# Patent

## Title
Protein delivery system to generate induced pluripotent stem (iPS) cells or tissue-specific cells

## Abstract

A novel protein delivery system to generate induced pluripotent stem (iPS) cells is described. The delivery system comprises a construct with a receptor binding domain that recognizes a receptor in a somatic cell, a translocation domain that allows the transfer of an inducer into the cytosolic space, and a cargo bearing domain to which the inducer is attached and facilitates transfer of the inducer into the cell.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 61/275,297, entitled “A NOVEL PROTEIN DELIVERY SYSTEM TO GENERATE INDUCED PLURIPOTENT STEM (iPS) CELLS OR TISSUE-SPECIFIC CELLS” and filed Aug. 27, 2009. This application also claims the benefit under 35 U.S.C. §120 of U.S. application Ser. No. 12/870,782, entitled PROTEIN DELIVERY SYSTEM TO GENERATE INDUCED PLURIPOTENT STEM (iPS) CELLS OR TISSUE-SPECIFIC CELLS and filed on Aug. 27, 2010, which is now U.S. Pat. No. 8,420,352, issued Apr. 16, 2013. Both applications listed above are incorporated herein by reference in their entireties.


FIELD OF THE INVENTION
This invention relates a protein delivery system for therapeutic purposes; more particularly, the protein delivery system can be utilized to deliver reprogramming factors for causing the differentiation of somatic cells into pluripotent stem (iPS) cells or tissue specific cells for regenerative medicine or disease treatment.
BACKGROUND OF THE INVENTION
In the past, pluripotent stem cells have been generated by means of nuclear transplant and cell fusion (Shinya Yamanaka, Pluripotency and Nuclear Reprogramming, Philos Trans R Soc Lond B Biol Sci. 363(1500): 2079-2087 (Jun. 27, 2008)). Both methods require embryonic stem cells, which pose ethical dilemmas for both research and therapeutic use. This issue is overcome by the recently discovered induced pluripotent stem (iPS) cells, which share the same attractive biological properties of embryonic stem (ES) cells (Yamanaka, A Fresh Look at iPS Cells. Cell 137:13-17 (S. 2009)).
Induced pluripotent stem cells were first produced from mouse fibroblasts in 2006 (Takahashi, Y. and S. Yamanaka, Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors, Cell 126: 663-676 (2006)) and human fibroblasts in 2007 (Yu Junying, et al., Induced Pluripotent Stem Cell Lines Derived from Human Somatic Cells, Science 318: 1917-1920 (2007), Takahashi, K., et al. Induction of Pluripotent Stem Cells From Adult Human Fibroblasts by Defined Factors, Cell 131: 861-872 (2007)) by forcing the expression of defined factors. Two sets of factors have been used to trigger the reprogramming of adult somatic cells to iPS cells in these studies: one includes Oct-3/4, Sox2, Klf4, and c-Myc (Takahashi, Cell 126: 663-676; Takahashi, Cell 131:861-872); and the other includes Oct4, Sox2, Nanog, and Lin28 (Junying, Cell 318:1917-1920). The reprogramming efficiency of these defined factors can be increased 10-fold by knocking down p53 activity.
In 2008, Melton's group demonstrated that by delivering a specific combination of three transcription factors, Ngn3 (also known as Neurog3), Pdxl, and Mafa, the differentiated pancreatic exocrine cells in adult mice were re-programmed into cells that closely resemble β-cells (Zhou, Q. et al., In Vivo Reprogramming of Adult Pancreatic Exocrine Cells to β-cells, Nature 455: 627-633 (2008)). The induced β-cells are morphologically indistinguishable from endogenous islet β-cells; they express genes essential for β-cell function and can ameliorate hyperglycemia by remodeling local vasculature and secreting insulin. This study suggests that adult somatic cells can be re-programmed to tissue specific cells directly by a specific combination of transcription factors without reversion to a pluripotent stem cell state.
The potential of iPS cells is enormous. However, the clinical application of iPS cells faces many obstacles (Yamanaka, Cell 137: 13-17; Miura, K. et al., Variation in the Safety of Induced Pluripotent Stem Cell Lines, Nature Biotechnology 27(8):743-745 (2009); Carpenter, M. et al., Developing Safe Therapies From Human Pluripotent Stem Cells, Nature Biotechnology 27: 606-613 (2009)). One major hurdle is directly related to the delivery vehicle for the reprogramming factors. Initially, the re-programming factor genes were introduced into somatic fibroblast by retro or lentiviral vectors (Takahashi, Cell 126: 663-676; Junying, Cell 318:1917-1920; Takahashi, Cell 131:861-8723-5). The process of re-programming through retro or lentiviral vectors had an efficiency of only  ̃0.05% (Okita, K. et al., Generation of Mouse Induced Pluripotent Stem Cells Without Viral Vectors, Science 322: 949-953; Yamanaka, S. Elite and Stochastic Models for Induced Pluripotent Stem Cell Generation, Nature 460: 49-52 (2009)).
Insertional activation of an oncogene is always a great concern of using retro or lentiviral vectors since these vectors randomly integrate into the host's genome. Although transgenes are largely silenced in iPS cells, reactivation of c-myc transgene could lead to tumorigenesis (Okita, K., et al., Generation of Germline-Competent Induced Pluripotent Stem Cells, Nature 448: 313-318 (2007)). Leaky expression of these transgenes may also inhibit complete iPS cell differentiation and maturation, leading to a greater risk of teratoma formation (Yamanaka, Cell 137:13-17). In addition, the transgenes could be re-activated and expressed in cells that are re-differentiated from the iPS cells, leading to a risk of re-programming differentiation status of iPS cells or tumor formation.
A non-integrating viral vector such as an adenoviral vector was later used to deliver these re-programming factor genes (Zhou, Nature 455: 627-633; Stadtfeld, M. et al., Induced Pluripotent Stem Cells Generated Without Viral Integration, Science 322: 945-949 (2008)). However, large amounts of adenoviral vectors, which may cause cytopathic effect on cells, are required for effective transduction into cell types that lack the adenovirus receptor CAR. In addition, low level expression of some adenoviral genes may affect the transduced cells if a “non-gutless” adenoviral vector is chosen as a delivery vehicle.
Re-programming can also be accomplished via direct plasmid transfection (Okita, Science 322: 949-953; Yu, J. et al. Human Induced Pluripotent Stem Cells Free of Vector and Transgene Sequences, Science 324: 797-801 (2009)), but it is more than 100-fold less efficient than that of a retroviral vector (Okita, Science 322: 949-953). Both adenoviral vector transduction and plasmid transfection may not exclude stable integration. The integration frequencies of adenoviral vectors are  ̃10−3 to 10−5 per cell (Harui, A. et al., Frequency and Stability of Chromosomal Integration of Adenovirus Vectors, J. Virology 73: 6141-6146 (1999)).
A comparison of methods to generate pluripotent stem cells is shown in table 1.







TABLE 1







Pros and Cons of three methods to create pluripotent stem cells












Use of
Application 





human 
to human
Immuno-
Chromosome



embryo
cells
rejection
abnormality





Nuclear transfer
Yes
unknown
Yes
normal


ES cell fusion
Yes
Yes
Yes
tetraploid


iPS Cells
No
Yes
No
vector insertion






(oncogene 






deregulation)









ES, embryonic stem cell; iPS, induced pluripotent stem cells.

In 1998, Frankel and Green independently observed that HIV-1 Tat protein can penetrate cells in a receptor-independent fashion (Frankel, A. and C. Pabo, Cellular Uptake of the Tat Protein from Human Immunodeficiency Virus, Cell 55: 1189-1193 (1988); Green, M. and P. Loewenstein, Autonomous Functional Domains of Chemically Synthesized Human Immunodeficiency Virus Tat Trans-Activator Protein, Cell 55:1179-1188 (1988)). The Tat protein transduction domain (PTD) that contains the short basic arginine-rich region (aa 48-57) of HIV-1 Tat is widely used to deliver variety of molecules including peptides both in vitro and in vivo (Schwarze, S. R. et al., In Vivo Protein Transduction: Delivery of a Biologically Active Protein Into Mouse, Science 285: 1569-1572 (1999); Lindsay, M. A. Peptide-Mediated Cell Delivery: Application in Protein Target Validation, Current Opinion in Pharmacology 2:587-594 (2002); Kwon, Y. D., et al., Cellular Manipulation of Human Embryonic Stem Cells by Tat-Pdxl Protein Transduction, Molecular Therapy 12: 28-32 (2005); Kim, D., Generation of Human Pluripotent Stem Cells by Direct Delivery of Reprogramming Proteins, Cell Stem Cell 4: 472-476 (2009); Wadia, J. S. et al., Transducible Tat-HA Fusogenic Peptide Enhances Escape of Tat-Fusion Protein After Lipid Raft Macropinocytosis, Nature Medicine 10:310-315 (2009); Zhou, H. et al., Generation of Induced Pluripotent Stem Cells Using Recombinant Proteins, Cell Stem Cell 4: 381-384.19-24 (2009)). This method to deliver molecules by a protein-based vehicle is called protein transduction. Binding of Tat-PTD to cell surface through ionic interaction leads to the internalization of Tat-fusion proteins by lipid raft-dependent macropinocytosis (Wadia, J. S., 20 Nature Medicine 10:310-315). The majority of the Tat-fusion protein, however, remains trapped in macropinosomes, indicating that the escape of peptides or proteins from macropinosomes is an inefficient process (Wadia, J. S., Nature Medicine 10:310-315). Recently, poly-arginine (11R or 9R) PTD fused to C-terminus of the 4 re-programming factors (Oct4, Sox2, Klf4, and C-Myc) successfully re-programmed mouse embryonic fibroblast (Zhou, H. et al., Generation of Induced Pluripotent Stem Cells using Recombinant Proteins, Cell Stem Cell 4: 381-384 (2009)) and human newborn fibroblast (Kim, D., Cell Stem Cell 4: 472-476) cells to iPS cells but with very low efficiency.

SUMMARY OF THE INVENTION
The present invention is a novel protein-based system for delivering reprogramming factors to adult somatic cells to generate iPS cells or tissue specific cells without using gene expression vectors. The system comprises a construct with a receptor binding domain, a translocation domain, a cargo bearing domain and an inducer. The receptor binding domain directs the construct to a somatic cell. The translocation domain facilitates the transport of the cargo bearing domain and inducer into the cell. The cargo bearing domain delivers the inducer into the cell. In one embodiment of the invention, the construct utilizes exotoxin domains for the binding, translocation, and cargo functions of the construct. Some of such exotoxins include C. difficile TcdA and TcdB toxins, C. botulinum BoNTs A through G and C2 toxins. The various domains of the construct can be swapped in order to accommodate various sizes of inducers and to direct the inducers to somatic cells having specific binding receptors.
The constructs can be used to deliver pluripotency inducers into somatic cells and cause the cells to become iPS cells. Another embodiment of the present invention further provides a method for generating iPS cells in which somatic cells are exposed to the construct bearing one or more inducers. The construct may also be utilized with other constructs, such as lentiviruses, small protein delivery systems, or small molecules to generate iPS cells



BRIEF DESCRIPTION OF THE DRAWINGS
The above and other features, aspects, and advantages of the present invention are considered in more detail, in relation to the following description of embodiments thereof shown in the accompanying drawings, in which:
FIG. 1A is a graphical representation of the C. difficile structure.
FIG. 1B is a graphical representation of various possible constructs using the C. difficile configuration.
FIGS. 2A through 2D are graphical representations of the various domains of constructs containing Oct4 and TcdB/TcdA, which are referenced as SEQ ID Nos. 1 through 4. Dashes represent transitions between domains, tags, and spacers.
FIGS. 3A through 3D are graphical representations of the various domains of constructs containing Sox2 and TcdB/TcdA, which are referenced as SEQ ID Nos. 5 through 8. Dashes represent transitions between domains, tags, and spacers.
FIGS. 4A through 4E are graphical representations of the various domains of constructs containing Oct4/Sox2 and C2I and C2II, which are referenced as SEQ ID Nos. 9 through 13. Underlined sequences are mutations that inactivate the active site of the toxin. Dashes represent transitions between domains, tags, and spacers.
FIG. 5A through 5F are graphical representations of the various domains of constructs containing Oct4/Sox2 and BoNTs and other RBDs, which are referenced as SEQ ID Nos. 14 through 19. Underlined sequences are mutations that inactivate the active site of the toxin. Dashes represent transitions between domains, tags, and spacers.
FIG. 6 is a time-course Western blot analysis showing recombinant TcdB protein expression by B. megaterium 2-3 hours after induction.
FIG. 7 is a picture showing mouse colorectal tumor CT26 cells exposed to TcdB and atoxic TcdB.
FIG. 8A is a graphical representation of a test construct of TcdB and TcdB with SNAP as a cargo.
FIG. 8B is and SDS-Page gel showing the purified SNAP-TcdB construct.
FIG. 8C shows a Western blot analysis of the purified samples using an anti-SNAP-tag antibody.
FIG. 8D is a chart showing that the SNAP-TcdB construct is as toxic as wild-type TcdB.



DETAILED DESCRIPTION
The invention summarized above may be better understood by referring to the following description, which should be read in conjunction with the accompanying claims and drawings in which like reference numbers are used for like parts. This description of an embodiment, set out below to enable one to build and use an implementation of the invention, is not intended to limit the invention, but to serve as a particular example thereof. Those skilled in the art should appreciate that they may readily use the conception and specific embodiments disclosed as a basis for modifying or designing other methods and systems for carrying out the same purposes of the present invention. Those skilled in the art should also realize that such equivalent constructs and cell lines do not depart from the spirit and scope of the invention in its broadest form.
As used herein, the term “construct” refers to a recombinant polypeptide having an amino acid sequence that includes an inducer sequence and a cargo delivery sequence. The construct may have other elements. For example, the construct may include a receptor binding domain (RBD), a translocation domain (TD), a cargo bearing domain (CBD), and a cleavage sequence (CS). As described in more detail below, the RBD allows the construct to identify and bind to a cell bearing the receptor. The TD allows the CBD, which may include an inducer, to be transported into the cytosol. The CBD may be an inactivated organic activity domain of a toxin. More specifically, it is understood that the CBD is rendered atoxic to the target cells by methods known in the art. In some embodiments, the CBD may be an inducer directly linked to the TD. The CS may be an intrinsic cysteine protease domain (CPD) or another protease sequence that allows the CBD carrying, for example, a pluripotency inducer to be released from the construct inside the cell.
An “inducer” is a transcription factor that among other properties may, when introduced in a somatic cell, aid in the transformation of the somatic cell into an iPS cell. The following inducers have been identified: Oct3/4, Sox2, Klf4, c-Myc, Nanog, lin28, hTERT (human telomerase), and SV40 large T-antigen. Other inducers may include MafA, Pdx-1, Ngn3, SV-40 T-ag, DPPA4, DPPA5, ZIC3, BCL-2, h-RAS, TPT1, SALL2, NAC1, DAX1, TERT, ZNF206, FOXD3, REX1, UTF1, p53 siRNA. In addition to the factors described above, inducers may also include p53 inhibitors, such as antibodies and antibody fragments that target p53, siRNAs, antisense RNA/DNA, and ribozymes.
A person of ordinary skill in the art understands that “substantially identical” homologs of the sequences described herein constitute exemplary embodiments of the present invention. Two amino acid sequences are “substantially identical” if (i) have only conservative amino acid substitutions that do not significantly affect the folding activity of the resulting polypeptide; (ii) the number of gaps between or insertions in, deletions of and substitutions of, is no more than 10%, preferably 5%, of the number of amino acid residues that occur over the length of the shortest of two aligned sequences; or (ii) no more than 30%, preferably 20%, more preferably 15%, or 10%, of the amino acid residues vary between the two sequences. Other methods as described by Houston et al. in United States Application Publication Number US2003/0161809A1 may also be used to determine whether two sequences are substantially identical.
Clostridium exotoxins are equipped with all the mechanisms for efficient cellular uptake and cytosolic delivery of their N-terminal enzymatic domain and can be used to deliver re-programming proteins to adult somatic cells. In particular, Clostridium exotoxins exhibit modular multi-domains that allow the toxin to efficiently translocate its N-terminus enzymatic domain, and anything attached to that N-terminus, into the cytosol. For example, Both Clostridium botulinum neurotoxin type D (BoNT-D) and Clostridium difficile toxin type B (TcdB) have demonstrated the ability to deliver a cargo appended to the N-terminus of the toxins into the cytosol (Bade, S. et al., Botulinum Neurotoxin Type D Enables Cytosolic Delivery of Enzymatically Active Cargo Proteins to Neurons Via Unfolded Translocation Intermediates, J. Neurochemistry 91: 1461-147225-26 (2004); Pfeifer, G. et al., Cellular Uptake of Clostridium difficile Toxin B, Translocation of the N-terminal Catalytic Domain Into the Cytosol of Eukaryotic Cells, J. of Boil. Chem. 278:44535-44541 (2003)).
I. C. difficile TcdA and TcdB Pluripotency Inducer iPS Cell Constructs
One embodiment of the present invention consists of a C. difficile toxin A (TcdA) and B (TcdB) construct having three functional domains as shown in FIG. 1: (a) the RBD binds target cells for endocytosis; (b) the TD promotes cytosolic delivery of the enzymatic domain of the toxin; and (c) the CBD, which is and inactive portion of the GT enzymatic domain, carrying an inducer. FIG. 1 A shows the primary structure of Clostridium difficile toxin with the N-terminal glucosyltransferase domain (GT), the intrinsic cysteine protease domain (CPD), the central translocation domain (TD), including a hydrophobic region (HR), and the C-terminal receptor binding domain (RBD). The CBD is located within the GT domain for engineering recombinant proteins.
As shown in FIG. 1B, various different constructs of TcdB mutants are made atoxic by truncation of the GT. Protein structure of the wild type TcdB, atoxic TcdB (aTcdB), and recombinant fusion proteins aTcdB-M1 aTcdB-M2, and aTcdB-M3. The aTcdB-M1 allows the inducer to be appended to the N-terminus of aTcdB. The aTcdB-M2 has the cargo attached to the truncated glucosyltransferase (GT) domain, and the aTcdB-M3 has the cargo replacing the entire GT domains.
The RBD of TcdA and TcdB RBDs bind to cell-surface carbohydrates, including Gal-α1, 3-Gal-β1, 4-GalNAc, as an initial step. These cell-surface carbohydrates are found in most somatic cells. Thus, the TcdA RBD will bind most somatic cells. After the RBD binds its receptor, the CBD is internalized by receptor-mediated endocytosis. The hydrophobic region (HR) in the TD enables the corresponding part of toxin to insert itself into the endosomal membrane to create a pore through which the CBD can translocate into the cytosol. The translocated can then be separated from the rest of the construct and released into cytosol by autoproteolysis, catalyzed by an intrinsic cysteine protease domain (CPD) located adjacent to the autocleavage site in the N-terminal part of TD as shown in FIG. 1B. As a result, as described herein, a construct that includes the RBD and TD of TcdA and TcdB can be used to introduce pluripotency inducers into somatic cells.
The CBD is an inactivated GT domain carrying an inducer. The GT domain can be inactivated using various methods known to a person of ordinary skill in the art. The GT domain can be truncated. The activity can also be demolished by amino acid substitutions in its sequence. In one embodiment of the present invention, the GT of TcdA is rendered inactive by the following deletions D285A and D287A, and the GT of TcdB is rendered inactive by the following deletions D286A and D288A. In some embodiments of the present invention various lengths of the GT domain may be utilized in which the portion of the domain responsible for the activity is deleted. The inactivation of the GT domain makes TcdA or TcdB atoxic and viable for use in a construct for generating iPS cells. The inactivated GT domain becomes a CBD to which an inducer can be attached. FIG. 1B shows various possible configurations of a construct in accordance with one embodiment of the present invention.
FIGS. 2A through 2D illustrate various constructs, in accordance with several embodiments of the present invention, that utilize the various domains of TcdB and Oct 4 and Sox 2. In FIG. 2A, the sequence of an Oct4-aTcdB construct (SEQ ID No 1) is provided with the following domains: Streptavidin Binding Peptide (SBP) (aa1-55); the Oct4 sequence (aa57-416); CBD (aa420-961); CPD (aa962-1185); TD (aa1186-2269), which includes a Hydrophobic Region (aa1374-1546); RBD (aa2270-2784); and a His tag (aa2785-2790). In FIG. 2B, the sequence of an Oct4-aTcdB(dGT) construct (SEQ ID No 2) is provided with the following domains: a Streptavidin Binding Peptide (SBP) (aa1-55); the Oct4 sequence (aa57-416); CBD (aa419-488); CPD (aa489-712); TD (aa713-1796), which includes a Hydrophobic Region (aa901-1073); RBD (aa1797-2311); and a His tag (aa2312-2317). It is understood that the SBP and His tag are simply tags for the construct, which may be interchanged with other tags or completely removed without affecting the constructs ability to cause an Oct 4 (the inducer) to be tranlocated into the cytosolic space.
In FIG. 2C, the sequence of an Oct4-aTcdA construct (SEQ ID No 3) is provided with the following domains: a Streptavidin Binding Peptide (SBP) (aa1-55); the Oct4 sequence (aa58-416); CBD (aa420-959); CPD (aa960-1187); TD (aa1188-2267), which includes a Hydrophobic Region (aa900-1376); RBD (aa2268-3128); and a His tag (aa3128-3134). In FIG. 2D, the sequence of an Oct4-aTcdA(dGT) construct (SEQ ID No 4) is provided with the following domains: a Streptavidin Binding Peptide (SBP) (aa1-55); the Oct4 sequence (aa58-416); CBD (aa419-486); CPD (aa487-714); TD (aa715-1794), which includes a Hydrophobic Region (aa903-1075); RBD (aa1795-2655); and a His tag (aa2656-2661).
Similarly, FIGS. 3A through 3D illustrate constructs utilizing TcdA, TcdB and Sox2. In FIG. 3A, the sequence of a Sox2-aTcdB construct (SEQ ID No 5) is provided with the following domains: a Streptavidin Binding Peptide (SBP) (aa1-55); the Sox2 sequence (aa58-373); CBD (aa377-918); CPD (aa919-1142); TD (aa1143-2226), which includes a Hydrophobic Region (aa1331-1503); RBD (aa2268-2741); and a His tag (aa2742-2747). In FIG. 3B, the sequence of an Sox2-aTcdB(dGT) construct (SEQ ID No 6) is provided with the following domains: a Streptavidin Binding Peptide (SBP) (aa1-55); the Sox2 sequence (aa58-373); CBD (aa376-445); CPD (aa446-669); TD (aa670-1753), which includes a Hydrophobic Region (aa858-1030); RBD (aa1754-2268); and a His tag (aa2269-2274).
In FIG. 3C, the sequence of a Sox2-aTcdA construct (SEQ ID No 7) is provided with the following domains: a Streptavidin Binding Peptide (SBP) (aa1-55); the Sox2 sequence (aa58-373); CBD (aa377-916); CPD (aa917-1144); TD (aa1145-2224), which includes a Hydrophobic Region (aa1333-1505); RBD (aa2225-3085); and a His tag (aa3086-3091). In FIG. 3D, the sequence of an Sox2-aTcdA(dGT) construct (SEQ ID No 8) is provided with the following domains: a Streptavidin Binding Peptide (SBP) (aa1-55); the Sox2 sequence (aa58-373); CBD (aa376-443); CPD (aa444-671); TD (aa672-1751), which includes a Hydrophobic Region (aa860-1032); RBD (aa1752-2612); and a His tag (aa2613-2618).
II. C. botolinum Neurotoxin Pluripotency Inducer iPS Cell Constructs
C. botulinum neurotoxin (BoNT) is another candidate of Clostridium exotoxins that can be used for pluripotent inducer delivery. The BoNT construct consist of a RBD that recognizes specific neuronal receptors. The heavy chain (Hc) of the BoNT toxins functions as the TD for the construct. The CBD of a construct in one embodiment of the present invention can be a C. botulinum neurotoxin light chain or a truncated light chain that has its non-palmitoylated preventing anchoring of the light chain to the membrane. The light chain is selected from of the BoNT type A through G to provide different longevity of the pharmacological effect of the inducer being delivered by the construct.
III. Clostridial C2 Toxin Constructs
The Clostridial C2 toxin has a binary toxin structure in which the C2II chain is responsible for cellular receptor binding, endocytosis, pore formation, and translocation of the C2I enzymatically active ADP-ribosylating chain. A construct in accordance with one embodiment of the present invention can be created by fusion of the pluripotency inducer to the C2II chain. Omission of part of the C2I chain renders the construct atoxic and safe for use as a promoter of generation of iPS cells. Alternatively, the C2I domain can be inactivated by modifications or deletions of the active site. Also, some of the residues may be altered as shown in the figures below.
FIGS. 4A through 4E show some representative examples of constructs that can be made in accordance with one embodiment of the present invention. In FIG. 4A, a basic C2II construct is presented with a SBP and a His tag (SEQ ID No. 9). FIG. 4B shows a construct having Oct4 and an inactivated C21 domain (SEQ ID No. 10). The C2I domain can be inactivated by point mutations at aa600, 602, 804, 805, and 807 of the construct. The SBP and Histidine tags are optional. FIG. 4C shows a construct having Oct4 and a truncated C2I (SEQ ID No. 11), which truncation renders it inactive. FIG. 4D shows a construct having Sox2 and an inactivated C2I sequence (SEQ ID No. 12). FIG. 4E shows a truncated C2I with Sox 2 (SEQ ID No 13).
IV. Clostridium Toxin Domain Swapping
The various Clostridium toxins bind to different cell types. The RBDs of the Clostridium toxins can be interchanged/swapped proving a variety of different constructs capable of delivering inducers to somatic cells. By domain swapping of the C-terminal RBD, one can create different constructs for cell-type or tissue-specific delivery. In one embodiment the RBD of TcdA or TcdB can be replaced with the RBD selected from BoNT for targeting neuronal cells. In another embodiment the RBD of TcdA or TcdB can be replaced with a ligand that binds to a cell-type or tissue-specific cell surface receptor and triggers the receptor-mediated internalization. In all, permutated protein-based delivery vehicles for different utility can be created by combination of the N-terminal domain swapping and C-terminal domain swapping.
Beyond a delivery vehicle itself, the C-terminus RBD of the BoNT can be used to replace the RBD of TcdA or TcdB and to create a protein delivery vehicle specifically targeting neuronal cells. Similarly, the RBD of TcdA or TcdB can be used to replace the BoNT's RBD resulting in non-cell-specific targeting of the inducer by the BoNT construct. Furthermore, the BoNT light chain can be used to replace the N-terminus of TcdB or TcdA to enhance desired biological function of cargo proteins that are fused to the N-terminus of BoNT light chain. The light chain is selected from BoNT type A to G, preferably the light chain of C. botulinum neurotoxin type A. Therefore, by using domain swap between Clostridium exotoxins, as shown herein, various pluripotency inducer delivery vehicles can be synthesized.
FIGS. 5A through 5D show some representative examples of constructs that can be made in accordance with one embodiment of the present invention. In FIG. 5A, Oct4 is attached to a BoNT/C1 as the TD and a TcdB RBD (SEQ ID No. 14). In the construct of FIG. 5A the activity of the light chain of BoNT/C1 can be destroyed by the modifications shown. FIG. 5B shows a Sox 2 with a BoNT/C1 TD and a TcdB RBD (SEQ ID No. 15). FIG. 5C shows a construct having Oct4 as the inducer, C2I as the TD and Transferrin as the RBD (SEQ ID No. 16). FIG. 5D is similar to FIG. 5C but utilizes Sox 2 instead of Oct4 (SEQ ID No. 17). FIG. 5E provides a further example of a construct in accordance with one embodiment of the present invention in which the inducer is Oct4, the CBD and TD are provided by BoNT/C1 and the RBD corresponds to ILGF (SEQ ID No. 18). FIG. 5F provides a further example of a construct in accordance with one embodiment of the present invention in which the inducer is Sox2, the CBD and TD are provided by BoNT/C1 and the RBD corresponds to ILGF (SEQ ID No. 19).
A person of ordinary skill in the art would recognize that the constructs can be made by standard methods. For example, the construct can be expressed in E. coli or algae systems by standard methods. Humphreys, David P. et al. High-Level Periplasmic Expression in Escherichia coli Using a Eukaryotic Signal Peptide: Importance of Codon Usage at the 59 End of the Coding Sequence, Protein Expression and Purification 20,252-264 (2000); Griswold, Karl E. et al., Effects of Codon Usage Versus Putative 50-mRNA Structure on the Expression of Fusarium solani cutinase in the Escherichia coli Cytoplasm, Protein Expression and Purification 27: 134-142 (2003); Mayfield, Stephen P et al., Chlamydomonas reinhardtii Chloroplasts as Protein Factories, Current Opinion in Biotechnology, 18:126-133 (2007).
V. Examples
Preparation of TcdB Construct
We have successfully demonstrated robust recombinant expression of C. difficile toxins using the B. megaterium expression system. Both the TcdA and TcdB genes were amplified from C. difficile (VPI 10463) genomic DNA by PCR. These PCR products were cloned into the B. megaterium expression vector pHis1522 (MoBiTec, Germany). We also created an atoxic TcdB gene by mutating amino acids critical for substrate binding in the glucosyltransferase domain. These constructs were used to transfect B. megaterium protoplasts (MoBiTec). Additionally, we also generated a complete synthetic genetic construct encoding TcdB to facilitate recombinant constructs in the future. A GT truncated mutant was also made from Ga14-TF/aTcdB with only 68 amino acids upstream of the self-cleavage site (-ΔGT68). Positive clones were selected for recombinant protein expression and purification of recombinant His-tagged recombinant TcdB (rTcdB) from bacterial lysate was performed by Ni-affinity chromatography following an ion-exchange fractionation. With these clones, we have performed small-scale time-course expression studies and found the peak expression for both recombinant proteins to be 2-3 hr after xylose induction as shown in FIG. 6.
We were able to obtain an average of 5-10 mg of highly purified recombinant proteins from the lysate of one liter of total bacterial culture. The purified rTcdB was functionally tested in mouse intestinal epithelial CT26 cells for their cytopathic and cytotoxic effects and glucosylation of Rac1. Exposure of cells to the highest dose possible of atoxic TcdB (aTcdB) for a prolonged period of time indicated that the mutant GT is virtually devoid of enzymatic activity ( ̃5 logs) and exhibited dramatically reduced cytotoxicity (>5 logs). FIG. 4 shows that no cytopathic effect was observed on those cells treated with aTcdB.
To verify that the recombinant TcdB is capable of delivering a fused cargo to cells, we appended a small enzyme (O6-alkylguanine-DNA alkyltransferase, called SNAP-tag by New England Biolabs) to the amino terminus of TcdB. We can efficiently express the chimeric fusion of SNAP-tag to active recombinant TcdB. The chimeric protein retains toxicity roughly equivalent to wild type TcdB, indicating that the cargo must have accessed the cell cytosol because it is appended to the glucosyltransferase domain of TcdB that causes the toxicity only after it reaches in the cytosol as shown in FIG. 7.
Demonstration of SNAP-TcdB Fusion Protein Construction, Purification, and Biological Functionality.
As shown in FIG. 8A, the domain structure of the N-modified SNAP-TcdB fusion is a 270 kD peptide in comparison to the wild-type which is 290 kD. The wild type TcdB construct had hexahistidine tag. The SNAP-TcdB construct was labeled with hexahistidine and streptavidin tags for affinity purification. FIG. 8B, shows the full-length SNAP-TcdB products recovered by streptavidin purification; the eluent (E) and flow through (FT) can be compared to the wild-type (W) samples, which yielded no products as expected. In FIG. 8C, a Western blot analysis of the purified samples show strong signals using an anti-SNAP-tag antibody. Finally, in FIG. 8D, Vero cells were exposed to different doses of purified SNAP-TcdB or wild-type TcdB for 1 hr and % of cell rounding was assessed. The results confirm that the addition of a fusion partner to TcdB does not adversely its biological function and remains able to delivery cargo to cells.
Construction & Functional Testing of the Reporter Constructs in Existing Cell-Based Systems.
The functional TcdB peptide described above can be used to develop the construct in accordance with one embodiment of the present invention. The construct can be constructed by adding the sequence of a pluripotency inducer to the N-terminal end of the aTcdB construct. The pluripotency inducer/TcdB construct can then be tested in one of two systems.
A mammalian reporter cell line used to detect the presence of a Ga14 DNA binding domain/NFkB activation domain-based chimeric transcription factor (Ga14-TF) can be modified to be used as a reporter for pluripotency inducer construct testing. The HEK293 cell line has a synthetic reporter construct integrated consisting of a Ga14-responsive promoter that expresses a bi-cistronic reporter gene containing the YFP Venus gene followed by a Gaussia luciferase gene (GLuc) gene. The Venus and Gluc are separated by a “self-cleavage” peptide 2A of the foot-and-mouth disease virus allowing independent expression of the two reporter proteins. Inclusion of both reporter genes permits instantaneous examination of cells microscopically for Venus fluorescence as well as detection of GLuc bioluminescence. With a Ga14 based transcription factor as a cargo appendage to aTcdB, this system can be used to test functional cellular delivery of a transcription factor/pluripotency inducer. The Ga14 DNA binding sequence promoter of the HEK293 Venus/Glue reporter system can be replaced the with an Oct4/Sox2 element repeat 4 times. Other pluripotency inducers can also be tested by replacing the Ga14 DNA binding sequence promoter of the HEK293 Venus/Gluc reporter system with their respective sequences.
A second Oct4/Sox2-based cell line reporter may be utilized to test the construct. This second system consists of the DNA-binding regions required for Oct4 and Sox2 for site-specific promoter activation (Ambrosetti D-C et al., Modulation of the Activity of Multiple Transcriptional Activation Domains by the DNA Binding Domains Mediates the Synergistic Action of Sox2 and Oct-3 on the Fibroblast Growth Factor-4 Enhancer, J. Biological Chem. 275:23387-23397 (2000)). The promoter reporter gene construct referred to as pCATSO3 contains six copies of the HMG and POU motifs present in the FGF-4 enhancer located upstream of a basal SV40 promoter. The HMG motif binds Sox2 and the adjacent POU motif binds Oct4 (Chakravarthy, H. et al., Identification of DPPA4 and Other Genes as Putative Sox2:Oct-3/4 Target Genes Using a Combination of in Silico Analysis and Transcription-Based Assays, J. Cellular Physiology, 216:651-662 (2008); Rizzino, A. Sox2 and Oct-3/4: A Versatile Pair of Master Regulators that Orchestrate the Self-Renewal and Pluripotency of Embryonic Stem Cells by Functioning as Molecular Rheostats, System Biology and Medicine, WIREs Syst Biol Med 1(2), (2009)). HeLa cells, pCATSO3 expresses very low levels of the reporter gene (CAT). This promoter reporter gene construct is highly sensitive to the presence of Sox2 and Oct4, which are not expressed in HeLa cells at baseline. When HeLa cells are transfected with pCATSO3 along with expression vectors for Sox2 and Oct4, there is a robust stimulation (>40-fold) of the reporter gene. Sox2 and Oct4 each stimulate the reporter gene  ̃3-fold. Given the synergistic response to Sox2 and Oct4, this assay can provide a highly sensitive test to gauge the ability of the Sox2- and Oct4-aTcdB fusion proteins to work together cooperatively.
Demonstration of Bioactive Oct4 & Sox2 Delivery by aTcdB to Oct4/Sox2 Reporter Cell Lines.
For a cellular-based differentiation assay, we will take advantage of the finding made by the Smith and Rizzino laboratories, which have shown that small increases ( ̃2-fold) in the levels of Oct4 or Sox2 trigger the differentiation of mouse ES cells (Niwa H, et al. Quantitative Expression of Oct-3/4 Defines Differentiation, Dedifferentiation or Self-Renewal of ES Cells, Nat Genet 24:372-376 (2000); Kopp, J. et al., Small Increases in the Levels of Sox2 Trigger the Differentiation of Embryonic Stem Cells, Stem Cells 26:903-911 (2008)). In the case where Oct4 is elevated, ES cells differentiate within 48 hrs into cells that express markers of extraembryonic endodemi and trophectoderm (Niwa H, Nat Genet 24:372-376). In the case where Sox2 is elevated, ES cells differentiate within 48 hrs into cells that express markers present in ectoderm, mesoderm and trophectoderm, but not endoderm (Kopp, J. Stem Cells 26:903-911). These differentiation markers are readily determined by RNA analysis using real-time RT-PCR (Kopp, J. Stem Cells 26:903-911). Using this assay, the individual functions of the Oct4-aTcdB fusion protein and the Sox2-aTcdB fusion protein can be assessed by determining their ability to drive the differentiation of ES cells into specific sets of differentiated cells.
For a cell-based transcription assay, we propose to test the ability of Oct4- and Sox2-aTcdB fusion proteins to stimulate the promoter/reporter gene construct that has been stably transfected into HeLa cells. This cell reporter system has been constructed and works well for this type of assay as described previously. The Sox2- and Oct4-aTcdB fusion proteins will be tested individually and in combination over a range of protein concentrations. For these studies, we will compare the ability of the Sox2- and Oct4-aTcdB fusion proteins to stimulate the promoter reporter gene construct with the ability of Sox2 and Oct4 delivered by lentiviral vectors to stimulate pCATSO3 (Nowling, T. et al., Transactivation Domain of the Transcription Factor Sox-2 and its Associated Coactivator, J. Biol. Chem. 275:3810-3818 (2000)).
Additionally, an Oct4/Sox2 reporter cell system closely related to the Ga14-TF cell line can be constructed and tested with direct plasmid transfection. A synthetic reporter construct containing Venus/GLuc expressed from the SO3 promoter, containing six copies of the HMG and POU motifs to bind Sox2/Oct4, can be cloned into a lentiviral vector. Both sox2 and oct4 genes can then be synthesized and cloned into a pcDNA mammalian expression plasmid. This system can be validated by co-transfecting HEK 293 cells with the reporter construct and both sox2 and oct4 expression plasmids. Activation of this reporter can be monitored by GLuc assay and Venus (YFP) bioluminescence. After confirming intracellular Oct4/Sox2 activity, Oct4 and Sox2 may be separately cloned into the aTcdB delivery plasmid for production of inducer constructs. The reporter can be further validated by plasmid transfection of one of the embryonic transactivators (i.e. Sox2) complemented by protein delivery of the other factor (i.e. Oct4) and vice versa to assay the reporter response to the individually delivered protein. Finally, the aTcdB inducer construct can be used to deliver both factors in order to test the combinatorial effect and efficiency of the iPS cell-generating system. For comparison, protein delivery peptides (e.g., Tat-Oct4) can be used to demonstrate increased efficiency of the pluripotency inducer constructs.
De-differentiation of Mouse Embryonic Fibroblasts to iPS Cells by Delivery of Sox2 & Oct4 via aTcdB
The efficiency of the fusion protein-mediated reprogramming can be compared to the efficiency of reprogramming observed when MEFs are infected with a lentiviral vector that expresses the four well-characterized reprogramming transcription factors (Cox, J. L., and Rizzino, A. Induced Pluripotent Stem Cells: What Lies Beyond the Paradigm Shift, Experimental Biology and Medicine 235:148-158 (2010)). MEFs have been reprogrammed using a lentiviral vector that expresses a polycistronic transcript that codes for Oct4, Sox2, Klf4 and c-Myc, which are separated from one another by self-cleaving peptides (2A from the foot and mouth disease virus). Each pluripotency inducer construct can be tested by infecting MEFs with an inducible lentiviral vector (Tet-on) that expresses other pluripotency inducers. For example, the pluripotency inducer construct containing Oct4 an Sox2 can be tested by infecting MEFs with an inducible lentiviral vector that expresses only Klf4 and c-Myc in response to doxycycline. These cells can then be used to assess the ability of the Sox2- and Oct4-aTcdB fusion proteins added to the medium daily along with doxycycline, to promote the formation of MEFs.
In addition to determining the efficiency of reprogramming (percentage of cells reprogrammed), the quality of the iPS cells generated can be assessed by examining a wide-range of well established properties of pluripotent stem cells, including: expression of endogenous pluripotency transcription factors (e.g. endogenous Sox2, Oct4, Nanog, UTF1), the demethylation of the endogenous Oct4 and Nanog genes, expression of the cell surface marker SSEA-1, and the ability of the iPS cells to differentiate into cells that express markers from each of the embryonic germ layers using embryoid bodies. Rizzino, A. Transcriptional Regulation in an In Vitro Model System for Mammalian Embryogenesis, In “Hormones and Growth Factors in Development and Neoplasia”, eds. R. B. Dickson and D. S. Salomon, John Wiley & Sons, New York, pp. 115-129 (1998)).
The invention has been described with references to a preferred embodiment. While specific values, relationships, materials and steps have been set forth for purposes of describing concepts of the invention, it will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the basic concepts and operating principles of the invention as broadly described. It should be recognized that, in the light of the above teachings, those skilled in the art can modify those specifics without departing from the invention taught herein. Having now fully set forth the preferred embodiments and certain modifications of the concept underlying the present invention, various other embodiments as well as certain variations and modifications of the embodiments herein shown and described will obviously occur to those skilled in the art upon becoming familiar with such underlying concept. It is intended to include all such modifications, alternatives and other embodiments insofar as they come within the scope of the appended claims or equivalents thereof. It should be understood, therefore, that the invention may be practiced otherwise than as specifically set forth herein. Consequently, the present embodiments are to be considered in all respects as illustrative and not restrictive.

## Claims

1. A method for generating induced Pluripotent Stem (iPS) cells, comprising:
contacting somatic cells with a construct that comprises a receptor binding domain, a translocation domain, a cargo bearing domain, and an inducer, wherein the receptor binding domain, translocation domain, and cargo domain are atoxic Clostridium toxin domains.

2. The method of claim 1, further comprising the step of assessing the pluripotency of the somatic cells after contact with the construct.

3. The method of claim 1, wherein the construct is transfected into the somatic cells during contact.

4. The method of claim 1, wherein the Clostridium toxin is a C. difficile toxin.

5. The method of claim 4, wherein the C. difficile toxin is TcdA.

6. The method of claim 4, wherein the C. difficile toxin is TcdB.

7. The method of claim 1, wherein said cargo bearing domain is an inactive exotoxin domain.

8. The method of claim 1, wherein the Clostridium toxin is a C. botulinum toxin.

9. The method of claim 8, wherein the C. botulinum toxin is selected from the group consisting of BoNTA, BoNTB, BoNTC, BoNTD, BoNTE, BoNTF and BoNTG.

10. The method of claim 8, wherein a receptor binding domain of the C. botulinum toxin is replaced with a non-specific receptor binding domain.

11. The method of claim 1, wherein the Clostridium toxin is a clostridial C2 toxin.

12. The method of claim 1, wherein the inducer is Oct3/4.

13. The method of claim 12, wherein the construct has a sequence comprising the amino acid sequence of SEQ ID NO: 1.

14. The method of claim 12, wherein the construct has a sequence comprising the amino acid sequence of SEQ ID NO: 2.

15. The method of claim 12, wherein the construct has a sequence substantially identical to a sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2, wherein no more than 20% of the amino acid residues vary between the construct sequence and SEQ ID NO:1 or SEQ ID NO: 2.

16. The method of claim 1, wherein the inducer is Sox2.

17. The method of claim 16, wherein the construct has a sequence comprising the amino acid sequence of SEQ ID NO: 5.

18. The method of claim 16, wherein the construct has a sequence comprising the amino acid sequence of SEQ ID NO: 6.

19. The method of claim 16, wherein the construct has a sequence substantially identical to a sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, wherein no more than 20% of the amino acid residues vary between the construct and SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7.

20. The method of claim 1, wherein the inducer is selected from the group consisting of Klf4, c-Myc, Nanog, lin28, hTERT (human telomerase), and SV40 large T-antigen.