in all progenitor cells

differentiating toward the exocrine and endocrine pancreas (Soria Differentiation 2001 ; 68(4-5):205-219; Hui et al., Eur J Endocrinol 2002; 146(2): 129-141) plays an essential role in normal pancreas development. Indeed, no pancreatic tissue exists in Pdx-1 knockout mice. The lack of a functional Pdx- 1 protein in humans results in agenesis of the pancreas. In adults, Pdx-1 expression is restricted to β-cells and about 20% of δ-cells, where it plays a key role in insulin gene expression (Soria

Differentiation 2001; 68(4-5):205-219; Hui et al., Eur J Endocrinol 2002; 146(2): 129- 141).

Other PTFs are expressed selectively in the endocrine cells in the developing pancreas where they may play a role in endocrine cell fate decisions. These pancreatic transcription factors contain homeodomains and can be divided into early factors, including neurogenein 3 (Ngn3), Nkx2.2, and Nkx6.1, that are coexpressed in endocrine progenitor cells and later factors (Pax4, Pax6, and Isl-1) that found in more mature cells (Soria Differentiation 2001 ; 68(4-5):205-219; Wilson et al., Mech Dev 2003; 120(l):65-80). The basic helix-loop-helix (bHLH) transcription factor Ngn3 is transiently expressed in endocrine progenitor cells during pancreas development and directly regulates Beta2/NeuroD. Ngn3 controls endocrine cell fate decisions in multipotent pancreatic endodermal progenitors (Gradwohl et al., Proc Natl Acad Sci U S A 2000; 97(4): 1607-1611; Gu et al., Development 2002; 129(10):2447-2457; Schwitzgebel et al., Development 2000; 127(16):3533-3542). Beta2/NeuroD is a bHLH protein Beta2/NeuroD that is a direct downstream target gene of Ngn3.

Beta2/NeuroD is expressed in pancreatic endocrine cells and activates insulin gene transcription. Pax4 and Pax6, are two homeodomain proteins expressed both in the developing gut and in the adult pancreas, where they function in the specification of different cell types. Pax4 is a key factor in the late-stage differentiation of insulin- producing β cells and somatostatin producing δ cells (Sosa-Pineda et al., Nature 1997; 386(6623):399-402). Pax4 is transiently expressed in developing β cells and is shut down by autoregulation (Sosa-Pineda et al., Nature 1997; 386(6623):399-402). . Transfection of mouse embryonic stem cells with Pax4 leads to marked increases in IPCs compared with Pdx-1 transfected cells (Blyszczuk et al., Proc Natl Acad Sci U S A 2003; 100(3):998-1003). In contrast, Pax6 is required in the generation of glucagon producing a cells (St Onge et al., Nature 1997; 387(6631):406-409). Nkx2.2 and Nkx6.1 function in the development of pancreatic β cells and they have a similar pattern of gene expression (Sander et al., Genes Dev 1997; 11 ( 13) : 1662- 1673) . Isl-1 is required for the differentiation of islet cells (Ahlgren et al., Nature 1997;

385(6613):257-260) because no endocrine cells are present in Isl-1 knockout mice.

Because pancreatic transcription factors such as Pdxl and Ngn3 exert upstream control over the commitment of stem or progenitor cells to differentiate into pancreatic endocrine cells (Ahlgren et al., Nature 1997; 385(6613):257-260) and Pax4 exerts a second-wave of commitment of the endocrine precursor cells to islet beta cells, these pancreatic transcription factors are useful for reprogramming cells (e.g., adult cells or embryonic stem cells),. In one embodiment, an adult cell that fails to express insulin is converted into an insulin-producing cell. For example, as reported herein, pancreatic transcription factors are used to generate insulin-producing cells from liver-derived cells or liver stem cells. Transcription factors were commonly thought of as cytosolic proteins without the ability to translocate from one cell to another. More recently, evidence indicates that some transcription factors behave as paracrine signaling molecules. Such transcription factors typically include a protein transduction domain. It was recently reported that the PDX-1 protein contains an antennapedia-like protein transduction domain that can transduce pancreatic duct and islet cells (Noguchi et al., Diabetes 2003; 52(7): 1732-1737). Protein-engineering can be used to provide other transcription factors that include one or more protein transduction domains.

Protein Transduction Domains

Protein transduction domains are short peptide sequences that enable proteins to translocate across the cell and nuclear membranes, leading to entry into the cytosol by means of atypical secretory and internalization pathways (Joliot et al., Nat Cell Biol 2004; 6(3): 189-196). In 1988 Green and Loewenstein discovered that the human immunodeficiency virus type 1 (HIV-1) TAT-protein, an 86-amino acid protein, could rapidly enter cells and was even capable of entering the cell nucleus (Green and Loewenstein PM. Cell 1988; 55(6): 1179-1188). Building on this observation, a minimal unidecapeptide protein transduction domain (corresponding to residues 47- 57 of HIV-1 TAT) was developed by Dowdy and co-workers (Schwarze et al., Science 1999; 285(5433): 1569-1572). This unidecapeptide sequence was used successfully to deliver an NH2-terminal ΤΑΤ-β-galactosidase fusion protein (120 kDa) to mouse tissues via intraperitoneal injections into mice (Schwarze et al., Science 1999; 285(5433): 1569-1572). The ΤΑΤ-β-galactosidase fusion protein retained biological activity. This general method has now been successfully used for the transduction of a variety of proteins. PTD-containing peptides or proteins are taken up by cells within 5 minutes at concentrations as low as 100 nM as assessed by direct labeling with fluorescence or by indirect immunofluorescence using antibodies. This uptake is independent of endocytotic mechanisms, transmembrane protein channels, and protein receptor binding. In addition, in vitro studies have

demonstrated that protein transduction domain-mediated translocation occurs at low temperatures and exhibits no strong cellular specificity.

Recombinant Polypeptide Expression

The invention generally provides recombinant glycosylated human Pdxl polypeptides and protein-based therapies useful for treating diabetes and other diseases, disorders, or injuries associated with a deficiency in the number or biological activity of a cell of interest. In general, a protein-based therapeutic comprises a recombinant glycosylated human Pdxl polypeptide comprising a protein transduction domain, where the protein transduction domain is capable of acting as a "molecular passport" to permit entry into cells of a biologically active transcription factor. The transcription factor acts to reprogram the cell. The reprogrammed cell has an altered transcriptional and/or translational profile, i.e., expresses an altered set of mRNAs and/or polypeptides expressed relative to an untreated control cell.

The present invention describes methods for producing recombinant Pdxl polypeptides in Pichia pastoris, a methylotrophic yeast. Pichia is capable of metabolizing methanol as the sole carbon source. The first step in the metabolism of methanol is the oxidation of methanol to formaldehyde by the enzyme, alcohol oxidase. Expression of this enzyme, which is coded for by the AOXl gene is induced by methanol. The AOXl promoter can be used for inducible polypeptide expression or the GAP promoter for constitutive expression of a gene of interest.

Once the recombinant polypeptide of the invention is expressed, it is isolated, as described herein or using any method known in the art (e.g., affinity

chromatography). In one example, an antibody (e.g., produced as described herein) raised against a polypeptide of the invention may be attached to a column and used to isolate the recombinant polypeptide. Lysis and fractionation of polypeptide-harboring cells prior to affinity chromatography may be performed by standard methods (see, e.g., Ausubel et al., supra). Alternatively, the polypeptide is isolated using a sequence tag, such as a hexahistidine tag, that binds to nickel column.

Once isolated, the recombinant protein can, if desired, be further purified, e.g., by high performance liquid chromatography (see, e.g., Fisher, Laboratory Techniques In Biochemistry and Molecular Biology, eds., Work and Burdon, Elsevier, 1980). Polypeptides of the invention, particularly short peptide fragments, can also be produced by chemical synthesis (e.g., by the methods described in Solid Phase Peptide Synthesis, 2nd ed., 1984 The Pierce Chemical Co., Rockford, 111.). These general techniques of polypeptide expression and purification can also be used to produce and isolate useful peptide fragments or analogs (described herein).

Pdx-1 Polypeptides and Analogs

Also included in the invention are recombinant glycosylated human Pdxl polypeptides and fragments thereof that are modified in ways that enhance their ability to reprogram a cell or their ability to induce regeneration. For example, variations in the sequence increase protein solubility or yield. The invention provides a modified recombinant glycosylated human Pdxl polypeptide having an enhanced ability to reprogram a liver-derived cell to an insulin-producing cell. In other examples, the rPDXl protein increases the regenerative capacity of a pancreatic cell. Alternatively, the alteration is in the protein transduction domain, and the altered domain increases transport of an operably linked protein into a cell or cellular compartment, such as the nucleus. In other embodiments, the alteration in the protein transduction domain reduces interference with a biological activity of an operably linked polypeptide.

The invention provides methods for optimizing a transcription factor or protein transduction domain amino acid sequence or nucleic acid sequence by producing an alteration in the sequence. Such alterations may include certain mutations, deletions, insertions, or post-translational modifications. The invention further includes analogs of any naturally-occurring polypeptide of the invention. Analogs can differ from a naturally-occurring polypeptide of the invention by amino acid sequence differences, by post-translational modifications, or by both. Analogs of the invention will generally exhibit at least 85%, more preferably 90%, and most preferably 95% or even 99% identity with all or part of a naturally-occurring amino, acid sequence of the invention. The length of sequence comparison is at least 5, 10, 15 or 20 amino acid residues, preferably at least 25, 50, or 75 amino acid residues, and more preferably more than 100 amino acid residues. Again, in an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e^"3 and e^"100 indicating a closely related sequence.

Modifications include in vivo and in vitro chemical derivatization of polypeptides, e.g., acetylation, carboxylation, phosphorylation, or glycosylation; such modifications may occur during polypeptide synthesis or processing or following treatment with isolated modifying enzymes. Analogs can also differ from the naturally-occurring polypeptides of the invention by alterations in primary sequence. These include genetic variants, both natural and induced (for example, resulting from random mutagenesis by irradiation or exposure to ethanemethylsulfate or by site-specific mutagenesis as described in Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual (2d ed.), CSH Press, 1989, or Ausubel et al., supra). Also included are cyclized peptides, molecules, and analogs which contain residues other than L-amino acids, e.g., D-amino acids or non- naturally occurring or synthetic amino acids, e.g., .beta, or .gamma, amino acids.

In addition to full-length polypeptides, the invention also provides fragments of any one of the polypeptides or peptide domains of the invention. As used herein, the term "a fragment" means at least 5, 10, 13, or 15 amino acids. In other embodiments a fragment is at least 20 contiguous amino acids, at least 30 contiguous amino acids, or at least 50 contiguous amino acids, and in other embodiments at least 60 to 80, 100, 200, 300 or more contiguous amino acids. Fragments of the invention can be generated by methods known to those skilled in the art or may result from normal protein processing (e.g., removal of amino acids from the nascent polypeptide that are not required for biological activity or removal of amino acids by alternative mRNA splicing or alternative protein processing events).

Assays for measuring functional activity include, but are not limited to, those described in the Examples below.

Therapeutic Methods

The invention provides for the treatment of diseases and disorders associated with a deficiency in cell number (e.g., a reduction in the number of pancreatic cells) or an insufficient level of cell biological activity (e.g., a deficiency in insulin production). For example, the invention provides compositions for the treatment of diabetic patients who lack sufficient levels of insulin due to a decrease in the number or activity of insulin producing pancreatic cells. In one embodiment, a rPDXl polypeptide is administered to a cell, tissue, or organ in situ to ameliorate a deficiency in β-cell number. Alternatively, the polypeptide is administered to cells in vitro and then the cells containing the rPDXl polypeptide (or nucleic acid molecules encoding them) are administered to the patient to ameliorate the disease, disorder, or injury. The polypeptide is delivered to those cells in a form in which it can be taken up by the cells, such that sufficient levels of protein are transduced to ameliorate a disease or disorder. In one embodiment, a therapeutic rPDXl polypeptide is delivered locally to a site where an increase in regeneration or where cellular reprogramming is desired. Administration may be my any means sufficient to result in a sufficient level of cellular transduction. While the particular level of transduction will vary depending on the therapeutic objective to be achieved, desirably at least 2, 5, 10, or 15% of the cell of a tissue are transduced. In other embodiments, at least 25%, 35%, or 50% of cells are transduced. In still other embodiments, at least 75%, 85%, 95% or more of cells are transduced. Preferably, levels of a polypeptide are altered by at least about 5%, 10%, 25%, 50%, 75% or more.

In various embodiments, rPDXl polypeptides are administered by local injection to a site of disease or injury, by sustained infusion, or by micro-injection under surgical conditions (Wolff et al., Science 247: 1465, 1990). In other embodiments, the rPDXl polypeptides are administered systemically to a tissue or organ of a patient having a deficiency in cell number that can be ameliorated by cell regeneration or reprogramming.

In another approach cellular transduction into the affected tissue of a patient is accomplished by transferring a rPDXl polypeptide of the invention into a cultivatable cell type ex vivo (e.g., an autologous or heterologous primary cell or progeny thereof), after which the cell (or its descendants) are injected into a targeted tissue at the site of disease or injury. In some embodiments, the cells are present in a cellular matrix that provides for their survival, proliferation, or biological activity. Another therapeutic approach included in the invention involves administration of a recombinant glycosylated human Pdxl polypeptide, biologically active fragment, or variant thereof. The present invention provides methods of treating disease and/or disorders or symptoms thereof which comprise administering a therapeutically effective amount of a pharmaceutical composition comprising a compound of the formulae herein to a subject (e.g., a mammal such as a human). Thus, one embodiment is a method of treating a subject suffering from or susceptible to a disease or disorder or symptom thereof characterized by a deficiency in cell number. The method includes the step of administering to the mammal a therapeutic amount of an amount of a composition of the invention sufficient to treat the disease or disorder or symptom thereof, under conditions such that the disease or disorder is treated.

In other embodiments, therapeutic polypeptides of the invention are produced in a cell transduced with a viral (e.g., retroviral, adenoviral, and adeno-associated viral) vector that is used for somatic cell gene therapy, especially because of their high efficiency of infection and stable integration and expression (see, e.g., Cayouette et al., Human Gene Therapy 8:423-430, 1997; Kido et al., Current Eye Research 15:833-844, 1996; Bloomer et al., Journal of Virology 71:6641-6649, 1997; Naldini et al., Science 272:263-267, 1996; and Miyoshi et al., Proc. Natl. Acad. Sci. U.S.A. 94: 10319, 1997). For example, a nucleic acid molecule, or a portion thereof, that encodes a therapeutic protein of the invention can be cloned into a retroviral vector and expression can be driven from its endogenous promoter, from the retroviral long terminal repeat, or from a promoter specific for a target cell type of interest (e.g., a cell of the central nervous system). Other viral vectors that can be used include, for example, a vaccinia virus, a bovine papilloma virus, or a herpes virus, such as Epstein-Barr Virus (also see, for example, the vectors of Miller, Human Gene Therapy 15-14, 1990; Friedman, Science 244: 1275-1281, 1989; Eglitis et al., BioTechniques 6:608-614, 1988; Tolstoshev et al., Current Opinion in Biotechnology 1:55-61, 1990; Sharp, The Lancet 337: 1277-1278, 1991; Cornetta et al., Nucleic Acid Research and Molecular Biology 36:311-322, 1987; Anderson, Science 226:401-409, 1984; Moen, Blood Cells 17:407-416, 1991; Miller et al., Biotechnology 7:980-990, 1989; Le Gal La Salle et al., Science 259:988-990, 1993; and Johnson, Chest

107:77S-83S, 1995). Retroviral vectors are particularly well developed and have been used in clinical settings (Rosenberg et al., N. Engl. J. Med 323:370, 1990;

Anderson et al., U.S. Pat. No. 5,399,346). Most preferably, a viral vector is used to administer the gene of interest systemically or to a cell at the site that requires cell reprogramming or an increase in regeneration. Formulation of Pharmaceutical Compositions

The administration of a composition of the invention for the treatment of a disease, disorder, or injury characterized by a deficiency in β cell number or biological activity may be by any suitable means that results in a concentration of the therapeutic that, combined with other components, is effective in ameliorating, reducing, or stabilizing the disease. For example, an amount that reduces or normalizes blood glucose levels in a subject. A therapeutic rPDXl polypeptide, polynucleotide, or cell comprising such agents may be contained in any appropriate amount in any suitable carrier substance, and is generally present in an amount of 1- 95% by weight of the total weight of the composition. The composition may be provided in a dosage form that is suitable for parenteral (e.g., subcutaneously, intravenously, intramuscularly, or intraperitoneally) administration route. The pharmaceutical compositions may be formulated according to conventional pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy (20th ed.), ed. A. R. Gennaro, Lippincott Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York). Desirably, the polypeptide may be modified or formulated to enhance polypeptide half-life, increase absorption, or provide for sustained release.

Pharmaceutical compositions according to the invention may be formulated to release the active compound substantially immediately upon administration or at any predetermined time or time period after administration. The latter types of compositions are generally known as controlled release formulations, which include (i) formulations that create a substantially constant concentration of the drug within the body over an extended period of time; (ii) formulations that after a predetermined lag time create a substantially constant concentration of the drug within the body over an extended period of time; (iii) formulations that sustain action during a

predetermined time period by maintaining a relatively, constant, effective level in the body with concomitant minimization of undesirable side effects associated with fluctuations in the plasma level of the active substance (sawtooth kinetic pattern); (iv) formulations that localize action by, e.g., spatial placement of a controlled release composition adjacent to or in the peritoneal cavity or at another site where distribution of the composition is desired; (v) formulations that allow for convenient dosing, such that doses are administered, for example, once every one to two days, or once every one to two weeks; and (vi) formulations that target an disease, disorder, or injury by using carriers or chemical derivatives to deliver the therapeutic agent to a particular cell type (e.g., liver cell or pancreatic cell) whose function is perturbed in a subject having the disease, disorder, or injury. For some applications, controlled release formulations obviate the need for frequent dosing during the day to sustain the plasma level at a therapeutic level.

Any of a number of strategies can be pursued in order to obtain controlled release in which the rate of release outweighs the rate of metabolism of the compound in question. In one example, controlled release is obtained by appropriate selection of various formulation parameters and ingredients, including, e.g., various types of controlled release compositions and coatings. Thus, the therapeutic is formulated with appropriate excipients into a pharmaceutical composition that, upon

administration, releases the therapeutic in a controlled manner. Examples include single or multiple unit tablet or capsule compositions, oil solutions, suspensions, emulsions, microcapsules, microspheres, molecular complexes, nanoparticles, patches, and liposomes.

If desired, therapeutic compositions of the invention are provided together with other agents that enhance the regeneration of a cell of interest or that enhance the reprogramming of a cell of interest. In one embodiment, the agents are growth factors, such as soluble growth factors. For example, a therapeutic rPDXl polypeptide is provided together with a soluble growth factors, such as PDGF,EGF, VEGF, bFGF, HGF, NGF, KGF) or is provided together with a beta cell promoting factor, such as nicotinamide, exentin 4, GLP-1, betacellulin, Islet neogenesis associated protein (INGAP), or Ghrelin.

Methods of Delivery

The pharmaceutical composition may be administered by injection, infusion or implantation (subcutaneous, intravenous, intramuscular, intraperitoneal, or the like) in dosage forms, formulations, or via suitable delivery devices or implants containing conventional, non-toxic pharmaceutically acceptable carriers and adjuvants. In one embodiment, a therapeutic composition of the invention is provided via an osmotic pump. The formulation and preparation of such compositions are well known to those skilled in the art of pharmaceutical formulation. Formulations can be found in Remington: The Science and Practice of Pharmacy, supra.

Compositions for parenteral use may be provided in unit dosage forms (e.g., in single-dose ampoules), or in vials containing several doses and in which a suitable preservative may be added (see below). The composition may be in the form of a solution, a suspension, an emulsion, an infusion device, or a delivery device for implantation or it may be presented as a dry powder to be reconstituted with water or another suitable vehicle before use. Apart from the active polypeptide therapeutic(s), the composition may include suitable parenterally acceptable carriers and/or excipients. The active polypeptide therapeutic (s) may be incorporated into an osmotic pump, microspheres, microcapsules, nanoparticles, liposomes, or the like for controlled release. Furthermore, the composition may include suspending, solubilizing, stabilizing, pH-adjusting agents, tonicity adjusting agents, and/or dispersing, agents.

As indicated above, the pharmaceutical compositions according to the invention may be in the form suitable for sterile injection. To prepare such a composition, the suitable active recombinant glycosylated human Pdxl polypeptide therapeutic(s) are dissolved or suspended in a parenterally acceptable liquid vehicle. Among acceptable vehicles and solvents that may be employed are water, water adjusted to a suitable pH by addition of an appropriate amount of hydrochloric acid, sodium hydroxide or a suitable buffer, 1,3-butanediol, Ringer's solution, and isotonic sodium chloride solution and dextrose solution. The aqueous formulation may also contain one or more preservatives (e.g., methyl, ethyl or n-propyl p- hydroxybenzoate). In cases where one of the compounds is only sparingly or slightly soluble in water, a dissolution enhancing or solubilizing agent can be added, or the solvent may include 10-60% w/w of propylene glycol or the like.

In one embodiment, a therapeutic composition of the invention (e.g., recombinant glycosylated human Pdxl polypeptide) is provided locally via a canula. For example, for reprogramming a liver-derived cell to insulin producing cell a composition of the invention is provided to the liver via the portal vein. More preferably, the composition is directed specifically to a single lobe of the liver by providing the composition (e.g., via a canula) to only one of the three branches of the portal vein, such that only one lobe of the liver comprises insulin producing cells. In other embodiments, a composition of the invention is provided via an osmotic pump. Desirably, the osmotic pump provides for the controlled release of the composition over 1-3 days, 3-5 days, 5-7 days, or for 2, 3, 4, or 5 weeks.

Combination Therapies

Compositions of the invention comprising a recombinant glycosylated human Pdxl polypeptides and may, if desired, be delivered in combination with any other polypeptide or polynucleotide therapeutic of the invention or with any conventional therapeutic known in the art. In one embodiment, a recombinant glycosylated human Pdxl polypeptide of the invention is used to reduce hyperglycemia in a diabetic subject. This therapeutic effect is desirable even if the therapeutic method does not entirely eliminate the patient's dependence on insulin. Accordingly, recombinant glycosylated human Pdxl polypeptides of the invention may be administered together with insulin to alleviate hyperglycemia or a symptom or complication thereof.

Desirably, a therapeutic rPDXl polypeptide of the invention reduces a patient's dependence on insulin by at least about 5, 10, or 15%, more desirably by at least about 20%, 25%, or even by 30%, or even more desirably by 50%, 75%, 85% or more. In other embodiments, the polypeptide therapeutic is combined with a polynucleotide of the invention (e.g., a polynucleotide encoding a pancreatic transcription factor). In other embodiments, compositions of the invention are used in combination with diet, weight loss, or oral, injectable, nasal or other insulin therapies to reduce and/or normalize blood glucose levels. Combinations of the invention may be formulated together and administered simultaneously or may be administered within twenty-four hours, within 2, 3, or 5 days, or within 1, 2, 3 or 5 weeks of each other.

Kits or Pharmaceutical Systems

The present compositions may be assembled into kits or pharmaceutical systems for use in ameliorating hyperglycemia. Kits or pharmaceutical systems according to this aspect of the invention comprise a recombinant glycosylated human Pdxl polypeptides and a carrier means, such as a box, carton, tube or the like, having in close confinement therein one or more container means, such as vials, tubes, ampoules, bottles and the like. The kits or pharmaceutical systems of the invention may also comprise associated instructions for using the agents of the invention. The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, "Molecular Cloning: A Laboratory Manual", second edition (Sambrook, 1989); "Oligonucleotide Synthesis" (Gait, 1984); "Animal Cell Culture" (Freshney, 1987); "Methods in Enzymology" "Handbook of Experimental Immunology" (Weir, 1996); "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987); "Current Protocols in Molecular Biology" (Ausubel, 1987); "PCR: The Polymerase Chain Reaction", (Mullis, 1994); "Current Protocols in Immunology" (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments will be discussed in the sections that follow.

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and therapeutic methods of the invention, and are not intended to limit the scope of what the inventors regard as their invention.

EXAMPLES

Example 1: Optimization of rPDXl expression conditions and strains

The resulting plasmid from the construction, pPICZa-rPDXI, was transformed into P. pastoris of the 15 single colonies that grew on YPD containing 500 μg eocin, eight clones were selected for testing the capacity of rPDXl protein expression by Western blot analysis (Fig. 1). All selected clones expressed rPDXl with a molecular weight of 50 kDa, which is slightly higher in yeast compared with native PDXI expressed in the rat insulinoma 13-cell line INS-1 (46 kDa) (Fig. 1, lane C), suggesting that the protein expressed in yeast might be glycosylated. There was no protein that reacted with anti-PDXI antibody in untransformed P. pastoris. Fig. 1 shows that the S I clone has the highest expression of the rPDXl and therefore was used for the subsequent time-course study. To determine the optimal time for rPDXl expression, the S I clone was seeded in the culture medium and samples were collected at 12, 24, 48. 72, and 96 hours. Fig. 2A shows that the intensity of rPDXl protein increased over time, with the strongest signal intensity at 96 hours. Therefore, a 96-hour incubation time was identified as optimal (Fig. 2A). To determine the secretory ability of the rPDXl in the yeast expression system, we examined the protein levels both in the culture medium and in the yeast cells by Western blot. Fig. 2B reveals that the majority of the rPDXl protein was cell- associated whereas only 10% of rPDXl was secreted into the culture medium.

Example 2: Large-scale fermentation and purification of rPDXl

To obtain the optimal fermentation conditions to scale-up protein production, the SI clone was used after 96 hours incubation in the presence of 1% methanol. The rPDXl protein in the supernatant was purified by a two-step method. First, rPDXl was precipitated with 60% saturated ammonium sulfate. The pellet containing rPDXl was dissolved in column buffer and subjected to heparin-gel affinity chromatography. The rPDXl was eluted from the column with elution buffer containing 1.5 M KCl. The purity of the rPDXl was assessed by SDS-PAGE (Fig. 4A). An average of about 120 pg of purified rPDXl was recovered from 1 L of culture medium. The purity of the purified rPDXl reached nearly 95% as confirmed by SDS-gel staining and Western blotting with anti-PDXl antibodies (Fig. 4B, ) lanes 2 and 3.

Example 3: Glycosylation analysis of purified rPDXl

To examine whether the rPDXl harvested from the yeast expression system underwent post-translational glycosylation, the purified rPDXl from supernatant was treated with PNGase F to remove N-linked glycosylation and subjected to SDS-PAGE and Western blotting analysis. As shown in Fig. 3, the pretreated rPDXl has a predicted molecular weight of approximately 50 kDa, whereas the treated rPDXl exhibited two distinct bands, one large band at 46 kDa and one faint band at 31 kDa (Fig. 3). The shift in the band post-PNGase F treatment indicates that the rPDXl was indeed glycosylated in the yeast expression system. P NGase F is known to strictly cut N-linked glycans. As previously mentioned, protein expressed in yeast is glycosylated by both O-or N-glycosidic linkages. The presence of two bands is likely due to the ability of PNGase F to remove only the N-form of glycosylation from the rPDXl protein. It is also possible that the presence of two bands indicates that the PNGase F may have digested rPDXl only partially. Regardless, it is clear that rPDXl was secreted from the P. pastoris and was glycosylated

post-translationally.

Example 4: Biological activity of rPDXl

There are several direct target genes for PDXI, including the insulin gene. To determine the biologic function of rPDXl, we transfected a rat insulin II promoter-luciferase reporter gene into human Huh-7 cells prior to treatment with different concentrations of purified rPDXl. Luciferase activity was detected after 24 hours of incubation with rPDXl. Fig. 5 shows that the cells treated with rPDXl showed an increase in expression of the insulin repor- ter gene (Fig. 5). A previous report indicates that rPDXl protein produced in E. coli can also activate thepsulin- luciferase reporter gene, but at much lower levels when compared to our rPDXl protein produced in yeast.

These data show that P. pastoris yeast can successfully be used as an expression system for generating biologically functional rPDXl protein, serving as an alternative to prokaryotic expression systems. As demonstrate herein the P. pastoris yeast expression system has the ability to secrete glycosylated and biologically active rPDXl into the culture medium. Furthermore, we developed a purification strategy and protocol for rPDXl purification from the culture medium of P. pastoris yeast cells. P. pastoris cells expressed a large quantity of rPDXl.

It may be possible to improve the expression level further by varying the nature of the signal peptide, deleting the nuclear localization signal (NLS), chaperone co-expression or varying expression conditions, which have proven effective in other cases.

In addition to its roles in embryonic development of the pancreas and the maintenance of β-cells in the adult pancreas, PDXI over-expression induces the adoption of an insulin-producing cell phenotype in hepatocytes. Its ability to direct β- cell differentiation and transdifferentiation to the β-cell phenotype gives it the potential for use in β-cell replacement therapy for Type 1 diabetes. Inducing the over- expression of PDXI and other pancreatic transcription factors to study their effects typically requires the use of viral (e.g. lentiviral) vectors for deliver into cells and incorporation into the genome. This approach inevitably entails some risk of insertional mutagenesis and raises issues for potential clinical application. PDX1 PTD, therefore, makes it especially attractive for use as a differentiation reagent for generating patient- specific pluripotent stem cells to differentiate into pancreatic p- cells because it can translocate directly into cells without requiring the use of viral vectors.

Protein therapy using PDX1 protein for diabetes provides hope for enhanced safety. Accumulating evidence shows that reactivating the developmental program for reprogramming non-pancreatic β-cells or for differentiating pancreatic stem/progenitor cells into insulin- secreting β-like cells has great clinical potential in diabetes. The ability to obtain biologically functional, near-native PDX1 protein in good yields is important for clinical application and for understanding the reprogramming process in Pancreatic β-cells.

The results described above were carried out with the following methods and materials.

Expression plasmid construction

Human PDXI cDNA (283 amino acids) was amplified by PCR on a plasmid encoding full-length human PDXI purchased from Origene technologies (USA). The sequence of the amplified gene was analyzed and confirmed by an ABI 3130xL Genetic Analyzer. In order to express the native N-terminus of PDXI, an Xhol site was introduced to allow in-frame cloning behind the a-mating factor pre-secretion signal of pPICZaA and a nucleotide sequence encoding the KeX2 gene cleavage site was placed upstream of the PDXI gene. The PCR products were cut with Xhol and Xbal and ligated into this site downstream of the alcohol oxidase 1 promoter (AOX1) in pPICZocA LInvitrogen, USA).

Transformation of P. pastoris and selection of transformants

Pichia pastoris X-33 was transformed with a linearized expression vector by digestion with Sacl. The transformation was performed using the lithium chloride method following the kit manual (Pichia Easycomp Transformation Kit, Invitrogen, USA). The transformant cells were plated on YPDS (1% yeast extract, 2% iQeptone, 2% dextrose, and 1 M o-sorbitol) plates containing 100 pg/ml of Zeocin. Approximately thirty Zeocin-resistant colonies were replated on a YPD (1% xeast extract, 2% peptone, and 2% dextrose) plate containing 500 pg/ml of Zeocin. After incubation at 30 C for 2-3 days, several colonies appeared on the YPD plate and eight of the larger colonies were selected for protein expression.

Expression and purification of human PDX1

Eight transformants were screened for expression of secreted rPDXl by

Western blot analysis of culture medium, and the Si clone was chosen for large scale expression and purification of rPDXl. SI cells were grown in 500 ml of BMGY (100 mM potassium phosphate, RH 6.0, 1.34% YNB, 4 x 10⁵ biotin, and 1% glycerol) for 48 hours at 28 C. The cells were harvested by centrifugation (1000 rpm, 25 min at 4 C) and resuspended in 1.0 L of BMMY (100 mM potassium phosphate, RH 6.0, 1.34% YN B, 4xl0^"5 biotin, and 0.5% methanol) in a 2 L flask for induction of AOXI. The yeast cells were grown in BMMY media for 96 hours at 28 °C with shaking. Every 24 hours methanol (100%) was added to a final concentration of 0.5% to maintain induction. Cell culture supernatant was harvested by centrifugation and was precipitated with 60% saturated ammonium sulfate. The precipitated proteins were collected and dissolved in buffer A (25 mM Hepes, pH 7.9, 20% glycerol, 0.1 M KCI, 0.2 mM EDTA, and 0.5 mM DTT), and dialyzed against the same buffer. The resulting sample was loaded onto a heparin-agarose column (Bio-Rad). The column was first washed with 0.2 M KCl in buffer A, and proteins were eluted with 0.1 M KCl in buffer A. T he elution fractions were analyzed using SDS-PAGE followed by Coomassie brilliant blue staining and Western blotting. The purified PDX1 was dialyzed against 10% glycerol in phosphate-buffered solution (PBS) and stored at - 80 °C.

N-glycanase digestion and Western blotting analysis

The rPDXl expression sample was digested for 5 hours at 37 °C with recombinant N- glycanase (PNGase F, New England BioLabs) according the manufacturer's instructions. The digested and undigested controls were subjected to SDS-PAGE on a 12% gel and transferred to a polyvinylidene fluoride (PVDF) membrane. The blot was probed with rabbit anti-PDXI polyclonal antibodies and developed using the Amersham Pharmacia Biotech ECL detection system.

Insulin-reporter luciferase assay

The rat insulin II promoter-luciferase reporter construct (pGL2- RIP2-Luc) was a gift from Dr. Carlotti ( Smith et al., Biotechnol. Bioeng. 79 (2002) 713-723.). Human hepatocellular carcinoma Huh-7 cells were seeded onto a 12- well plate and grown for 24 hour in Dulbecco's modified Eagle's medium (DMEM) containing 10% FBS at 5% CO₂ and 37 °C. One microgram of pGL2- RIP2-Luc reporter plasmid and 0.01 pg of internal control vector HSV-TK-hRluc luciferase, pRL-TK, Promega) were used to co-transfect these cells using Lipofectamine 2000 reagent (Invitrogen, USA). Following 12 hours transfection, the medium was replaced with medium containing rPDXl protein or PBS with 10% glycerol (protein storage solution) for negative controls. After 24 hours protein treatment, cell lysates were collected and assayed for luciferase activities using Dual-Glo-Assay-S

Other Embodiments

From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.

The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.

Claims

What is claimed is:

1. A recombinant human Pd l polypeptide having a molecular weight of about 50 kDA, comprising N- and O-linked glycosylation post-translational modifications, wherein the protein has the ability to penetrate cell membranes and activate insulin expression.

2. A method for producing a glycosylated human Pdxl polypeptide, the method comprising contacting a Pichia pastoris cell comprising an expression vector encoding rPDXl with culture media comprising 100 mM potassium phosphate, pH 6.0, 1.34% YNB, 4 x 10⁵ biotin, and 1% glycerol for 48 hours at 28 C;

resuspending the cells in said culture media further comprising 0.5% methanol, and culturing the cells for 96 hours at 28 °C; and

adding 100% methanol every 24 hours to the culture to maintain the culture at 0.5% methanol throughout the culture period, thereby producing glycosylated human Pdxl polypeptide.

3. The method of claim 2, further comprising purifying the Pdxl polypeptide, the purification method comprising precipating proteins from the cell culture supernatant by with 60% saturated ammonium sulfate, collecting the precipitated proteins, dissolving the proteins in a buffer comprising 25 mM Hepes, pH 7.9, 20% glycerol, 0.1 M KCI, 0.2 mM EDTA, and 0.5 mM DTT, purifying the proteins over a heparin-agarose column, and eluting the proteins with 0.1 M KCl in buffer A.

4. A recombinant human Pdxl polypeptide produced by the method of claim 2 and/or 3.

5. A Pichia pastoris cell comprising an expression vector encoding human rPDXl.

6. A method for reprogramming a cell, the method comprising:

(a) contacting a cell with a recombinant human Pdxl polypeptide of claim 1 or produced according to the method of claim 4; and (b) altering the expression level of at least one pancreatic transcription factor polypeptide in the cell, thereby reprogramming the cell.

7. The method of claim 6, wherein the cell is selected from the group consisting of adipocytes, bone marrow derived cells, epidermal cells, endothelial cells, fibroblasts, hematopoietic cells, hepatocytes, myocytes, neurons, pancreatic cells, and their progenitor cells or stem cells.

8. The method of claim 6, wherein the cell is contacted in vitro or in vivo.

9. The method of claim 6, wherein the alteration is an increase in the level of a polypeptide that is not detectably expressed in a corresponding control cell.

10. The method of claim 9, wherein the reprogrammed cell expresses insulin.

11. A method for generating an insulin producing cell, the method comprising:

(a) contacting a cell with a recombinant human Pdxl polypeptide of claim 1 or produced according to the method of claim 4; and

(b) increasing the expression of insulin in the cell, thereby generating an insulin producing cell.

12. The method of claim 11, wherein the cell is selected from the group consisting of adipocytes, bone marrow derived cells, epidermal cells, endothelial cells, fibroblasts, hematopoietic cells, hepatocytes, myocytes, neurons, pancreatic cells, and their progenitor cells or stem cells.

13. The method of claim 11, wherein the cell is a hepatocyte or liver- derived stem cell.

14. The method of claim 11, wherein the cell of step (a) fails to express a detectable level of insulin prior to contact with the recombinant human Pdxl polypeptide.

15. The method of claim 11, wherein the method further comprises contacting the cell with a transcription factor is selected from the group consisting of Ngn3, Pax4, NeuroDl, Nkx2.2, Nkx6.1, Isll, Pax6, and MafA.

16. A method of ameliorating hyperglycemia in a subject in need thereof, the method comprising:

(a) contacting a cell of the subject with a recombinant human Pd l polypeptide of claim 1 or produced according to the method of claim 4; and

(b) increasing the expression of insulin in the cell, thereby generating an insulin producing cell.

17. The method of claim 16, wherein the cell is selected from the group consisting of adipocytes, bone marrow derived cells, epidermal cells, endothelial cells, fibroblasts, hematopoietic cells, adipocytes, epidermal cells, endothelial cells, fibroblasts, hematopoietic cells, hepatocytes, myocytes, neurons, pancreatic cells, and their progenitor cells or stem cells.

18. The method of claim 16, wherein the cell is a hepatocyte or liver- derived stem cell.

19. The method of claim 16, wherein the subject has type 1 or type 2 diabetes.

20. The method of claim 16, wherein the cell of step (a) fails to express a detectable level of insulin prior to contact with the fusion protein.

21. The method of claim 16, wherein the method reduces blood glucose level in the subject.

22. The method of claim 16, wherein the method normalizes blood glucose level in the subject.

23. A pharmaceutical composition comprising a recombinant human Pd l polypeptide of claim 1 or produced according to the method of claim 4 in a pharmaceutically acceptable excipient.

24. A kit for treating hyperglycemia, the kit comprising an effective amount of a recombinant human Pdxl polypeptide of claim 1 or produced according to the method of claim 4 and instructions for use thereof.