GENERATION OF BRAIN AND SPINAL CORD NEURONS, CARDIAC MYOCYTES, RENAL NEPHRONS AND HEPATOCYTES USING REG PEPTIDES, PEPTIDOMIMETICS, SMALL MOLECULES AND STIMULATORY ANTIBODIES TO REG RECEPTOR

Abstract

Peptides that are bioactive regions and optimized bioactive regions of the human and mammalian Reg gene proteins and that are capable of generation of tissues such as brain, spinal cord, heart, liver, and kidney are described. In particular, 7-15-amino acid Reg peptides and optimized Reg peptides are disclosed which are capable of in vivo and ex vivo transformation of progenitor cells, progenitor tissue and stem cells into specialized cells and tissues, including functioning brain and spinal cord neurons, cardiac myocytes, liver hepatocytes and renal nephrons. Methods of in vivo and ex vivo transformation of progenitor cells into differentiated cells and tissues are also described.

Claims

1. An isolated or modified peptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27.

2. A pharmaceutical formulation comprising the peptide of claim 1.

3. The pharmaceutical formulation of claim 2, wherein the formulation is a soluble liposome or nanoparticle preparation.

4. The pharmaceutical formulation of claim 2, wherein the formulation comprises a targeting agent for targeted administration to heart, brain, spinal column, liver, or kidney.

5. A method of treating a subject in need of one or more differentiated cells or tissue types, comprising administering to the subject a peptide having Reg Receptor binding activity, wherein the amount of peptide is effective for forming differentiated cells or tissues from progenitor cells in the subject in vivo.

6. The method of claim 5, wherein the peptide having Reg Receptor binding activity has an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27.

7. The method of claim 5, wherein the one or more differentiated cells or tissue types are heart, liver, brain, spinal column, or kidney cells or tissues.

8. The method of claim 5, wherein the peptide is administered directly to the heart, liver, brain, spinal cord or kidney of a subject.

9. The method of claim 5, wherein the peptide is administered by way of intravenous, subcutaneous, intra-arterial, or intrathecal delivery.

10. The method of claim 5, wherein the subject has a condition selected from the group consisting of heart disease, myocardial infarction, stroke, acute brain injury, neurodegenerative disease, spinal cord injury, peripheral neuropathy, acute and chronic kidney disease and liver disease.

11. A method of treating a subject in need of one more differentiated cells or tissues, the method comprising: culturing a plurality of progenitor cells ex vivo; contacting the progenitor cells with a peptide having Reg Receptor binding activity, wherein the amount of peptide is effective for transforming the progenitor cells to differentiated cells or tissues in culture; and administering the one or more differentiated cells or tissues to the subject.

12. The method of claim 11, wherein the peptide having Reg Receptor binding activity has an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27.

13. The method of claim 11, wherein the progenitor cells are selected from the group consisting of neural stems cells, tissue stem cells, mesenchymal stem cells, totipotent embyronic stem cells, multipotent stem cells, pluripotent stem cells, including embryonic cells, adult somatic stem cells, human adult bone-marrow derived stem cells, umbilical cord stems cells, human amniotic membrane-derived mesenchymal cells, mammalian stem cells, cord cells, and ectodermal stem cells.

14. The method of claim 11, wherein the differentiated cells or tissues are selected from the group consisting of brain, spinal cord, heart, kidney and liver cells or tissues.

15. The method of claim 11, wherein the differentiated cells or tissues are administered directly to the heart, liver, brain, spinal cord or kidney of a subject.

16. The method of claim 11, wherein the subject has a condition selected from the group consisting of heart disease, myocardial infarction, stroke, acute brain injury, neurodegenerative disease, spinal cord injury, peripheral neuropathy, acute and chronic kidney disease and liver disease.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0119] The file of this patent contains at least one photograph or drawing executed in color. For a fuller understanding of the nature and advantages of the present invention, reference should be made to the following detailed description taken in connection with the accompanying drawings, in which:

[0120] FIGS. 1A-C are graphics depicting the 3-dimensional structures of human Reg1a, human Reg3a and Hamster Reg 3gamma by Swiss-Prot folding algorithms with the Regenerative Peptides contained within the Red circled binding arm. Regenerative peptides are contained within the Red circled binding arm.

[0121] FIG. 2 is a set of sequences which demonstrate peptide homology between human Reg1a, Reg1b, Reg3a and Reg 4 and the Hamster Reg3gamma. The common sequences are highlighted in red.

[0122] FIG. 3 is the 919-amino acid sequence of the Reg Receptor with the 20-amino acid binding domain (SEQ ID NO: 6) presented in this invention is bolded.

[0123] FIG. 4 demonstrates the results of studies generating stimulatory antibodies to a 20-amino acid binding site within the Reg Receptor.

[0124] FIG. 5 provides the protocol for development of stimulatory antibodies to the binding domains within the Reg Receptor

[0125] FIG. 6 is the documentation of titer controls and norms for the antibody studies.

[0126] FIGS. 7A-C are images of an SDS PAGE gel and Western blot results which demonstrates the results from studies conducted to demonstrate Reg Receptor expression and purification utilizing 293T cells that were transfected with Reg Receptor expression plasmid DNA. FIG. 7A demonstrates the use of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to illustrate the high purity of the Reg Receptor. FIGS. 7B and 7C are the Western blot results showing that the purified protein is the Reg Receptor using the antibody to the FLAG epitope (FIG. 7B) and Reg Receptor (CD104) (FIG. 7C).

[0127] FIG. 8 is a graph which shows the 7-amino acid Regenerative peptide, the 8-amino acid Regenerative peptide and the 9-amino acid Regenerative peptide all bind directly to Reg Receptor.

[0128] FIG. 9 is an image of a Western Blot which demonstrates a confirmation study to evaluate the translocation of the Reg Receptor from the cytoplasmic membrane of human extra-islet exocrine tissue. The Western blot analysis identified he presence of the Reg receptor on the cytoplasmic membrane and the movement of the Reg Receptor from the cytoplasmic membrane through the cytoplasm to the nucleus in the presence of the Regenerative peptides 7aa (SEQ ID NO: 3), Baa (SEQ ID NO: 4) and 9aa (SEQ ID NO: 5).

[0129] FIG. 10 is a table which demonstrates the results showing upregulated transcription factors stimulated by the Regenerative peptides acting through their receptor in an in-vivo mouse study of injected with peptides SEQ ID NO: 4, SEQ ID NO:5, SEQ ID NO: 8, SEQ ID NO:1 and SEQ ID NO:16

[0130] FIG. 11 is a set of sequences which demonstrate the 7-amino acid human Reg sequences (SEQ ID NO: 3) that has 100% homology with Reg sequences found in other mammals including chimpanzee, rat, mouse, golden hamster, guinea pig, rabbit, pig, sheep, cow, white-cheeked gibbon, Sumatran orangutan, Lowland gorilla, white-tufted-eared marmoset, European domestic ferret, which have not been described in the prior art as being a peptide which has not been described in the prior art as being a peptide that interacts with the Reg receptor for the generation of new neurons, cardiac myocytes, nephrons and hepatocytes

[0131] FIG. 12 is a set of sequence which demonstrate the 8-amino acid human Reg sequence (SEQ ID NO: 4) that has 100% homology with Reg sequences found in other mammals including chimpanzee, rat, golden hamster, guinea pig, rabbit, pig, sheep, cow, white-cheeked gibbon, Sumatran orangutan, Lowland gorilla, white-tufted-eared marmoset, which have not been described in the prior art as being a peptide that interacts with the Reg receptor that can generate for generation of as a peptide for generation of new neurons, cardiac myocytes, nephrons and hepatocytes.

[0132] FIG. 13 is a set of sequence which demonstrate the 9-amino acid human Reg sequences (SEQ ID NO: 14) that has 100% homology with Reg sequences found in other mammals including chimpanzee, rat, mouse, golden hamster, white-cheeked gibbon, Lowland gorilla, and white-tufted-eared marmoset, which have not been described in the prior art as being a peptide that interacts with the Reg receptor that can generate for generation of being a peptide for generation of new neurons, cardiac myocytes, nephrons and hepatocytes.

[0133] FIG. 14 is a set of sequence which demonstrates the 20-amino acid human sequence has been identified within the Reg receptor that has 100% homology with Reg receptor sequences found in other mammals including chimp, rat, mouse, guinea pig, rabbit, dog, cow, opossum, galago, white-cheeked gibbon, Sumatran orangutan, macaque, Lowland gorilla, white-tufted eared marmoset, horse and Tasmanian devil from which stimulatory antibodies and small molecules can be generated to interact with this receptor site resulting in new neurons, hepatocytes, myocytes and nephrons.

[0134] FIGS. 15A and B are microscopic images which show immunofluorescent staining of Reg receptor on the cell surface of human pancreatic exocrine ductal cells. In utilizing Cy3 immunofluorescent staining of Reg receptor in human pancreatic ductal cells in standard medium, there is immunofluorescent staining of Reg receptor, which is well-defined at the cell borders indicating surface expression of Reg receptor on the cytoplasmic membrane of cells (FIG. 15A). FIG. 15B demonstrates the difference in Reg receptor staining when cells are exposed to Reg.

[0135] FIG. 16 shows the protocol for the stability testing in human plasma for the Regenerative Peptides SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO:3 and SEQ ID NO:16

[0136] FIG. 17 is a table which shows the Regenerative Peptides SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO:3, SEQ ID NO:16 and the incubation time points used in the stability testing in human plasma.

[0137] FIG. 18A is a table which shows the results of the Regenerative Peptides SEQ ID NO:1 incubated in human plasma.

[0138] FIG. 18B is a table which shows the results of the Regenerative Peptides SEQ ID NO: 8 incubated in human plasma.

[0139] FIG. 18C is a graph which shows the results of the Regenerative Peptides SEQ ID NO:1 and SEQ ID NO: 8 incubated in human plasma. SEQ ID NO: 8 is significantly more resistant to proteolysis in human plasma compared to SEQ ID NO:1

[0140] FIG. 19A is a table which shows the results of the Regenerative Peptides SEQ ID NO:16 incubated in human plasma.

[0141] FIG. 19B is a table which shows the results of the Regenerative Peptides SEQ ID NO:3 incubated in human plasma.

[0142] FIG. 19C is a graph which shows the results of the Regenerative Peptides SEQ ID NO:16 and SEQ ID NO: 3 incubated in human plasma. SEQ ID NO: 16 is significantly more resistant to proteolysis in human plasma compared to SEQ ID NO:3

DETAILED DESCRIPTION OF THE INVENTION

[0143] Reference will now be made in detail to various exemplary embodiments of the invention. It is to be understood that the following discussion of exemplary embodiments is not intended as a limitation on the invention. Rather, the following discussion is provided to give the reader a more detailed understanding of certain aspects and features of the invention.

[0144] Over the past decade, the regenerating gene (Reg or REG) family has emerged among many species, including humans, as a potential key initiating factor in the process of new cell formation within an organ following acute injury, including the brain, heart and liver. Although Reg is typically expressed during embryogenesis when organs are being populated for the first time and downregulated after fetal development, the Reg genes are upregulated within the brain, pancreas, heart and liver when there is acute injury with the formation of new neurons, islets, cardiac myocyte, renal nephron and hepatocyte neogenesis within the injured organs (Levetan. J Diabetes. 2010 June; 2(2):76-84, Levetan Endocr Pract. 2008; 14(9). After fetal development, the Reg genes are usually undetectable, but are upregulated in response to acute organ injury.

[0145] FIGS. 1A-C are illustrations showing the similarities and differences between three dimensional structures of human Reg1a, Reg3a and the Hamster Reg3 gamma peptides based on their primary -amino acid sequences and by SwissProt folding algorithms. FIG. 1A shows the three dimensional structure for Human Reg1a. FIG. 1B shows the three dimensional structure for Reg3a. FIG. 1C shows the three dimensional structure for the hamster Reg3gamma. The circled region of the protein that contains the Regenerative peptides described in this invention, which bind to Reg Receptor. Red circled arm indicates the bioactive region and homologous sequence of the Reg proteins that is the binding arm to the Reg receptor.

[0146] FIG. 2 is an alignment of the 166-amino acid sequence for Reg1a, the 166-amino acid sequence for Reg1b, the 175-amino acid sequence for Reg3 alpha, the 158-amino acid sequence for Reg4, and the mammalian Reg3gamma protein have been shown in the prior art to interact with the Reg receptor, resulting in downstream generation of new adult cells. FIG. 2 shows the identification of the 100% homologous sequence within the human Reg 1a, Reg1b, Reg3a, Reg4 and the Hamster Reg3gamma peptides that is located within the binding arm of the protein that binds to the Reg Receptor. This exact sequence is also found in the human, chimpanzee, rat, mouse, golden hamster, guinea pig, rabbit, pig, sheep, cow, white-cheeked gibbon, Sumatran orangutan, Lowland gorilla, white-tufted-eared marmoset, and European domestic ferret.

[0147] FIG. 3 shows the 919-amino acid Reg Receptor (SEQ ID NO: 7) and the 20-amino acid domain (SEQ ID NO: 6) in red that was identified in this discovery that is a binding domain for the Regenerative Peptides, and from which stimulatory antibodies to this region of the receptor have been generated.

[0148] FIGS. 4-6 were from studies undertaken by this inventor to develop stimulatory antibodies to the 20-amino acid bioactive region of the 919-amino acid Reg Receptor/EXTL-3 to accelerate the downstream regulation of transcription factors to develop new cells. Progression of beta cell formation by stimulating potential binding sites on the Reg Receptor. For the production of in stimulatory antibodies and identification and confirmation of the 20-amino acid binding region of EXTL-3, many sequences were evaluated within the N-terminal portion of the Reg receptor/Receptor within amino acids 1-332 of (SEQ ID NO:7) to find the region to which antibodies were produced, which this inventor identifies as the binding domain of the Reg peptides. Consistently, in Enzyme ImmunoAssay studies measuring titers from peptide sequences within SEQ ID NO:7, resulted in very high polyclonal antibodies being raised to a 20-amino acid Reg receptor sequence (SEQ ID NO: 8) (amino acids 117-136). The results of the Enzyme ImmunoAssay are summarized in FIG. 4. FIG. 5 demonstrates the standard protocol used for development of antibodies to peptide regions within Reg receptor. Data sets were taken from the bleed after the day 0 and day 21 boosts. The animals were injected with a peptide of SEQ ID NO: 6 and were conjugated to keyhole limpet hemocyanin (KLH). The screening antigen is not conjugated to KLH so that the response solely to the peptide and not to KLH can be identified. The 50% titer is a dilution value where the signal is half-way between the peak and the baseline, so the higher the dilution value (titer), the greater the response to the antigen. The positive control is an internal control that was generated from ovalbumin antibodies in rabbit. At a dilution of 1:750,000, the absorbance fell within a range of 0.45 to 0.9. In the case of the response to SEQ ID NO: 6, there was a high response (FIG. 4). The test bleed taken 31 days after the day 0 and 21 day boosts for CD 153 showed a 50% titer of 36,000 which is an average response, and CD 154 showed a 50% titer of 125,000 which is a high response according to the titer reference range shown in FIG. 6. Studies are underway to evaluate the efficacy of the antibody generated with and without the presence of Reg peptide demonstrating that the antibodies to the Reg peptide interaction with Reg receptor to generate new neurons, cardiac myocytes, nephrons and hepatocytes.

[0149] FIGS. 7A-C demonstrates the results from studies conducted to demonstrate Reg Receptor expression and purification utilizing 293T cells that were transfected with Reg Receptor expression plasmid DNA. Cells were collected after 72 hours. The Reg Receptor was tagged with FLAG epitope and FLAG resin was utilized to purify out the Reg Receptor. As shown in FIG. 7C, the Reg Receptor was highly purified. The Reg Receptor was purified by Anti-Flag M2 affinity gel. Target protein was confirmed by 4-12% SDS-PAGE and Western-blot. FIG. 7A demonstrates the use of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to illustrate the high purity of the Reg Receptor. FIGS. 7B and 7C are the Western blot results showing that the purified protein is the Reg Receptor using the antibody to the Reg Receptor (CD104). The Reg Receptor is shown in FIG. 7C to be highly purified. To then demonstrate the direct binding of Regenerative Peptides to the Reg Receptor, the purified Reg receptor was coated onto 96 well plate by using bicarbonate coating buffer, pH 9.6; 4 C. overnight at concentration 3 ug/ml, 100 ul per well. Plates were coated overnight coated plate and washed three times with 0.5 TBST and blocked with 3% BSA and rotated at room temperature for 1 hour. After blocking, plates were washed three times with 0.5 TBST. Peptides were then diluted with TBST buffer and added into wells in duplicate then left to bind at room temperature for 1 hour. After washing three times, 100 ng/ml strep-HRP was added into plate at 100 ul/well, and rotated at room temperature for one hour. ABST reagents were warmed to room temperature, mixed immediately before using. Then 100 ul was added to each well and read after 25 minutes reaction and absorbance was evaluated at 405 nm by a Spectramax M5 plate reader. The purified Reg Receptor was coated on plates. Then plates were blocked with BSA solution. Subsequently, the Regenerative peptides were added into the wells, and HRP-straptavidin and its substrates were added into the wells to reveal the interaction between Receptor and the peptide.

[0150] FIG. 8 shows the 7-amino acid Reg peptide (SEQ ID NO: 3), the 8-amino acid Reg peptide (SEQ ID NO: 4) and the 9-amino acid Reg peptide all show binding (SEQ ID NO: 5) to the Reg Receptor. The scrambled control peptide did not bind to Reg Receptor (FIG. 8).

[0151] FIG. 9 demonstrates a confirmation study utilizing Western blot analysis to evaluate the translocation of the Reg Receptor from the cytoplasmic membrane of human extra-islet exocrine tissue inclusive of ductal cells containing progenitor. Western blot analysis identified the presence of the Reg receptor on the cytoplasmic membrane and the movement of the Reg Receptor from the cytoplasmic membrane through the cytoplasm to the nucleus in the presence of the Regenerative peptides (30 minute treatment). Cytoplasmic extracts were obtained in 10 mM HEPES (pH 8.0), 1 mM EDTA, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 200 mM sucrose and 0.5% Nonidet P-40. Nuclear extracts were obtained in 20 mM HEPES (pH 7.9), 0.75 mM MgCl2, 210 mM NaCl, 50 mM KCl, 1 mM EDTA, 10% glycerol, and 0.5 mM DTT. Both extraction buffers contained 0.5 mM PMSF, 1 g/ml leupeptin, 1 g/ml aprotinin, 2.5 mM Na4P2O7, 1 mM -glycerophosphate, and 1 mM Na3VO4. Reg Receptor extracts were size fractionated on SDS-polyacrylamide gels and transferred to nitrocellulose. After blocking in 3% milk in Tris-buffered saline (pH 7.4), blots were sequentially incubated with rabbit anti-human Reg Receptor antibody overnight at 4 C. and appropriate horseradish peroxidase-conjugated secondary antibody. Secondary signals were developed with chemiluminescence substrate and analyzed by autoradiography.

[0152] Fractions and quality control utilized the fractions with two antibodies, GAPDH as a cytosol molecule and Lamin B is a nuclear molecule. Both GADPH and Lamin B were demonstrated in this invention to serve as excellent controls for the nuclear and cytosolic fractions (FIG. 9). Evaluation was conducted to determine the impact and interaction of the Reg Receptor with the Regenerative peptides and the human 14-amino acid (SEQ ID NO: 2) peptide. This invention demonstrates in FIG. 9 that the 8-amino acid (SEQ ID NO: 4) Regenerative Peptide resulted in the translocation of the Reg Receptor from the cytoplasm to the nucleus both in the shorter and longer exposure times.

[0153] FIG. 10 demonstrates the in vivo efficacy of Reg peptides on pancreatic transcription factors NGN3, Pdx1, and Sox9 compared to uninjected and vehicle alone controls with cohorts of 10 mice (adult, age 8 weeks, mixed male and female) will be injected intraperitoneally with selected candidate Reg peptides at 2-3 dosages/animal daily across 2-3 time courses (an average of 60 mice/peptide tested). An additional 20 animals will serve as negative (uninjected and vehicle alone) controls. Pancreata were subjected to rapid dissection and denaturant RNA preparation followed by real-time, quantitative RT-PCR analysis of gene expression markers for beta cell development and differentiation, including Ngn3, Pdx1 and Sox9. All Reg peptides had a significant fold increase in transcription factors ranging from a 1.33 fold rise to a 3.44-fold rise compared to vehicle and control transcription factors, indicating the ability to raise pancreatic transcription factors in mice.

[0154] FIG. 11 shows the identification of the Regenerative Peptide 7-amino acid human Reg sequence (SEQ ID NO: 3) that has 100% homology with sequences found in other mammals including chimpanzee, rat, mouse, golden hamster, guinea pig, rabbit, pig, sheep, cow, white-cheeked gibbon, Sumatran orangutan, Lowland gorilla, white-tufted-eared marmoset, European domestic ferret which has not been described in the prior art as being a peptide that interacts with the Reg receptor that can generate new neurons, cardiac myocytes, nephrons and hepatocytes in times of injury when the Reg receptor is upregulated in such organs. This invention evaluated GenBank, Basic Local Alignment Search Tool (BLAST) algorithm and UniProtKB which produced by the UniProt Consortium which consists of groups from the European Bioinformatics Institute (EBI), the Swiss Institute of Bioinformatics (SIB) and the Protein Information Resource (PIR).

[0155] FIG. 12 shows the identification of the Regenerative Peptide 8-amino acid human Reg sequence (SEQ ID NO: 4) that has 100% homology with sequences found in other mammals including chimpanzee, rat, mouse, golden hamster, guinea pig, rabbit, pig, sheep, cow, white-cheeked gibbon, Sumatran orangutan, Lowland gorilla, and white-tufted-eared marmoset which have not been described in the prior art as being a peptide that interacts with the Reg receptor that can generate new neurons, cardiac myocytes, nephrons and hepatocytes. This invention evaluated GenBank, Basic LocalAlignment Search Tool (BLAST) algorithm and UniProtKB which produced by the UniProt Consortium which consists of groups from the European Bioinformatics Institute (EBI), the Swiss Institute of Bioinformatics (SIB) and the Protein Information Resource (PIR)

[0156] FIG. 13 shows the identification of the Regenerative Peptide 9-amino acid human Reg sequence (SEQ ID NO: 5) that has 100% homology with sequences found in other mammals including chimpanzee, rat, golden hamster, white-cheeked gibbon, Sumatran orangutan, Lowland gorilla, white-tufted-eared marmoset, which have not been described in the prior art as being a peptide that interacts with the Reg receptor that can generate for generation of new neurons, cardiac myocytes, nephrons and hepatocytes due to its ability to bind to This invention evaluated GenBank, Basic Local Alignment Search Tool (BLAST) algorithm and UniProtKB, which produced by the UniProt Consortium which consists of groups from the European Bioinformatics Institute (EBI), the Swiss Institute of Bioinformatics (SIB) and the Protein Information Resource (PIR).

[0157] FIG. 14 demonstrates the 20-amino acid human sequence (SEQ ID NO: 6) that has been identified within the human Reg receptor that has 100% homology with Reg receptor sequences found in other mammals including chimp, rat, mouse, guinea pig, rabbit, dog, cow, opossum, galago, white-cheeked-gibbon, Sumatran orangutan, macaque, Lowland gorilla, white-tufted eared marmoset, horse and Tasmanian devil from which stimulatory antibodies and small molecules to this receptor site can be generated to result in new neurons, hepatocytes, myocytes and nephrons. This invention evaluated GenBank, Basic Local Alignment Search Tool (BLAST) algorithm and UniProtKB which produced by the UniProt Consortium which consists of groups from the European Bioinformatics Institute (EBI), the Swiss Institute of Bioinformatics (SIB) and the Protein Information Resource (PIR).

[0158] FIGS. 15A and 15B shows immunofluorescent staining of Reg receptor on the cell surface of human pancreatic exocrine ductal cells. In utilizing Cy3 immunofluorescent staining of Reg receptor in human pancreatic ductal cells in standard medium, there is immunofluorescent staining of Reg receptor, which is well-defined at the cell borders indicating surface expression of Reg receptor on the cytoplasmic membrane of cells (FIG. 15A). FIG. 15B demonstrates the difference in Reg receptor staining when cells are exposed to Reg peptide (SEQ ID NO: 2).

[0159] FIGS. 16-19C demonstrate the methods for stability testing of Regenerative Peptides SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 3 and SEQ ID NO:16 and the incubation time points used in the stability testing in human plasma. FIG. 16 shows the protocol for the stability testing in human plasma for the Regenerative Peptides SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO:3 and SEQ ID NO:16. FIG. 17. shows Peptides and Incubation time points used in Stability testing of Regenerative Peptides in Human Plasma. FIGS. 18A-C shows the results of the Regenerative Peptides SEQ ID NO:1 and SEQ ID NO: 8 incubated in human plasma. SEQ ID NO: 8 is significantly more resistant to proteolysis in human plasma compared to SEQ ID NO:1. FIGS. 19A-C show the results of the Regenerative Peptides SEQ ID NO: 16 and SEQ ID NO: 3 incubated in human plasma. SEQ ID NO: 16 is significantly more resistant to proteolysis in human plasma compared to SEQ ID NO: 3.

BRIEF DESCRIPTION OF THE SEQUENCES

[0160] As used herein, Peg-40KD-maleimide includes:

[0161] Methoxy-PEG-(CH2)3NHCO(CH2)2-MAL, Mw 40,000

[0162] Chemical Name: -[3-(3-Maleimido-1-oxopropyl)amino]propyl--methoxy, polyoxyethylene

[0163] CAS#: 883993-35-9

[0164] As used herein, 3-Mpa=3-mercaptopropionic acid, CAS#: 107-96-0

[0165] SEQ ID NO: 1

[0166] This is the 15-amino acid peptide found within the mammalian Reg protein

TABLE-US-00023 Ile-Gly-Leu-His-Asp-Pro-Ser-His-Gly-Thr-Leu-Pro- Asn-Gly-Ser

[0167] SEQ ID NO: 2

[0168] This is the 14-amino acid peptide found within the human Reg protein

TABLE-US-00024 Ile-Gly-Leu-His-Asp-Pro-Thr-Gln-Gly-Thr-Glu-Pro- Asn-Gly

[0169] SEQ ID NO: 3

[0170] This is the 7-amino acid peptide found within the human and mammalian Reg protein

TABLE-US-00025 Trp-Ile-Gly-Leu-His-Asp-Pro

[0171] SEQ ID NO: 4

[0172] This is the 8-amino acid peptide found within the human and mammalian Reg protein

TABLE-US-00026 Val-Trp-Ile-Gly-Leu-His-Asp-Pro

[0173] SEQ ID NO: 5

[0174] This is the 9-amino acid peptide found within the human and mammalian Reg protein

TABLE-US-00027 Asn-Val-Trp-Ile-Gly-Leu-His-Asp-Pro

[0175] SEQ ID NO: 6

[0176] This is a 20-amino acid peptide found within the human Reg receptor also known as EXTL-3

TABLE-US-00028 Cys-Lys-Lys-Ser-Ile-Glu-Asn-Ala-Lys-Gln-Asp-Leu- Leu-Gln-Leu-Lys-Asn-Val-Ile-Ser

[0177] SEQ ID NO: 7

[0178] Is the 919-amino acid human Reg receptor, also known as EXTL-3, exostosin-like 3 (public accession number NP_001431).

TABLE-US-00029 MTGYTMLRNGGAGNGGQTCMLRWSNRIRLTWLSFTLFVILVFFPLIAHYY LTTLDEADEAGKRIFGPRVGNELCEVKHVLDLCRIRESVSEELLQLEAKR QELNSEIAKLNLKIEACKKSIENAKQDLLQLKNVISQTEHSYKELMAQNQ PKLSLPIRLLPEKDDAGLPPPKATRGCRLHNCFDYSRCPLTSGFPVYVYD SDQFVFGSYLDPLVKQAFQATARANVYVTENADIACLYVILVGEMQEPVV LRPAELEKQLYSLPHWRTDGHNHVIINLSRKSDTQNLLYNVSTGRAMVAQ STFYTVQYRPGFDLVVSPLVHAMSEPNFMEIPPQVPVKRKYLFTFQGEKI ESLRSSLQEARSFEEEMEGDPPADYDDRIIATLKAVQDSKLDQVLVEFTC KNQPKPSLPTEWALCGEREDRLELLKLSTFALIITPGDPRLVISSGCATR LFEALEVGAVPVVLGEQVQLPYQDMLQWNEAALVVPKPRVTEVHFLLRSL SDSDLLAMRRQGRFLWETYFSTADSIFNTVLAMIRTRIQIPAAPIREEAA AEIPHRSGKAAGTDPNMADNGDLDLGPVETEPPYASPRYLRNFTLTVTDF YRSWNCAPGPFHLFPHTPFDPVLPSEAKFLGSGTGFRPIGGGAGGSGKEF QAALGGNVPREQFTVVMLTYEREEVLMNSLERLNGLPYLNKVVVVWNSPK LPSEDLLWPDIGVPIMVVRTEKNSLNNRFLPWNEIETEAILSIDDDAHLR HDEIMFGFRVWREARDRIVGFPGRYHAWDIPHQSWLYNSNYSCELSMVLT GAAFFHKYYAYLYSYVMPQAIRDMVDEYINCEDIAMNFLVSHITRKPPIK VTSRWTFRCPGCPQALSHDDSHFHERHKCINFFVKVYGYMPLLYTQFRVD SVLEKTRLPHDKTKCFKFI.

[0179] SEQ ID NO: 8

[0180] This is the optimization of the mammalian 15-amino acid Reg peptide by blocking with an n-terminal acetyl group and a c-terminal amide group.

TABLE-US-00030 Ac--Ile-Gly-Leu-His-Asp-Pro-Ser-His-Gly-Thr-Leu- Pro-Asn-Gly-Ser-NH2

[0181] SEQ ID NO: 9

[0182] This is a pegylated version of the mammalian 15-amino acid Reg peptide

TABLE-US-00031 Ac--Ile-Gly-Leu-His-Asp-Pro-Ser-His-Gly-Thr-Leu- Pro-Asn-Gly-Ser (Peg-40KD-maleimide)-NH2

[0183] SEQ ID NO:10

[0184] This is a pegylated version of the mammalian 15-amino acid Reg peptide

TABLE-US-00032 Peg-40KD-maleimide-3-Mpa-Ile-Gly-Leu-His-Asp-Pro- Ser-His-Gly-Thr-Leu-Pro-Asn-Gly-Ser-NH2 (3-Mpa =3- mercaptopropionic acid)

[0185] SEQ ID NO:11

[0186] This is a cyclized versions of the mammalian 15-amino acid Reg peptide to include but are not limited to:

##STR00006##

Cyclic amide bond between side chain of Asp on position 1 and Lys on position 17

[0187] SEQ ID NO: 12

[0188] This is the optimization of the 14-amino acid human Reg peptide by blocking with an n-terminal acetyl group and a c-terminal amide group.

TABLE-US-00033 Ac--Ile-Gly-Leu-His-Asp-Pro-Thr-Gln-Gly-Thr-Glu- Pro-Asn-Gly--NH2

[0189] SEQ ID NO: 13

[0190] This is a pegylated version of the human 14-amino acid Reg peptide

TABLE-US-00034 Ac--Ile-Gly-Leu-His-Asp-Pro-Thr-Gln-Gly-Thr-Glu- Pro-Asn-Gly (Peg-40KD-maleimide)-NH2

[0191] SEQ ID NO:14

[0192] This is a pegylated version of the human 14-amino acid Reg peptide

TABLE-US-00035 Peg-40KD-maleimide-3-Mpa-Ile-Gly-Leu-His-Asp-Pro- Thr-Gln-Gly-Thr-Glu-Pro-Asn-Gly-NH2 (3-Mpa-3- mercaptopropionic acid)

[0193] SEQ ID NO:15

[0194] Cyclized versions of the human 14-amino acid Reg peptide to include but are not limited to:

##STR00007##

Cyclic amide bond between side chain of Asp on position 1 and Lys on position 16

[0195] SEQ ID NO: 16

[0196] This is the optimization of the 7-amino acid human and mammalian Reg peptide by blocking with an n-terminal acetyl group and a c-terminal amide group.

TABLE-US-00036 Ac--Trp-Ile-Gly-Leu-His-Asp-Pro--NH2

[0197] SEQ ID NO: 17

[0198] This is a pegylated version of the human and mammalian 7-amino acid Reg peptide

TABLE-US-00037 Ac-Trp-Ile-Gly-Leu-His-Asp-Pro (Peg-40KD- maleimide)-NH2

[0199] SEQ ID NO:18

[0200] This is a pegylated version of the human and mammalian 7-amino acid Reg peptide

TABLE-US-00038 Peg-40KD-maleimide-3-Mpa-Trp-Ile-Gly-Leu-His-Asp- Pro-NH2 (3-Mpa =3-mercaptopropionic acid)

[0201] SEQ ID NO:19

[0202] Cyclized versions of the human and mammalian 7-amino acid Reg 3 peptide to include but are not limited to:

##STR00008##

Cyclic amide bond between side chain of Asp on position 1 and Lys on position 9

[0203] SEQ ID NO: 20

[0204] This is the optimization of the 8-amino acid human and mammalian Reg peptide by blocking with an n-terminal acetyl group and a c-terminal amide group.

TABLE-US-00039 Ac-Val-Trp-Ile-Gly-Leu-His-Asp-Pro--NH2

[0205] SEQ ID NO: 21

[0206] This is a pegylated version of the human and mammalian 8-amino acid Reg protein

TABLE-US-00040 Ac-Val-Trp-Ile-Gly-Leu-His-Asp-Pro (Peg-40KD- maleimide)-NH2

[0207] SEQ ID NO:22

[0208] This is a pegylated version of the human and mammalian 8-amino acid Reg peptide

TABLE-US-00041 Peg-40KD-maleimide-3-Mpa-Val-Trp-Ile-Gly-Leu-His- Asp-Pro-NH2 (3-Mpa =3-mercaptopropionic acid)

[0209] SEQ ID NO:23

[0210] Cyclized versions of the human and mammalian 8-amino acid Reg peptide to include but are not limited to:

##STR00009##

Cyclic amide bond between side chain of Asp on position 1 and Lys on position 10

[0211] SEQ ID NO: 24

[0212] This is the optimization of the human and mammalian 9-amino acid Reg peptide by blocking with an n-terminal acetyl group and a c-terminal amide group.

TABLE-US-00042 Ac-Asn-Val-Trp-Ile-Gly-Leu-His-Asp-Pro--NH2

[0213] SEQ ID NO: 25

[0214] This is a pegylated version of the human and mammalian 9-amino acid Reg peptide

TABLE-US-00043 Ac-Asn-Val-Trp-Ile-Gly-Leu-His-Asp-Pro (Peg-40KD- maleimide)-NH2

[0215] SEQ ID NO:26

[0216] This is a pegylated version of the human and mammalian 9-amino acid Reg peptide

TABLE-US-00044 Peg-40KD-maleimide-3-Mpa-Asn-Val-Trp-Ile-Gly-Leu- His-Asp-Pro-NH2 (3-Mpa =3-mercaptopropionic acid)

[0217] SEQ ID NO:27

[0218] Cyclized versions of the human and mammalian 9-amino acid Reg peptide to include but are not limited to:

##STR00010##

Cyclic amide bond between side chain of Asp on position 1 and Lys on position 11

[0219] The Reg peptides may be produced through recombinant molecular biology techniques or solid phase synthesis techniques. Recombinant molecular biology techniques include those described in Molecular Cloning: A Laboratory Manual, Green and Sanbrook, 2012. Solid-phase synthesis techniques are described in Merrifield, in J. Am. Chem. Soc., 15:2149-2154 (1963), M. Bodanszky et al., (1976) Peptide Synthesis, John Wiley & Sons, 2d Ed.; Kent and Clark-Lewis in Synthetic Peptides in Biology and Medicine, p. 295-358, eds. Alitalo, K., et al. Science Publishers, (Amsterdam, 1985); as well as other reference works known to those skilled in the art such. A summary of peptide synthesis techniques may be found in J. Stuart and J. D. Young, Solid Phase Peptide Synthelia, Pierce Chemical Company, Rockford, Ill. (1984), which is incorporated herein by reference. The synthesis of peptides by solution methods may also be used, as described in The Proteins, Vol. II, 3d Ed., p. 105-237, Neurath, H. et al., Eds., Academic Press, New York, N.Y. (1976). Appropriate protective groups for use in such syntheses will be found in the above texts, as well as in J. F. W. McOmie, Protective Groups in Organic Chemistry, Plenum Press, New York, N.Y. (1973), which is incorporated herein by reference. In general, these synthetic methods involve the sequential addition of one or more amino acid residues or protected amino acid residues to a growing peptide chain. Normally, either the amino or carboxyl group of the first amino acid residue is protected by a suitable, selectively removable protecting group. A different, selectively removable protecting group is utilized for amino acids containing a reactive side group, such as lysine. Block synthesis techniques may also be applied to both the solid phase and solution methods of peptide synthesis. Rather than sequential addition of single amino acid residues, preformed blocks comprising two or more amino acid residues in sequence are used as either starting subunits or subsequently added units rather than single amino acid residues. Alternative or additional peptide synthesis methods and techniques can be found in Peptide Chemistry: A Practical Textbook: 2nd Edition, Miklos Bodanszky, 1993.

[0220] Reg peptides of the invention may also be synthesized by solid-phase peptide synthesis using procedures similar to those described by Merrifield, 1963, J. Am. Chem. Soc., 85:2149. During synthesis, N--protected amino acids having protected side chains are added stepwise to a growing polypeptide chain linked by its C-terminal and to an insoluble polymeric support, i.e., polystyrene beads. The proteins are synthesized by linking an amino group of an N--deprotected amino acid to an -carboxyl group of an N--protected amino acid that has been activated by reacting it with a reagent such as dicyclohexylcarbodiimide. The attachment of a free amino group to the activated carboxyl leads to peptide bond formation. The most commonly used N--protecting groups include Boc, which is acid labile, and Fmoc, which is base labile. Details of appropriate chemistries, resins, protecting groups, protected amino acids and reagents are well known in the art and so are not discussed in detail herein (See, Atherton et al., 1989, Solid Phase Peptide Synthesis: A Practical Approach, IRL Press, and Bodanszky, 1993, Peptide Chemistry, A Practical Textbook, 2nd Ed., Springer-Verlag).

[0221] Purification of the resulting peptides is accomplished using conventional procedures, such as preparative HPLC using gel permeation, partition and/or ion exchange chromatography. The choice of appropriate matrices and buffers are well known in the art and so are not described in detail herein.

[0222] Inert polymer molecules such as high molecular weight polyethyleneglycol (PEG) can be attached to a peptide of this disclosure or an analog or derivative thereof with or without a multifunctional linker either through site-specific conjugation of the PEG to the N- or C-terminus of the protein or via epsilon-amino groups present on lysine residues. Linear or branched polymer derivatization that results in minimal loss of biological activity can be used. The degree of conjugation can be closely monitored by SDS-PAGE and mass spectrometry to ensure proper conjugation of PEG molecules. Unreacted PEG can be separated from peptide-PEG conjugates by size-exclusion or by ion-exchange chromatography.

[0223] Protocols for blocking peptides with acetyl and amide groups are known in the art and can be found in a number of protein protocol textbooks known in the art. Specific examples include those published in Methods in Molecular Biology, Vol. 35: Peptide Synthesis Protocols, Chapter 8: Site-Specific Chemical Modification Procedures, Edited by M W Pennington and B M Dunn, 1994, as well as U.S. Pat. Nos. 4,708,934, 5,503,989, U.S Patent Application Publication No. US 20060127995. Alternative or additional protein modification procedures can be found in Peptide Chemistry: A Practical Textbook: 2nd Edition, Miklos Bodanszky, 1993. Further, peptide cyclization (Davies, J. Peptide Sci. 9: 471-501 (2003)) and pegylation (Roberts, Advanced Drug Delivery Reviews (2002) 54:459-476 and Veronese, Biomaterials (2001) 22(5):405-17) methods have been reviewed. Protocols for creating maleimide-activated PEG constructs may be found in Schumacher et al., In Situ Maleimide Bridging of Disulfides and a New Approach to Protein PEGylation, Bioconjugate Chem., 2011, 22 (2), pp 132-136, Doherty et al., Site-Specific PEGylation of Engineered Cysteine Analogs of Recombinant Human Granulocyte-Macrophage Colony-Stimulating Factor, Bioconjug Chem. 2005; 16(5): 1291-1298, US Patent Application Publication No. 20090298746 A1, European Patent No. EP 1881850 B1, European Patent No. EP 2178900 B1.

Embodiments

[0224] Embodiments of the invention include but are not limited to the following:

[0225] Embodiment 1A: Regenerative Peptides which comprise the following 7-15-amino acid Reg sequences and 7-15-amino acid optimized Reg Peptides sequences from the human and mammalian Reg peptides that are utilized for both direct production of nerve cells, cardiac myocytes, liver cells and kidney cells via in vivo formation by the usage of the 7-15 optimized and native peptides in times of acute loss of cells from the brain, spinal cord, heat, kidney and liver with such peptides used for the formation of new neurons, including motor neurons, cardiac myocytes, liver hepatocytes and kidney nephrons and include: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27 and may be delivered to a patient in need of new neurons, myocytes, hepatocytes, and nephrons by Routes of delivery of therapies described include, but are not limited to oral, intravenous, intra-arterial, subcutaneous delivery and intrathecal delivery and through the spinal canal for generation central neurons and for spinal cord injuries. Also, therapy may be given by organ specific targeting and may include direct administration liver via the umbilical and hepatic artery, femoral or radial arterial delivery to the heart or directly to the arteries supplying the heart, as is done in cardiac catheterizations, and intrathecal delivery and through the spinal canal for central nervous system as is done for antibiotic delivery for central nervous system infections and for chemotherapy delivery for central lesions and delivered to the renal artery via the femoral artery with a small puncture in the groin. A catheter is inserted through the artery and directed toward the renal artery. Once the catheter is positioned in the artery supplying blood to the kidney, above sequences cam be injected into the renal artery for the formation of new nephrons.

[0226] Embodiment 2A: Regenerative Peptides which comprise the following 7-15-amino acid Reg sequences and 7-15-amino acid optimized Reg Peptides sequences from the human and mammalian Reg peptides that are utilized for production of nerve cells, cardiac myocytes, liver cells and kidney cells via ex vivo formation by the usage of the 7-15 optimized and native peptides generated outside of the body using ex vivo cells. Which include, but are not limited neural stems cells, tissue stem cells, mesenchymal stem cells, totipotent embyronic stem cells, multipotent stem cells, pluripotent stem cells, including embryonic cells, adult somatic stem cells, human adult bone-marrow derived stem cells, umbilical cord stems cells, human amniotic membrane-derived mesenchymal cells, mammalian stem cells, cord cells, ectodermal stem or any other tissue or cell types that can be transformed by human and mammalian 7-15-amino acid Reg peptide sequences into functional neurons, myocytes, hepatocytes and nephrons. from which Reg peptides can transform such tissue into adult functioning cells for usage in times of acute loss of cells from the brain, spinal cord, heart, kidney and liver with such peptides used for the formation of new neurons, including motor neurons, cardiac myocytes, liver hepatocytes and kidney nephrons and include: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27 and then neurons, myocytes, nephrons, hepatocytes may be delivered to a patient in need of new neurons, myocytes, hepatocytes, and nephrons by Routes of delivery of therapies described include, but are not limited to oral, intravenous, intra-arterial, subcutaneous delivery and intrathecal delivery and through the spinal canal for generation central neurons and for spinal cord injuries. Also, therapy may be given by organ specific targeting and may include direct administration liver via the umbilical and hepatic artery, femoral or radial arterial delivery to the heart or directly to the arteries supplying the heart, as is done in cardiac catheterizations, and intrathecal delivery and through the spinal canal for central nervous system as is done for antibiotic delivery for central nervous system infections and for chemotherapy delivery for central lesions and delivered to the renal artery via the femoral artery with a small puncture in the groin. A catheter is inserted through the artery and directed toward the renal artery. Once the catheter is positioned in the artery supplying blood to the kidney, above sequences cam be injected into the renal artery for the formation of new nephrons. This modality of delivery includes, but is not limited to: oral, intravenous, subcutaneously, intra-arterial, intrathecal and into the spinal column and targeted delivery to the brain, spinal column, kidney liver or delivered directly or indirectly to the brain, spinal column, heart and liver and may include targeted therapy to the given organ, which may be delivered orally, intravenously, intra-arterially, intrathecally or directly into the spinal column at the site of injury.

[0227] Embodiment 3A: Peptidomimetics, small molecules and stimulatory antibodies used in vivo to stimulate the 20-amino acid region on the 919-amino acid Reg Receptor/EXTL-3 (SEQ: ID 6) to generate of new neurons, cardiac myocytes, nephrons and hepatocytes. Administration of peptidomimetics includes formulations that stimulate SEQ ID 6 on the EXTL-3 receptor resulting in formation of new cardiac myocytes, nephrons, hepatocytes and neurons. This modality of delivery includes, but is not limited to: oral, intravenous, subcutaneously, intra-arterial, intrathecal and into the spinal column and targeted delivery to the brain, spinal column, kidney liver or delivered directly or indirectly to the brain, spinal column, heart and liver and may include targeted therapy to the given organ, which may be delivered orally, intravenously, intra-arterially, intrathecally or directly into the spinal column at the site of injury.

[0228] Embodiment 4A: Peptidomimetics, small molecules and stimulatory antibodies used ex vivo to stimulate the 20-amino acid region on the 919-amino acid Reg Receptor/EXTL-3 (SEQ: ID 6) to generate of new neurons, cardiac myocytes, nephrons and hepatocytes to transform neural stems cells, tissue stem cells, mesenchymal stem cells, totipotent embyronic stem cells, multipotent stem cells, pluripotent stem cells, including embryonic cells, adult somatic stem cells, human adult bone-marrow derived stem cells, umbilical cord stems cells, human amniotic membrane-derived mesenchymal cells, mammalian stem cells, cord cells, ectodermal stem or any other tissue or cell types into functional neurons, myocytes, hepatocytes and nephrons. The modality of delivery of peptidomimetics, small molecules and antibodies to stimulate (SEQ ID 6) includes, but is not limited to: oral, intravenous, subcutaneously, intra-arterial, intrathecal and into the spinal column and targeted delivery to the brain, spinal column, kidney liver or delivered directly or indirectly to the brain, spinal column, heart and liver and may include targeted therapy to the given organ, which may be delivered orally, intravenously, intra-arterially, intrathecally or directly into the spinal column at the site of injury.

[0229] Embodiment 5A: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27 are used to generate neurons, hepatocytes, myocytes and nephrons in vivo and ex vivo and may be used in conjunction with other medications used to treat heart disease, liver disease, neurological diseases and renal diseases.

[0230] Embodiment 6A: Peptidomimetics, small molecules and stimulatory antibodies used ex vivo to stimulate the 20-amino acid region on the 919-amino acid Reg Receptor/EXTL-3 (SEQ: ID 6) to generate of new neurons, cardiac myocytes, nephrons and hepatocytes either in vivo or ex-vivo and may be used in conjunction with other medications used to treat heart disease, liver disease, neurological diseases and renal diseases.

[0231] Embodiment 1B: An isolated or modified peptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27.

[0232] Embodiment 2B: A pharmaceutical formulation comprising the peptide of embodiment 1B.

[0233] Embodiment 3B: The pharmaceutical formulation of embodiment 2B, wherein the formulation is a soluble liposome or nanoparticle preparation.

[0234] Embodiment 4B: The pharmaceutical formulation of embodiment 2B, wherein the formulation comprises a targeting agent for targeted administration to heart, brain, spinal column, liver, or kidney.

[0235] Embodiment 5B: A method of treating a subject in need of one more differentiated cells or tissue types, comprising administering to the subject a peptide having Reg Receptor binding activity, wherein the amount of peptide is effective for forming differentiated cells or tissues in the subject in vivo.

[0236] Embodiment 6B: The method of embodiment 5B, wherein the peptide having Reg Receptor binding activity has an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27.

[0237] Embodiment 7B: The method of embodiment 5B, wherein the one or more cell or tissue types are heart, liver, brain, spinal column, or kidney cells or tissues.

[0238] Embodiment 8B: The method of embodiment 5B, wherein the peptide is administered directly to the heart, liver, brain, spinal cord or kidney of a subject.

[0239] Embodiment 9B: The method of embodiment 5B, wherein the peptide is administered by way of intravenous, subcutaneous, intra-arterial, or intrathecal delivery.

[0240] Embodiment 10B: The method of embodiment 5B, wherein the subject has a condition selected from the group consisting of heart disease, myocardial infarction, stroke, acute brain injury, neurodegenerative disease, spinal cord injury, peripheral neuropathy, acute and chronic kidney disease and liver disease.

[0241] Embodiment 11B: The method of transforming progenitor cells to differentiated tissue cells, comprising:

[0242] culturing a plurality of progenitor cells ex vivo; and

[0243] contacting the progenitor cells with a peptide having Reg Receptor binding activity,

[0244] wherein the amount of peptide is effective for transforming progenitor cells to differentiated cells or tissues in culture.

[0245] Embodiment 12B: The method of embodiment 11B, wherein the peptide having Reg Receptor binding activity has an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27.

[0246] Embodiment 13B: The method of embodiment 11B, wherein the progenitor cells are selected from the group consisting of neural stems cells, tissue stem cells, mesenchymal stem cells, totipotent embyronic stem cells, multipotent stem cells, pluripotent stem cells, including embryonic cells, adult somatic stem cells, human adult bone-marrow derived stem cells, umbilical cord stems cells, human amniotic membrane-derived mesenchymal cells, mammalian stem cells, cord cells, and ectodermal stem cells.

[0247] Embodiment 14B: The method of embodiment 11B, wherein the differentiated tissue cells are selected from the group consisting of brain, spinal cord, heart, kidney and liver tissue cells.

[0248] Embodiment 15B: A method of treating a subject in need of one more differentiated cells or tissues, the method comprising:

[0249] culturing a plurality of progenitor cells ex vivo;

[0250] contacting the progenitor cells with a peptide having Reg Receptor binding activity, wherein the amount of peptide is effective for transforming the progenitor cells to differentiated cells or tissues in culture; and

[0251] administering the one or more differentiated cells or tissues to the subject.

[0252] Embodiment 16B. The method of embodiment 15B, wherein the peptide having Reg Receptor binding activity has an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27.

[0253] Embodiment 17B. The method of embodiment 15B, wherein the progenitor cells are selected from the group consisting of neural stems cells, tissue stem cells, mesenchymal stem cells, totipotent embyronic stem cells, multipotent stem cells, pluripotent stem cells, including embryonic cells, adult somatic stem cells, human adult bone-marrow derived stem cells, umbilical cord stems cells, human amniotic membrane-derived mesenchymal cells, mammalian stem cells, cord cells, and ectodermal stem cells.

[0254] Embodiment 18B. The method of embodiment 15B, wherein the differentiated cells or tissues are selected from the group consisting of brain, spinal cord, heart, kidney and liver cells or tissues.

[0255] Embodiment 19B. The method of embodiment 15B, wherein the differentiated cells or tissues are administered directly to the heart, liver, brain, spinal cord or kidney of a subject.

[0256] Embodiment 20B. The method of embodiment 15B, wherein the subject has a condition selected from the group consisting of heart disease, myocardial infarction, stroke, acute brain injury, neurodegenerative disease, spinal cord injury, peripheral neuropathy, acute and chronic kidney disease and liver disease.

EXAMPLES

[0257] The following examples serve to further illustrate the invention and should not be used to limit the invention.

Example 1

[0258] A patient presents with an acute myocardial infarction as evidenced by EKG and cardiac isoenzymes. The patient undergoes cardiac catheterization and prior to injection of contrast is given 60 mg of SEQ ID NO: 8, which will be delivered into the myocardium and bind to the upregulated EXTL-3/Reg receptor in the location of the acute injury.

Example 2

[0259] A patient suffers a neck injury from being thrown from a horse with the spinal cord severed at Cervical Disk 7 and has immediate paralysis. Sixty mg of SEQ ID NO: 8, is delivered directly via injection to the severed cord under fluoroscopy and delivered every day for 14 days.

Example 3

[0260] A patient undergoes resection of the liver for removal of a large metastatic lesion from colon cancer. An oral hepatic targeted nanoparticle encapsulation of SEQ ID NO: 8 in a dosage of 60 mg and is given to the patient twice daily.

Example 4

[0261] A patient with marked renal insufficiency with a creatinine of 4.0 is given 60 mg of SEQ ID NO: 8 delivered renal artery via the femoral artery with a small puncture in the groin. A catheter is inserted through the artery and directed toward the renal artery. Once the catheter is positioned in the artery supplying blood to the kidney, SEQ ID NO: 8 is injected into the renal artery. This is repeated monthly.

[0262] The present invention has been described with reference to particular embodiments having various features. In light of the disclosure provided above, it will be apparent to those skilled in the art that various modifications and variations can be made in the practice of the present invention without departing from the scope or spirit of the invention. One skilled in the art will recognize that the disclosed features may be used singularly, in any combination, or omitted based on the requirements and specifications of a given application or design. When an embodiment refers to comprising certain features, it is to be understood that the embodiments can alternatively consist of or consist essentially of any one or more of the features. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention.

[0263] It is noted in particular that where a range of values is provided in this specification, each value between the upper and lower limits of that range is also specifically disclosed. The upper and lower limits of these smaller ranges may independently be included or excluded in the range as well. The singular forms a, an, and the include plural referents unless the context clearly dictates otherwise. It is intended that the specification and examples be considered as exemplary in nature and that variations that do not depart from the essence of the invention fall within the scope of the invention. Further, all of the references cited in this disclosure including published patents, published patent applications, books, and journal articles are each individually incorporated by reference herein in their entireties and as such are intended to provide an efficient way of supplementing the enabling disclosure of this invention as well as provide background detailing the level of ordinary skill in the art.