USOO8722625B2

(12) United States Patent (10) Patent No.: US 8,722,625 B2 Culiat (45) Date of Patent: May 13, 2014

(54) TREATMENT OF CARDIOVASCULAR Zhang, X. et al., “Nell-1 induces acrania-like cranioskeletal deformi DSORDERS USING THE CELL ties during mouse embryonic development'. Lab Invest 86(7): 633 644 (2006). DIFFERENTATION SIGNALNG PROTEIN Zhang, X. et al., “Overexpression of Nell-1, a craniosynostosis NELL1 associated gene, induces apoptosis in osteoblasts during craniofacial development”, J Bone Miner Res. 18(12): 2126-2134 (2003). (75) Inventor: Cymbeline T. Culiat, Oak Ridge, TN Ting, K. et al., “Human NELL-1 expressed in unilateral coronal (US) synostosis”, J Bone Miner Res. 14(1): 80-90 (1999). Aghaloo, T. et al., “Nell-1-induced bone regeneration in calvarial defects”, Am J Pathol. 169(3):903-915 (2006). (73) Assignee. UT-Battelle, LLC, Oak Ridge, TN (US) Zhang, X. et al., “Craniosynostosis in transgenic mice overexpress ing Nell-1 "...J Clin Invest. 110(6): 861-870 (2002). (*) Notice: Subject to any disclaimer, the term of this Cowan, C.M. et al., “Nell-1 induced bone formation within the dis patent is extended or adjusted under 35 tracted intermaxillary Suture', Bone 38(1): 48-58 (2006). U.S.C. 154(b) by 877 days. Desai, J. et al., “Nell-deficient mice have reduced expression of extracellular matrix proteins causing cranial and vertebral defects'. (21) Appl. No.: 12/284,667 Hum Mol Genet. 15(8): 1329-1341 (2006). Maeda, K. et al., “Brain specific human genes, NELL1 and NELL2, (22) Filed: Sep. 24, 2008 are predominantly expressed in neuroblastoma and other embryonal neuroepithelial tumors”, Neurol Med Chir (Tokyo) 41(12): 582-588 (65) Prior Publication Data (2001). Luce, M. J. et al., “The neuronal EGF-related genes NELL1 and US 2009/OO87415A1 Apr. 2, 2009 NELL2 are expressed in hemopoietic cells and developmentally regulated in the B lineage'. Gene 231(1-2): 121-126 (1999). Kuroda, S. et al., “Biochemical characterization and expression Related U.S. Application Data analysis of neural thrombospondin-1-like roteins Nell and NELL2”. (60) Provisional application No. 60/995,854, filed on Sep. Biochem Biophy's Res Commun. 265(1): 79-86 (1999). 28, 2007, provisional application No. 61/079,446, Watanabe, T.K. et al., “Cloning and characterization of two novel human cDNAs (NELL1 and NELL2) encoding proteins with six filed on Jul. 10, 2008. EGF-like repeats”. Genomics 38:273-276 (1996). Shen, Y. et al., “Knock Down of NELL2 in Wilms' Tumor Cell Line', (51) Int. Cl. Journal of the William Jarvie Society, vol. 49, p. 4.1(2006), Abstract. A6K38/00 (2006.01) Santini, M. P. et al., “Signalling pathways in cardiac regeneration'. A6 IK38/02 (2006.01) Novartis Found Symp. 274: 228-238 (2006). A6 IK 4.8/00 (2006.01) Rubart, M. et al., “Cell-based approaches for cardiac repair'. Ann NY (52) U.S. Cl. AcadSci, 1080: 34-38 (2006). USPC ...... 514/16.4: 514/44 R: 514/1.1 Ott, H. C. et al., “From cardiac repair to cardiac regeneration—ready to translate?”, Expert Opin Biol Ther: 6(9): 867-878 (2006). (58) Field of Classification Search Rosenthal, N. et al., “Growth factor enhancement of cardiac regen USPC ...... 514/16.4, 1.1, 44 R eration”. Cell Transplant I5(Suppl I): S41-S45(2006). See application file for complete search history. Kuroda, S. et al., “Involvement of epidermal growth factor-like domain of NELL proteins in the novel protein-protein interaction (56) References Cited with protein kinase C.”. Biochem Biophy's Res Commun. 265(3): 752-757 (1999). U.S. PATENT DOCUMENTS Haider, H. K.H., “Bone marrow cells for cardiac regeneration and repair: current status and issues'. Expert Rev Cardiovasc Ther: 4(4): 7,052,856 B2 5/2006 Ting 7,910,542 B2 3/2011 Culiat 557-568 (2006). 2004/0186423 A1 9, 2004 Cafferata Orlic, D. et al., “Transplanted Adult Bone Marrow Cells Repair 2006.0025367 A1 2/2006 Simari Myocardial Infarcts in Mice'. Ann N Y Acad Sci 938(1):221 2006.0053503 A1 3, 2006 Culiat et al. 230(2001). 2006/011 1313 A1 5/2006 Ting et al. Lu, S.S. et al., “The osteoinductive properties of Nell-1 in a rat spinal 2006/0228392 A1 10/2006 Ting fusion model”, The Spine Journal 7(1): 50-60 (2007). 2006, O292670 A1 12/2006 Ting et al. The Reporter, No. 78 (Jun. 2006), published by Oak Ridge National 2007/0134291 A1 6/2007 Ting et al. Laboratory, accessible on line at http://www.ornil.gov/info/reporter? 2009, O142312 A1 6, 2009 Culiat no78 une06 dw.htm. 2011/0236325 A1 9, 2011 Culiat et al. (Continued) FOREIGN PATENT DOCUMENTS Primary Examiner — Taeyoon Kim WO 02/100.426 A1 12/2002 (74) Attorney, Agent, or Firm — Edna I. Gergel WO 2004/072100 A1 8, 2004 WO 2009/042859 A1 4/2009 WO 2011091244 T 2011 (57) ABSTRACT It has been identified in accordance with the present invention OTHER PUBLICATIONS that Nell 1 is essential for normal cardiovascular development Liu, L. et al., “Characterizing the Role of the Nell Gene in Cardio by promoting properformation of the heart and blood vessels. vascular Development'. Office of Workforce Development for Teach The present invention therefore provides therapeutic methods ers and Scientist, (2007), Abstract. for treating cardiovascular disorders by employing a Nell Tsutsumi, S. et al., “The Novel Gene Encoding a Putative protein or nucleic acid molecule. Transmembrane Protein is Mutated in Gnathodiaphyseal Dysplasia (GDD), Am. J. Hum. Genet 74: 1255-1261 (2004). 14 Claims, 6 Drawing Sheets US 8,722.625 B2 Page 2

(56) References Cited Lee, M. etal. “Effect of Nell-1 Deliveryon Chondrocyte Proliferation and Cartilaginous Exracellular Matrix Deposition' Tissue Eng. Part OTHER PUBLICATIONS A (2010) pp. 1791-1800, vol. 16, No. 5. Abstract entitled “Characterizing the Role of the Nell Gene in Liu, L. et al., “Characterizing the Role of the Nell 1 Gene in Cardio Cardiovascular Development” from the 2007 American Association vascular Development” Oak Ridge Science Semester Poster Presen for the Advancement of Science (AAAS) Annual Meeting entitled tation, ORNL, Oak Ridge, TN. Presentation on Aug. 11, 2006 "Science and Technology for Sustainable Well-Being” that occurred (Poster). on Feb. 15-19, 2007, pp. A125. Rinchik, E.M. et al., “Functional Annotation of Mammalian Aghaloo.T. et al., “A Study of the Role of Nell-1 Gene Modified Goat Genomic DNA Sequence by Chemical Mutagenesis: A Fine-Struc Bone Marrow Stromal Cells in Promoting New Bone Formation” ture Genetic Mutation Map of a 1-to 2-cMSegment of Mouse Chro Molecular Therapy (2007) pp. 1872-1880, vol. 15, No. 10. mosome 7 Corresponding to Human Chromosome 1 lp14p15” PNAS Bareggi. R. et al., “Protein Kinase C (PKC) Isoenzymes Exhibit (2002) pp. 844-849, vol. 99, No. 2. Specific Expression in the Vertebral Column of Human Fetuses” Chen, W. et al., “Nfatc2 is a Primary Response Gene of Nell-1 Journal of Biological Reseasrch (1995) pp. 83-91, vol. LXXI. Regulating Chondrogenesis in ATDC5 Cells' Journal of Bone and Cowan, et al., “Synergistic Effects of Nell-1 and BMP-2 on the Mineral Research (2011) pp. 1230-1241, vol. 26, No. 6. Osteogenic Differentiation of Myoblasts,” Journal of Bone Mineral Liu et al., “Characterizing the Role of the Nell Gene in Cardiovas Reseasrch (2007) pp. 918-930, vol. 22. cular Development.” U.S. Department of Energy Journal of Under Culiat, C.T. et al., “Nell 1: A Candidate Gene for ENU-Induced graduate Research, 2007, pp. 63-70. Recessive Lethal Mutations at the 17R6 Locus and Potential Mouse Franke et al., “Systematic Association Mapping Identifies NELL1 as Models for Human Neonatal Unilateral . . . .” International Mamma a Novel IBD Disease Gene.” PLoS One, 2007, pp. 1-13, vol. 8(e691) lian Genome Society, 15th International Mouse Genome Conference and Supplemental documents. (2001). Culiat et al., “Nell 1: A Candidate Gene for ENU-Induced Recessive Desai, J. et al., “Nell1. A Gene Coding for a Novel PKC-Binding Lethal Mutations at the 17R6 Locus and Potential Mouse Models for Protein Isa Candidate for Late-Gestation Recessive Lethal Mutations Human Neonatal Unilateral Coronal Synostosis (UCS).” Jan. 27-31. at the I7R6 Locus' 16th International Mouse Genome Conference, 2002 (Abstract). San Antonio, TX. Presentation on Nov. 17-21, 2002 (Abstract). Desai et al., “Nelll-Deficient Mice Have Reduced Expression of Desai, J. et al., “Characterization of Mouse Nell 1: A Gene Coding for Extracellular Matrix Proteins Causing Cranial and Vertebral a Novel PKC-Binding Protein' Women in Science Meeting, ORNL. Defects.” 20th International Mouse Genome Conference, Charles Oak Ridge, TN. Presentation on May 1, 2006 (Abstract). ton, SC, Presentation on Nov. 14, 2006 (Abstract). U.S. Patent May 13, 2014 Sheet 1 of 6 US 8,722,625 B2

U.S. Patent May 13, 2014 Sheet 2 of 6 US 8,722,625 B2

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Figure 4A-4C

U.S. Patent May 13, 2014 Sheet 5 of 6 US 8,722,625 B2

U.S. Patent May 13, 2014 Sheet 6 of 6 US 8,722,625 B2

igaret A-7. US 8,722,625 B2 1. 2 TREATMENT OF CARDOVASCULAR order is prevented or is delayed; or alternatively, its progres DSORDERS USING THE CELL sion is slowed down, the extent of the injury is reduced, and DIFFERENTATION SIGNALING PROTEIN the recovery is accelerated. NELL1 In one embodiment, the present invention provides a method of treating a cardiovascular disorder by administering CROSS REFERENCE TO RELATED a Nell1 protein or functional derivatives thereof to a subject in APPLICATIONS need of the treatment. Nell1 proteins suitable for use in the present method include wild type Nell 1 proteins from any This application asserts the priority of U.S. provisional mammalian species, as well as functional derivatives thereof. application Ser. No. 60/995,854 filed Sep. 28, 2007, and U.S. 10 Nell proteins, as well as functional derivatives thereof, can provisional application Ser. No. 61/079,446, filed Jul. 10, be recombinantly produced or purified from a mammalian 2008, the contents of which are incorporated herein by refer body or tissue. In another embodiment, the present invention provides a CCC. method of treating a cardiovascular disorder by administering STATEMENT REGARDING FEDERALLY 15 a nucleic acid molecule encoding a Nelll protein to a subject SPONSORED RESEARCH in need of the treatment. The nucleic acid molecule can be provided in an expression vector, including viral vectors and This invention was made with Government support under non-viral vectors, suitable for effecting the expression of the Contract No. DE-AC05-00OR22725 between the United Nell protein in the targeted tissue or cells. States Department of Energy and UT-Battelle, LLC. The In accordance with the present invention, a Nell protein, Government has certain rights in this invention. functional derivatives thereof, or nucleic acid molecule can be combined with an appropriate pharmaceutically accept FIELD OF THE INVENTION able carrier for administration. Administration can be con ducted in any practical and convenient manner, including by The present invention relates in general to therapeutic 25 ingestion, injection or implantation, for example. methods for treating cardiovascular disorders. More specifi In a specific embodiment, a Nell 1 protein, functional cally, the present invention relates to therapeutic treatments of derivatives thereof, or Nell-encoding nucleic acid molecule cardiovascular disorders by employing the cell differentiation is used in combination with cell-based therapy for the repair signaling protein Nell1, as well as functional derivatives and regeneration of damaged cardiac muscles and blood ves thereof. 30 sels. For example, a Nell1 protein, functional derivatives thereof, or Nell-encoding nucleic acid molecule can be BACKGROUND OF THE INVENTION administered together with cardiomyocytes for repopulation of cells in the injured site. Alternatively, a Nell 1 protein, Despite many available methods of treatment, cardiovas functional derivatives thereof, or Nell-encoding nucleic acid cular disease is one the major causes of death each year in the 35 molecule can be administered together with stem cells iso U.S. Thus, there is still a need for more effective agents to lated from adult bone marrow for regeneration of damaged prevent and treat cardiac tissue injury, especially cardiac tis cardiac muscles and blood vessels. Sue injury resulting from ischemia/reperfusion. The Nell1 gene codes for a secreted trimeric protein that BRIEF DESCRIPTION OF THE DRAWINGS stimulates bone and cartilage precursor cells (osteoblasts and 40 chondrocytes) to differentiate into mature bone and cartilage FIGS. 1A-1B show the cardiovascular defects in mice tissue (Zhang et al., 2002: Desai et al., 2006). Nell-1 is a without Nell1 function (Nell1' mutation). Homozygote protein kinase C (PKC) 3-binding protein. The Nell1 cDNA fetuses at E18 days of gestation (Top) show decreased blood and amino acid sequences from a variety of mammalian spe circulation (arrows) and unexpanded lungs compared to het cies, including human, rat and mouse, have been reported. 45 erozygotes (bottom) and wild type animals (not shown). Overexpression of Nell 1 has been reported to cause pre Fetuses were unable to breathe after birth or after caesarean mature fusion of the growing cranial bone fronts, resulting in recovery. craniosynostosis in humans and transgenic mice carrying a FIGS. 2A-2B (in color) demonstrate that Nell1 protein is rat Nell 1 transgene. A Nell knock-out mouse was also shown required for blood vessel formation and establishment of a to exhibit several bone- and cartilage-related defects. There 50 complex vascular network. The loss of Nell1 function has been no characterization, however, of the impact of Nell1. resulted in a significant reduction of the number of blood if any, on cardiovascular development. vessels and extensive branching of the vasculature in Nell1 mutants (FIG. 2B) compared to (FIG. 2A) normal fetuses. SUMMARY OF THE INVENTION The decrease in blood vessel formation was observed 55 throughout the fetal body. It has been identified in accordance with the present inven FIGS. 3A-3B illustrate severe cardiovascular defects and tion that Nell1 is essential for normal cardiovascular devel neonatal lethality associated with the complete loss of Nell1 opment by promoting properformation of the heart and blood function in the mouse. The cardiovascular defects resulting vessels. The present invention therefore provides therapeutic from the complete loss of Nell1 function in Nell1' was methods for treating cardiovascular disorders by employing a 60 associated with decreased blood circulation into the heart Nell protein, functional derivatives thereof or nucleic acid muscles and predominance of increased numbers of imma molecule. ture cardiomyocytes. The dense packing of Smaller cardi Cardiovascular disorders or conditions contemplated by omyocytes in the mutant (FIG. 3B) was very apparent in the the present invention are diseases that involve the heart or denser/darker staining with haematoxylin and eosin, com blood vessels (arteries and veins), including in particular 65 pared to the wild type (FIG. 3A). These cardiovascular myocardial infarction (or “MI). By treating a cardiovascular defects are evident in E18.5 day fetuses recovered by caesar disorder or condition with the present methodology, the dis Ca. US 8,722,625 B2 3 4 FIGS. 4A-4C illustrate a strategy for treatment of heart cellular matrix, main structural components of heart muscle, muscle injury after MI in rodents using direct injection of and proper functioning of genes for heart metabolism and stem cells or drugs to the border Zone. contraction. FIG. 5 provides an alignment of the human (SEQID NO: Accordingly, the present invention provides methods for 2) and murine (SEQID NO: 4) Nell1 proteins. The functional treating cardiovascular conditions or disorders by employing domains of the human Nell1 protein are found in the essen a Nell1 protein, functional derivatives thereof, or Nell1 tially same regions as those identified in the murine Nell1 nucleic acid molecules. protein. The term “condition, as used herein, refers to a disease or FIGS. 6A-6D. NELL 1 Protein Treatment of Damaged ailment. The term “disorder, as used herein, refers to a con Heart Tissue in Mice with Myocardial Infarction (MI). (6A) 10 dition in which there is a disturbance of normal functioning. Untreated mouse hearts with MI due to the loss of blood The term “cardiovascular as used herein, refers to the heart and/or blood vessels. Supply from a ligation of the left anterior descending coronary Accordingly, the term “cardiovascular condition” or “car artery had a readily visible creamy white looking damaged diovascular disorder, as used herein, refers to diseases or tissue on the surface of the heart (17 days post MI-induction). 15 aliments that involve the heart, blood vessels (e.g., arteries All Nell1 protein treated hearts had lesseramount of damaged and veins). Generally, Such diseases or aliments result in an tissue as illustrated in FIG.7B to 7D. The damaged sections abnormality in the cardiac structure, cardiac muscle, and/or (outlined by blue lines) in controls were typically at least 50% cardiac function. The cardiovascular condition or disorder while the treated hearts had barely visible (6B) to as high as can be acute or chronic. 30% infarcts observed (6D). The term “cardiovascular condition' or “cardiovascular FIGS. 7A-7F. Reduction of Damaged Heart Tissue disorder can be used interchangeably throughout the speci Incurred From Myocardial Infarction in Nell1-treated Hearts. fication. Examples of a cardiovascular disease include aneu Longitudinal sections of normal hearts stained with either rysms, angina, atherosclerosis, cardiomyopathy, congestive haematoxylin and eosin (7A) or masson-trichome (7B) show heart failure, coronary artery disease, and myocardial infarc intense staining of the heart muscle and reveals a very com 25 tion, among others. Further examples of cardiovascular con pact organization of the muscle tissues in the right and left ditions include, for instances, blood vessels that have been ventricles (rv and lv respectively), and the interventricular revascularized. Such patients generally have a stent placed in septum separating the two ventricular chambers (IVS). After a blood vessel (e.g., artery, etc.) a myocardial infarction event, the muscle tissues died due to A cardiovascular condition especially suitable for being a lack of oxygenated blood Supply and the deterioration of the 30 treated with the method of the present invention is myocardial infarction (or “MI). MI, also known as a “heart attack’ or muscle architecture was evident by the large gaps in the tissue “heart failure', is a medical condition that occurs when the and the decreased intensity of the staining (7C; 17 days post blood supply to a part of the heart is interrupted. MI is often MI). Hearts with MI that were treated with the Nell1 protein caused by partial or complete occlusion of one or more of the had lesser damage in the heart tissue from the Surface to just 35 coronary arteries, usually due to rupture of an atherosclerotic before the middle of the heart (7D and 7E). In some hearts the plaque. The occlusion of the coronary artery results in cardiac improvement was manifested even deeper into the middle ischemia. The resulting ischemia or oxygen shortage causes section of the heart (7F). damage and potential death of heart tissue. The term “treating” or “treatment a disease, as used DETAILED DESCRIPTION OF THE INVENTION 40 herein, refers to preventing or delaying the onset of the dis ease, or when the disease does occur, retard the progression or The present invention relates to therapeutic methods for ameliorate the symptoms of the disease, reduce the extent of treating cardiovascular conditions or disorders by employing tissue injury or damage, or promote recovery of the injured the cell differentiation signaling protein Nell1, as well as tissue and regeneration of new functional tissue or cells. functional derivatives thereof. 45 The Subject Suitable for receiving a treatment in accor The present invention is based on the Surprising discovery dance with the present invention includes any mammalian by the inventor that the Nell1 protein is essential for normal subject in need of the treatment. In one embodiment, the cardiovascular development by promoting proper formation Subject is a human Subject. A Subject in need of treatment of the heart and blood vessels. The inventor discovered that includes both subjects who have been determined to have a loss of Nell resulted in several tissue and organ changes 50 higher risk of developing a cardiovascular disease, and Sub typical of cardiac muscle injury, including heart enlargement, jects who have a cardiovascular disease, as well as Subjects tissue hypertrophy, decreased blood vessel formation and who have recently experienced a cardiovascular event such as blood circulation. The inventor observed that microscopic MI. examination of Nell 1-deficient hearts showed heart enlarge In one embodiment, the method of the present invention is ment and cardiomyopathy, conditions associated with events 55 achieved by administration of a Nell1 protein to a subject in of myocardial infarction (“MI). Although the basic vascula need of the treatment. ture system was observed during embryo development even “A Nell1 protein’ as used herein, includes wild type (i.e., without a functional Nell1, the amount and complexity naturally occurring) Nell 1 proteins of any mammalian origin, (branched network) was significantly reduced in Nell1 such as human, murine, rat and the like. Preferred Nell1 mutants. The therapeutic application of Nell1 for heart 60 proteins for use in the present invention include human Nell1 muscle regeneration is therefore dependent not only on the protein (SEQID NO: 2), murine Nell1 protein (SEQID NO: protein’s abilities to signal muscle cell maturation, but also in 4), and rat Nell1 protein (SEQID NO: 6). its capabilities to Support the construction of the highly “A Nell protein’ as used herein, also includes functional branched vasculature that is required to Sustain new heart derivatives of a wild type Nell 1 protein. A “functional deriva muscle formation and maintenance of heart function. The 65 tive' refers to a modified Nell protein which has one or more inventor also observed that microarray experiments indicate amino acid substitutions, deletions or insertions as compared that Nell 1 is essential for the proper formation of heart extra to a wild type Nell1 protein, and which retains substantially US 8,722,625 B2 5 6 the activity of a wild type Nell1 protein. By “substantially” is protein can be fused with GAL4 activating domain and the meant at least 50%, at least 75%, or even at least 85% of the regulatory domain of PKC can be fused with the GAL4 DNA activity of a wild type Nell1 protein. According to the present binding domain. The activity of beta-galactosidase in yeast invention, in order for the functional derivative to substan cells can be detected. tially retain the activity or function of a wild type Nell1 In addition, one can also test the ability of a Nell1 deriva protein, the functional Nell1 derivative shares a sequence tive to stimulate differentiation of precursor cells, which are identity with the wild type Nell1 protein of at least 75%, at in the cardiomyocyte lineage, towards mature cardiomyo least 85%, at least 95% or even 99%. cytes. Maturity of cardiomyocytes can be assessed cellularly The structure of Nell1 proteins has been characterized (see, (histology) and molecularly (expression of cardiac-specific e.g., Kuroda et al., 1999a; Kuroda et al., 1999b, Desai et al., 10 proteins or extracellular matrix materials). Still further, a 2006). For example, the murine Nell1 protein (SEQ ID NO: Nell derivative can be tested for its ability to drive osteoblast 4) is a protein of 810 amino acids, having a secretion signal precursors to mature bone cells, by detecting expression of peptide (amino acids #1 to 16), an N-terminal TSP-like mod late molecular bone markers or mineralization (i.e., calcium ule (amino acids #29 to 213), a Laminin G region (amino deposits). By comparing the activity of a Nell 1 derivative acids #86 to 210), von Willebrand factor C domains (amino 15 with that of a wild type Nell1 protein in one or more of the acids #273 to 331 and 699 to 749), and a Ca"-binding EGF assays such as those described above, one should be able to like domains (amino acids #549 to 586). determine whether such derivative retains substantially the The secretion signal peptide domain of Nell1 protein is an activity or function of a wild type Nell 1 protein. amino acid sequence in the protein that is generally involved A Nell protein or functional derivative thereof may be in transport of the protein to cell organelles where it is pro prepared by methods that are well known in the art. One such cessed for secretion outside the cell. The N-terminal TSP-like method includes isolating or synthesizing DNA encoding the module is generally associated with heparin binding. Von Nell protein, and producing the recombinant protein by Willebrand factor C domains are generally involved with expressing the DNA, optionally in a recombinant vector, in a oligomerization of Nell 1. Laminin G domains of Nell1 pro Suitable host cell, including bacterial, yeast, insect or mam tein are generally involved in adherence of Nell 1 protein to 25 malian cells. Such suitable methods for synthesizing DNA specific cell types or other extracellular matrix proteins. The are, for example, described by Caruthers et al. 1985. Science interaction of Such domains with their counterparts is gener 230:281-285 and DNA Structure, Part A: Synthesis and ally associated with, for example, processes such as differen Physical Analysis of DNA, Lilley, D.M.J. and Dahlberg, J. E. tiation, adhesion, cell signaling or mediating specific cell-cell (Eds.), Methods Enzymol., 211, Academic Press, Inc., New interactions in order to promote cell proliferation and differ 30 York (1992). entiation. The Ca"-binding EGF-like domains of Nell1 binds Examples of suitable Nell 1 nucleic acid sequences include protein kinase C beta, which is typically involved in cell SEQ ID NOs: 1, 3, and 5. A Nell1 protein or functional signaling pathways in growth and differentiation. derivative may also be made synthetically, i.e. from indi The amino acid sequence of Nell1 protein is very highly vidual amino acids, or semisynthetically, i.e. from oligopep conserved, especially across mammalian species. For 35 tide units or a combination of oligopeptide units and indi example, the murine Nell1 protein shares about 93% vidual amino acids. Suitable methods for synthesizing sequence identity with the human Nell1 protein (SEQID NO: proteins are described by Stuart and Young in “Solid Phase 2), which, in turn, shares about 90% sequence identity with Peptide Synthesis.” Second Edition, Pierce Chemical Com the rat Nell1 protein (SEQID NO: 4). Those skilled in the art pany (1984), Solid Phase Peptide Synthesis, Methods Enzy can use any of the well-known molecular cloning techniques 40 mol., 289, Academic Press, Inc., New York (1997). Examples to generate Nell derivatives having one or more amino acid of suitable Nell1 amino acid sequences include SEQID NOs: Substitutions, deletions or insertions, taking into consider 2, 4, 6, and derivatives thereof. ation the functional domains (e.g., secretion signal peptide In another embodiment, the method of the present inven sequence, N-terminal TSP-like module, Laminin G region, tion is achieved by administration of a nucleic acid molecule von Willebrand factor C domain) of Nell1. See, for example, 45 encoding a Nell 1 protein or functional derivative to a subject Current Protocols in Molecular Cloning (Ausubel et al., John in need of the treatment. Wiley & Sons, New York). Suitable nucleic acid molecules for use in the present The minimum length of a Nell 1 functional derivative is invention include nucleic acid molecules having a nucleotide typically at least about 10 amino acids residues in length, sequence as set forth in SEQ ID NO: 1 (encoding the wild more typically at least about 20 amino acid residues in length, 50 type human Nell protein), SEQID NO:3 (encoding the wild even more typically at least about 30 amino acid residues in type murine Nell 1 protein), and SEQID NO: 5 (encoding the length, and still more typically at least about 40 amino acid rat wild type Nell protein), as well as degenerate sequences residues in length. As stated above, wild type Nell 1 protein is thereof. As used herein, the term “degenerate sequence' approximately about 810 amino acid residues in length. A refers to a sequence formed by replacing one or more codons Nell functional derivative can be at most about 810 amino 55 in the nucleotide sequence encoding wild type Nell protein acid residues in length. For example, a Nell functional with degenerate codes which encode the same amino acid derivative can be at most at most about 820, 805, 800, 790, residue (e.g., GAU and GAC triplets each encode the amino 780, 750, 600, 650 600, 550, etc. amino acid residues in acid residue Asp). length. In some embodiments, nucleic acid molecules for use in Once a Nell1 protein derivative is made, such protein can 60 the methods of the present invention are provided in an be tested to determine whether such derivative retains sub expression vector. Expression vectors for use in the present stantially the activity or function of a wild type Nell1 protein. methods include any appropriate gene therapy vectors, such For example, the ability of a Nell1 derivative to bind PKC beta as nonviral (e.g., plasmid vectors), retroviral, adenoviral, her can be tested. Suitable assays for assessing the binding of pes simplex viral, adeno-associated viral, polio viruses and Nell1 to PKC beta is described in e.g., Kuroda et al. (1999b). 65 vaccinia vectors. Examples of retroviral vectors include, but For example, protein-protein interaction can be analyzed by are not limited to, Moloney murine leukemia virus (Mo using the yeast two-hybrid system. Briefly, a modified Nell1 MuDV), Harvey murine sarcoma virus (HaMuSV), murine US 8,722,625 B2 7 8 mammary tumor virus (MuMTV), and Rous Sarcoma Virus vascular defects, thus minimizing heart muscle injury or (RSV)-derived recombinant vectors. A Nell 1-coding nucle stimulating tissue repair processes in the heart after MI. otide sequence can be placed in an operable linkage to a Other delivery systems and methods include, but are not promoter in the expression vector, wherein the promoter limited to: a) catheter-based devices that permit site specific directs the expression of the Nell1 protein in the targeted drug delivery to the heart muscle, b) via a thorascopic opening tissue or cells, and includes both a constitutive promoter and (Small minimally invasive wound in the thoracic cavity; simi a tissue or cell-specific promoter. lar to laparascopic methods) through which a scope and A Nell 1 protein, functional derivative thereof or Nell 1 guided injection device containing Nelll protein, derivative encoding nucleic acid molecule can be combined with a phar thereof, or nucleic acid molecule is introduced, c) ultrasonic 10 based drug delivery methods (see, for example, Mayer et al., maceutically acceptable carrier and prepared in formulations Advanced Drug Delivery Reviews, 2008, 60:1177-1192 and Suitable for administration to a Subject by injections, implan Bekeredjian et al., Ultrasound in Medicine and Biology, tations, inhalations, ingestions and the like. Pharmaceutically 2005, 31:687-691), and d) infusion into the pericardial space acceptable carriers are described hereinabove and include (see, for example, Xiao et al., Am. J Physiol, Heart Circ. oils, water, Saline solutions, gel, lipids, liposomes, resins, 15 Physiol., 2008, 294:H12212-12218). porous matrices, binders, fillers and the like, or combinations Important general considerations for design of delivery thereof. The carrier can be liquid, semi-solid, e.g. pastes, or systems and compositions, and for routes of administration, Solid carriers. Except insofar as any conventional media, for protein/peptide drugs may apply. For example, the appro agent, diluent or carrier is detrimental to the recipient or to the priate delivery system for Nell1 protein and/or functional therapeutic effectiveness of the active ingredients contained derivatives thereof will depend upon its particular nature, the therein, its use the present invention is appropriate. Examples particular clinical application, and the site of action. of carriers include oils, water, saline solutions, gel, lipids, Formulations for oral delivery or systemic delivery, for liposomes, resins, porous matrices, binders, fillers, patches, instance, may require certain considerations due to, for and the like, or combinations thereof. The carrier can also be example, instability of Nell 1 protein and/or functional deriva a controlled release matrix that allows optimum release of a 25 tives thereof in the gastrointestinal tract, or exposure of Nell1 Nell protein or nucleic acid admixed therein. protein and/or functional derivatives thereof to proteases. The term “therapeutically effective amount’ means the Any method known to those skilled in the art can be utilized dose required to prevent or delay the onset, slow down the to address such considerations. progression or ameliorate the symptoms of the disorder. Pre For example, for oral delivery, an absorption-enhancing cise dosages depend on the disease state or condition being 30 agent can be utilized. A wide variety of absorption-enhancing treated and other clinical factors. Such as weight and condi agents have been investigated and/or applied in combination tion of the subject, the subject's response to the therapy, the with protein compositions for oral delivery and for delivery type of formulations and the route of administration. As a by other routes (van Hoogdalem, Pharmac. Ther. 44, 407-43, general rule, a Suitable dose of a Nell composition (i.e., 1989: Davis, J. Pharm. Pharmacol. 44(Suppl. 1), 186-90, including a Nell1 protein or nucleic acid) for the administra 35 1992). Most commonly, typical enhancers fall into the gen tion to adult humans ranges from about 0.001 mg to about 20 eral categories of (a) chelators, such as EDTA, Salicylates, mg per kilogram of body weight. In some embodiments, a and N-acyl derivatives of collagen, (b) Surfactants, such as suitable dose of a Nell1 composition for the administration to lauryl sulfate and polyoxyethylene-9-lauryl ether, (c) bile adult humans is in the range of about 0.01 mg to about 5 mg salts, such as glycholate and taurocholate, and derivatives, per kilogram of body weight. However, the precise dosage to 40 Such as taurodihydrofusidate, (d) fatty acids, such as oleic be therapeutically effective and non-detrimental can be deter acid and capric acid, and their derivatives, such as acylcar mined by those skilled in the art. nitines, monoglycerides, and diglycerides, (e) non-surfac A Nell 1 protein, functional derivative thereof, or nucleic tants, such as unsaturated cyclic ureas, (f) saponins, (g) cyclo acid molecule can be administered to the Subject in any prac dextrins, and (h) phospholipids. tical and convenient manner. Suitable routes of administra 45 Alternatively, Nell 1 protein and/or functional derivative tion include the oral, nasal, topical, transdermal, and thereof, can be administered in combination with other drugs parenteral (e.g., intravenous, intraperitoneal, intradermal, or substances that directly inhibit proteases and/or other Subcutaneous or intramuscular) route. In addition, a Nell potential sources of enzymatic degradation of proteins. Yet protein, functional derivative thereof, or nucleic acid mol another alternative approach to prevent or delay gastrointes ecule can be introduced into the body, by injection or by 50 tinal absorption of Nell1 protein and/or functional derivative Surgical implantation or attachment, proximate to a prese thereof is to incorporate them into a delivery system that is lected tissue or organ site such that the Nell material is able designed to protect the protein from contact with the pro to enter the site by direct diffusion. For example, a Nell1 teolytic enzymes in the intestinal lumen and to release the protein, functional derivative thereof, or nucleic acid can be Nell protein and/or functional derivatives thereof at the site provided in a patch or gel like Substances, which, upon 55 of cardiovascular injury. A more specific example of this administration (by e.g., injection or implantation) can be strategy is the use of biodegradable microcapsules or micro taken up directly by tissues as a result of diffusing from a site spheres, both to protect a protein from degradation, as well as of high concentration to one where there is very low level of to effect a prolonged release of active protein (see, for the substance. If Nell1 protein, functional derivative thereof, example, Deasy, in Microencapsulation and Related Pro or nucleic acid molecule is administered locally, the formu 60 cesses, Swarbrick, ed., Marcell Dekker, Inc.: New York, lation is such that the Nell 1 protein, functional derivative 1984, pp. 1-60, 88-89, 208-11). thereof, or nucleic acid molecule does not diffuse and In a specific embodiment, a Nell 1 protein, functional adversely affect Surrounding organs. derivative thereof, or nucleic acid molecule is administered to Alternatively, a Nell1 protein, or functional derivative directly repair heart muscle after MI. Delivery can be per thereof, can be administered directly to injured and damaged 65 formed via direct delivery to or near the injured heart muscle tissue (e.g., infarct and Surrounding border Zones). Such site (infarct and border Zones) by injection, by catheter, via administration, can be applied, for example, to treat cardio absorbable biomatrix (i.e. biocompatible porous) material, US 8,722,625 B2 10 and the like, and combinations thereof. According to this ing in a severe truncation of the Nelll protein product and a embodiment, the Nell 1 composition is administered to the marked reduction insteady state levels of the Nell 1 transcript. Subject after the initial inflammatory responses Subsides— usually within 72 hours, within 48 hours, within 36 hours, EXAMPLE 2 within 24 hours, or even within 18 hours of MI, in order to minimize the extent of the injury and achieve better therapeu Heart Defects in Nell 1* Mutant Mouse tic efficacy. There is a flood of inflammatory responses imme diately after heart muscle injury. It is believed to be optimal to Formalin-fixed specimens were analyzed by heart length administer Nell1 after this initial defensive response of the and width measurements. These measurements were com Surrounding tissue. Regenerative processes, which naturally 10 pleted on wild type, heterozygous, and mutant mice at the begins after the inflammatory response slows down, are 18.5-day embryonic stage. Further observations were made where Nell 1 is likely to work best. using standard histological methods (haematoxylin and cosin Further according to the present invention, a Nell1 protein, staining on mouse Sagittal sections). functional derivative thereof, or Nell-encoding nucleic acid Nell1“ mice were observed to have significantly enlarged molecule can be used independently or in conjunction with 15 hearts based on length and width measurements. As shown in additional therapeutic compositions useful for treating a car Table 1, length measurements for all three genotypes did not diovascular condition. differ significantly. However, based on the statistical T-test, In a specific embodiment, a Nell 1 protein, functional the width measurements for mutant mice was significantly derivative thereof, or Nell-encoding nucleic acid molecule is greater compared to the width for wildtype and heterozygous used together with stem cells for the repair and regeneration mice, this confirming presence of an abnormal heart pheno of damaged cardiac muscles and blood vessels. type in mutant mice. Cell-based therapies for the repair and regeneration of Examination of the haematoxylin and eosin-stained slides damaged cardiac muscles and blood vessels utilize implanta showed dramatically reduced blood flow out of the heart. As tion of cells (such as cardiomyocytes), or introduction of stem shown in FIGS. 1-2, wild-type and heterozygote mice showed cells isolated from adult bone marrow to develop new cardiac 25 muscle in the area of implantation. See, e.g., Orlic et al., 2001; arteries filled with blood whereas blood was not a very promi Rubart et al., 2006; Ott et al., 2006; Rosenthal et al., 2006. nent feature in slides of mutant mice. Therefore, the loss of Without being bound by theory, the use of Nell1 increases the Nell function resulted in a significant reduction of the num efficiency of cell-based therapies for the repair and regenera ber of blood vessels and extensive branching of the vascula tion of damaged cardiac muscles and blood vessels. 30 ture in mutants as compared to wild type fetuses. The According to the present invention, a Nelll protein or decrease in blood vessel formation was observed throughout nucleic acid molecule can be co-delivered with the appropri the fetal body. ate cells, e.g., cardiomyocytes or adult stem cells, directly to In addition, a larger number of immature heart cells and the damaged sites of a subject using biological matrices or lesser extracellular matrix were observed in mutant mice as direct injection methods already in practice for cell-based 35 compared to wildtype mice (FIG.3A-3B). The dense packing therapies. of smaller cardiomyocytes in the mutant (FIG. 3B) was very In another embodiment, a Nell1 protein, functional deriva apparent in the denser/darker staining with haematoxylin and tive thereof, or Nell-encoding nucleic acid molecule is used eosin, compared to the wild type (FIG. 3A). in vitro to stimulate or promote the development and differ entiation of stem cells into cardiomyocytes useful for the 40 TABLE 1 repair and regeneration of damaged cardiac muscles and Measurements of Nell 1* Hearts Indicating Heart Enlargement blood vessels. See, for instance, example 7. Measurement (mm) of E18.5 fetal heart width and length of NelI1 This invention is further illustrated by the following heterozygote and homozygote mutant mice compared with wild-type littermates. There is significant enlargement of fetal hearts in examples, which are not to be construed in any way as impos homozygote mutant compared to the heterozygotes and normal mice. ing limitations upon the scope thereof. The terms and expres 45 sions which have been employed in the present disclosure are Homozygote Heteozygote Wild-type used as terms of description and not of limitation, and there is Nell16R Nell 16R +Nell 16R ++ no intention in the use of Such terms and expressions of Width 3.3 2.8 2.7 excluding any equivalents of the features shown and 2.5 2.8 2.8 described or portions thereof. It is to be understood that 50 2.8 2.3 2.8 various modifications are considered to be included within 2.3 2.7 2.5 2.8 2.8 2.7 the scope of the invention. All the publications mentioned in 3.0 2.5 2.3 the present disclosure are incorporated herein by reference. 3.2 2.5 2.2 3.0 2.5 2.5 EXAMPLE1 55 2.8 2.2 2.8 2.8 2.5 2.7 3.3 2.2 2.7 Nell 1* Mutant Mouse 3.0 2.2 3.0 2.5 2.5 2.3 The Nell1' mutant mouse was used in the experiments 2.5 described in the following examples. Generation, breeding 60 3.0 and maintenance of this mutant mouse is described in U.S. 2.5 Published Application 2006/0053503, which is incorporated No. of Fetuses 17 11 14 Average 2.853 2.530 2.476 herein by reference. Briefly, the mutant mouse contains a Length 3.2 3.7 2.7 recessive neonatal-lethal point mutation in the Nell1 gene, 2.8 3.2 2.7 originally induced by N-ethyl-N-nitrosourea (ENU). Nell1 65 2.8 2.8 3.3 has T to Abase change that converts a codon for cysteine into 2.7 3.2 3.0 a premature stop codon (TGT to TGA; Cys(502)Ter), result US 8,722,625 B2 11 12 TABLE 1-continued lungs. Mutant mice did not survive birth. The severity of the heart and blood vessel defects were likely to be the cause of Measurements of Nell 1* Hearts Indicating Heart Enlargement the death of the fetuses during the birth process reported Measurement (mm) of E18.5 fetal heart width and length of NelI1" heterozygote and homozygote mutant mice compared with wild-type earlier (Desai et al., 2006). Fetuses that were recovered by littermates. There is significant enlargement of fetal hearts in caesarean were unable to breathe as depicted in the collapsed homozygote mutant compared to the heterozygotes and normal mice. lung in the mutants. 3.0 3.2 3.3 3.2 3.0 3.0 EXAMPLE 3 3.2 3.0 2.5 3.3 3.3 3.0 10 ECM Genes Affected by Nell Influence Heart 3.0 2.8 3.3 Development 2.8 3.0 3.2 3.2 2.8 3.3 3.2 2.7 A comprehensive gene expression analysis using public 3.2 2.8 database (UCSC Genome Browser, Mouse Genome Infor 2.8 3.0 15 matics, Integrated Cartilage Gene Database, PubMed) was 2.5 3.0 conducted to investigate the relationship between cardiovas 2.8 cular development and each of the 28 extracellular matrix No. of Fetuses 17 11 14 (ECM) genes which were shown previously (Desai et al., Average 2.984 3.091 2.988 2006) to exhibit reduced expression in Nell1“ mutant mouse bodies. Of the 28 ECM genes studied, the bioinformatics T-Test p-values analysis showed that the majority of genes with reduced Mutant: Mutant: Heterozygote: expression in Nell-1 deficient mice are normally expressed in Wild-type Heterozygote Wild-type the heart (79% of the analyzed ECM genes; 22/28), blood Width O.OO12442891 O.OO46893426 0.6143698.331 vessels (71%; 20/28) and bone marrow (61%: 17/28) (See Length O.93S153O349 O.2470911230 O.3514701862 25 Table 2). The Mouse Genome Informatics database refer enced several genes (Col15a1, Osf-2, Bmprla, Pkd1, Mfge8. Ptger4. Notch3) that have been mutated in mice and actually These above cardiovascular defects were evident in E18.5 manifest abnormalities in cardiovascular development. day fetuses recovered by caesarean. Additionally, wild type Mouse mutations in Some of these genes display heart and heterozygote mice had spongy lungs that filled their deformities commonly associated with heart enlargement, as entire thoracic cavity, while mutant mice had compact, dense shown in Table 3 below. TABLE 2 Expression profile of genes in the Nell1 pathway and association with mutant mouse phenotypes.

# abnormal Gene Expression bone heart # total Symbol Gene Name heart vascular blood marrow phenotype' mutants'

Tnxb enascin O O 11 11 2 Prg4 proteoglycan 4 33 2 12 1 ThbS3 hrombospondin 3 O O 12 2 Colsa collagen 5alpha 3 subunit Neurog2 neurogenin 2 5 Colsa.1 procollagen type V, alpha 1 O O 10 O 1 1 Colöa1 procollagen Type VI aloha 1 O 6 12 O 1 Col15a1 procollagen type XV, alpha 1 O 9 10 2 1 1 Pacsin3 PKC and casein kinase O O Substrate in neurons 3 Tnc enascin c O O 21 1 3 Col12a1 procollagen type XII, alpha 1 O 2 O Chad chondroadherin 6 6 OSf2- Osteoblast specific factor 2 O O O 1 2 pending Col17a.1 procollagen type XVII alpha 1 Prkcc protein kinase C 2 Prkch protein kinase C, eta symbol O O 10 O 1 Bk- brain and kidney protein pending Ptk9. PTK9L protein tyrosine O O 10 O kinase 9-like Npdc1 neural proliferation, O O 1 differentiation and control gene Bmprla bone morphogenetic protein O 2 10 2 4 receptor type 1a Pk1 polycystic kidney disease I O 27 10 12 7 12 homolog US 8,722,625 B2 13 14 TABLE 2-continued Expression profile of genes in the Nell1 pathway and association with mutant mouse phenotypes.

# abnormal Gene Expression bone heart # total Symbol Gene Name heart vascular blood marrow phenotype' mutants' Tnfrsf11b tumor necrosis factor (ligand) 10 34 12 3 Mfges milkfat globule-EGF factor 8 10 12 10 10 1 5 protein Matn3 matrilin 3, cartilage matrix 28 1 protein Bmp7 bone morphogenetic protein 10 10 8 type 7 Matn2 matrilin 2, cartilage matrix 10 10 10 10 2 protein 2 Ptger4 prostaglandin E receptor 4 10 10 10 3 4 Notch3 notch gene homolog 3 10 30 4 # of Genes 22 2O 13 17 7 2O Percentage 79% 71% 46% 61% 25% 71.9%

TABLE 3 constitution and orientation in heart muscle after a heart attack, thereby preventing oralleviating heart muscle damage Mutated genes causing heart defects associated with enlargement and Subsequent loss of heart function (or death) resulting from MI. Gene Defect 25 Col6a1 Dilated descending aorta EXAMPLE 5 Bmprla Persistent truncus arteriosus Outflow tract formation abnormalities Pk1 Vascular leaksruptures The data presented here were based on studies of the Endocardial cushion defects Nell1 mutant mouse. Rodent Nell1 studies are believed to Abnormal atrial septum morphology 30 translate accurately to the human situation. The complete Double outlet right ventricle mouse Nell coding sequence has been reported (Genbank Abnormal septation Bmp7 Lack of endocardial cushion formation Accession No. AY622226; Desai et al., 2006). A comparison Ptger4 Dilated left ventricle of this sequence with the most current human Nell1 gene in Patent ductus arteriosus the public genome databases (UCSC Genome Browser and Congestive heart failure 35 NCBI) indicates a very high homology of 87% gene sequence identity. The corresponding 810-amino acid residue polypep tides have a 93% identity in their amino acid sequences (FIG. EXAMPLE 4 5). When one considers conservative substitution of similar amino acids, the human and mouse Nell 1 proteins are 97% Gene Expression in Nell 1* Mutant Mouse 40 conserved. This remarkable degree of gene and protein struc ture conservation suggests the conservation of functions and To define the involvement of Nell1 in the known molecular fundamental mechanisms of Nell 1-mediated pathways in pathways that govern heart structure and function, a compre human and mouse. hensive gene expression analysis was conducted in the entire 45 EXAMPLE 6 mouse genome (-30,000 genes) of normal fetal hearts and those dissected from Nell1'. This analysis consisted of 50 Animal Model for Assessing Therapeutic Efficacy of mutant fetal hearts separated into 4 pools of 10-13 hearts and Nel11 for MI 35 normal hearts separated into three pools of 10-12 hearts (18.5 days of gestation). RNAs were extracted from the 50 The efficacy of the Nell1 protein for regenerating cardiac pooled tissues, processed for microarray analysis on the Illu muscle after damage induced by a myocardial infarction (MI) mina Mouse V6 chips and scanned with Illumina Beadstation is tested in a widely used and accepted in Vivo animal model. 500GX. Data was analyzed with the BeadStudio software and Myocardial infarction is induced in a murine in vivo model by Gene Ontology Tree machine. At least 345 genes were iden blocking the main blood supply line to the left ventricle. The tified that were differentially expressed between normal and 55 Surgical procedures for generating this model are described in mutant samples (at p value=0.001 for the microarray detec detail by several publications (Patten et al., 1998; Tarnavski et tion and differential p values; denotes a very high statistical al., 2004; Ahn et al., 2004). significance). Table 4 lists a representative sampling of genes Briefly, mice are anesthetized, restrained in a Supine posi influenced by Nell 1 that already have established functions in tion, and intubated with pure oxygen regulated by a small cardiovascular conditions. Table 4 also provides the literature 60 animal ventilator. A thoracotomy is performed under a dis references for the specific studies that have demonstrated secting scope, at the fourth or fifth intercostal space of the left these gene functions. side, between the heart and lung margins. The thoracic Sur Table 4 shows a number of genes in the Nell pathway that gical hole is enlarged using retractors and the pericardial sac have been implicated in the processes that ensue after heart is gently torn with fine forceps. failure. The ability of Nell 1 to stimulate proteins that control 65 The left anterior descending coronary artery (LAD) is visu cell differentiation and proper secretion of the cardiac ECM alized and ligated by passing a tapered microSurgical needle strongly suggests that this protein can restore proper ECM (/4 circle, 140 microns) with a black silk monofilament suture US 8,722,625 B2 15 16 (size 7 or 8) underneath the coronary artery and tying the EXAMPLE 8 suture to completely stop the blood flow in the artery. A small polyethylene tubing (PE 10) 2-3 mm is placed between the tie Animal Model for Assessing Therapeutic Efficacy of and the LD to minimize cutting and severely injuring the Nell1 for Myocardial Ischemia and Reperfusion artery. Injury Myocardial infarction is confirmed by observing for The efficacy of the Nell1 for regenerating cardiac muscle blanched or white appearance of the left vertical that corre after damage induced by myocardial ischemia and reperfu spond to the muscles that have lost blood supply and the sion injury is tested in a widely used and accepted in vivo alteration of the wave pattern (pronounced ST wave eleva 10 animal model. Myocardialischemia and reperfusion injury is tion) in an electrocardiogram. Since the LAD provides the induced in an in vivo murine model as follow: blood supply to the left ventricle, this surgically-induced 1. After anesthesia, intubation and hook-up to a mouse ECG myocardial infarction will cause the death of myocardial machine, the chest cavity of the mouse is opened at the tissue (necrosis) in the left ventricular wall and the anterior intercostal space (usually 4” or 5") and the opening is section of the interventricular section. The size of the myo 15 retracted to reveal the left side of the heart and to locate the cardial infarction lesions/infarcts can be controlled by the LAD artery. The pericardial sac is torn gently with forceps exact position of the ligation along the LAD. Ligation at a and the LAD is positioned for easy access. All Surgical high position (atrioventricular junction) will reduce blood steps are done under a dissecting microscope. flow to a larger area and make larger infarcts while ligations 2. A tapered needle (4 circle 140 microns) with a size 8 silk at lower areas will make medium or Small lesions. Ligature or monofilament Suture is partially passed underneath the position is kept constant for any given experimental group to artery. A Small tubing 1-1.5" in length (e.g. polyethylene keep the infarction size constate. size 10 tubing) is placed on top and parallel to the LAD After myocardial infarction induction, the thoracic and artery and perpendicular to the length of the needle. The skin wounds are sutured and mice are allowed to recover from Suture is then pulled and a Surgical tie is made Such that the 25 tubing is tied with the artery located beneath it. anesthesia on a heating pad or with heat lamps. 3. The interruption of blood flow to the left ventricular heart To test the ability of Nell1 to repair cardiac tissue damage muscles is easily visualized by a blanched or white appear due to an acute myocardial infarction event, purified Nell1 ance of the affected region (where infarct develops). The protein are delivered directly into the Surrounding tissue ECG will confirm the ischemia by the alteration of the around the visible infarct and within the infarct. Direct deliv 30 wave pattern (e.g. ST segment elevation, T wave anoma ery of Nell 1 protein is performed by reopening the original lies) compared to the normal pattern. The change indicates thoracic wound used to induce the infarct. that the LAD is successfully ligated and restricted blood Nell1 and functional derivatives thereof containing EGF flow to the left ventricle has functionally induced an like domains and/or the von Willebrand like domain of Nell1 ischemic event. are administered at 2-3 points along one side of the infarct 35 4. The chest cavity and the skin are Sutured Such that one end border Zone. In some animals, direct delivery of Nell1 protein of the tubing is sticking out of the thoracic area above the is administered via microinjection, application of Nell in a sutured skin. After the desired amount of time of ischemia, gel or microspray, via nanoparticles, or time-release patches. the tubing is gently pulled out to relax the knot/ligated In others, it is administered via a Nell protein expression suture thereby allowing reperfusion of blood into the vector (continuous delivery). Administration of Nell 1 is per 40 affected area. formed after the initial Surge of inflammatory response trig 5. Reperfusion is indicated by the return of the ECG pattern to gered by cardiac damage and at the time heart tissue attempts normal or near normal pattern. Different groups of mice innate regenerative mechanisms (approximately 4-5 hrs after with varying times of occlusion before reperfusion are MI). The effects of Nell1 administration are evaluated by made. standard histology and immunohistochemistry techniques for 45 6. Varying concentrations of Nell1 protein are administered detection of proteins associated with cardiac tissue regenera via intraperitoneal injection or using a catheter device that tion (Orlic et al., 2001). is placed before the chest cavity is closed after LAD liga tion and ischemia. The catheter device allows for con trolled delivery so that Nell1 protein can be delivered EXAMPLE 7 50 immediately after reperfusion or given time points after reperfusion is induced. In other models, Nell 1 protein is In Vitro Stem Cell Therapy administered by reopening the Surgical Sutures and re entry to the chest cavity and direct Nell1 delivery by micro injection or gel patch. A promising approach in the field of heart muscle regen 55 eration after MI is the introduction of either embryonic or EXAMPLE 9 adult mesenchymal stem cells into the damaged heart. How ever, data indicate that although new heart muscle cells can be Animal Model for Assessing Therapeutic Efficacy of regenerated that the new tissue may not necessarily display Nell1 for Cardiac Hypertrophy the full functional capacity of mature heart tissue (contractil 60 ity). The use of Nell1 protein as a therapeutic for cardiac hyper To promote full functional capacity of mature heart tissue, trophy is tested in a widely used and accepted in vivo animal Nell 1 protein and functional derivatives thereof containing model. Cardiac hypertrophy is generated by physical/surgical EGF like domains and/or the von Willebrand like domain of means pressure-overload. Nell1 are co-delivered with stem cells to the injured heart 65 In the in vivo pressure overload animal model, the aorta of muscle using the same strategies currently in use for stem cell a mouse/rat or large animal is banded to reduce the diameter delivery. and thus the blood in the left ventricle builds up pressure and US 8,722,625 B2 17 18 induces hypertrophy of the left ventricle (Tarnavski et al are measured in Nell-treated and controls during the time 2004). This type of animal model mimics the human condi that untreated mutant mice show the severe symptoms of tion of aortic stenosis where the narrowing of the aortic valve cardiomyopathy, generally at 4-5 months in Nov mice. restricts blood flow from the left ventricle to the aorta. The After cardiovascular functional/physiological studies, the persistent increased pressure in the left ventricle leads to mice are sacrificed and hearts are dissected and fixed for increase in muscle mass (hypertrophy) of the walls. This morphological and histological evaluation Such as: total heart model is generated as follows: size, chamber sizes (especially left ventricle), heart valve 1. Mice are anesthesized and a 5 mm transverse incision is structure, chordae tendinae, interventricular septum, heart made at the level of the left armpit, 2 mm away from the muscle cell (cardiomyocyte) size and appearance, vessels sternal border. A small incision (5mm) is made at the 2" 10 going in and out of the heart etc. intercostal space and opened with microretractors. 2. The thymus and fat covering the aortic area are pushed EXAMPLE 11 away and the pericardial sac is gently torn. The ascend ing portion of the aorta is located and bluntly dissected NELL 1 Protein Treatment of Heart Muscle Damage from the pulmonary trunk and forceps is placed under 15 neath the ascending aorta from Myocardial Infarction 3. A 7-0 silk suture is placed around the aorta and a loose knot is made. A 25 or 27 gauge needle (outer diameter of The ability of Nell 1 protein to trigger cellular pathway(s) 0.51 mm) that is bent into an L shape is placed through for regeneration of damaged heart muscle was demonstrated the loose loop, positioned above and parallel to the aorta in an in vivo mouse model. A heart attack or myocardial and a second knot is tied securely. The needle is retracted infarction was generated in 4-5 month old adult mice (strain to yield a constricted aorta (60-80% constriction for a 27 C57Bl/6J) by surgically tying the left anterior descending gauge). Two more knots are tied. (LAD) coronary artery, which is the main blood supply line to 4. The chest cavity is closed by suturing ribs and then the the left ventricle (lv) and the interventricular septum (IVS). skin wound. 25 The left ventricle pumps oxygenated blood through the aorta Nell1 protein and functional derivatives thereof containing into the rest of the body while the IVS divides the right and EGF like domains and/or the von Willebrand like domain of left ventricles of the heart. LAD ligation in animal models Nell1 are administered as an injectable after the onset of results in the damage and Subsequent death of the heart hypertrophic changes and heart function anomalies detected muscle tissue. Table 5 Summarizes the results of treating by ECG. Times of administration are tested as one high dose 30 mouse hearts with the purified human NELL 1 protein on the after hypertrophy is diagnosed or at lower doses given mul third day post-MI event. The NELL1 protein was diluted in tiple times (weekly) after hypertrophy is diagnosed. Efficacy phosphate buffered saline (PBS) and was delivered directly of the treatment is evaluated by quantitative measurements of onto the damaged heart muscle as a very concentrated micro Ventricular and heart size, physiological monitoring by ECG drop, while the mice were under anaesthesia and intubation and other heart visualization tools, molecular markers for 35 for about an hour. Three mice were treated with 312 ng and heart failure etc. as described earlier. four mice with 624 ng purified NELL 1 protein. Four mice underwent the same cardiac Surgery but were given a micro EXAMPLE 10 drop of PBS on the damaged heart tissue and served as con trols. In addition to these controls, over 20 MI mice were Animal Model for Assessing Therapeutic Efficacy of 40 previously generated and studied to obtain consistency in MI Nell1 for Cardiomyopathy Surgical and post-Surgical techniques. These earlier “con trols’ displayed the same characteristics as controls repre The use of Nell1 protein as a therapeutic for cardiomyopa sented in Table 5. All treated and untreated mice were main thy is tested in a widely used and accepted in vivo animal tained for an additional 14 days before they were sacrificed to model. The in vivo mouse model of cardiomyopathy is gen 45 collect hearts and other major organs (a total of 17 days erated by gene-targeted approaches Such as knock-outs or post-MI). Heart size measurements indicated slight increases over-expression of a single gene, wherein the homozygotes in both heart width and depth in Nell1-treated hearts. (two mutant gene copies) and/or heterozygotes (one mutant Remarkably ALL treated mice showed dramatically lesser copy) can Survive to the juvenile or adult stage. Suitable in visible areas of the infarcted tissue on the surface of the heart. Vivo mouse models of cardiomyopathy contain knock-outs or 50 In 6 out of 7 hearts the damaged tissue was only visible under over-expression of genes and pathways (e.g., (extracellular the microscope after they were fixed in buffered formalin. matrix and matricellular proteins, tenascins, thrombospon FIG. 6A-6D show the range of improvement observed in dins, matrilins, etc.) that are controlled by the Nell signaling NELL 1-treated hearts, from barely visible to about 30% inf protein. A specific example of an appropriate Small animal arct sizes in comparison to the usual 50-90% infarct sizes seen model is the targeted knockout of the mouse Nov (Ccm3) gene 55 in controls. FIG. 7A-7D present histological analysis of sec reported by Heath et al. (BMC Developmental Biology 2008: tioned hearts stained with Masson-Trichome and further con 8:18). firmed that there is decreased damage at the cellular level in Briefly, Nov (Ccm3) mutant mice are generated. Imaging of the NELL1-treated hearts compared to the controls. At 17 hearts by echocardiograms and electrocardiograms are con days post-MI, heart muscle tissue is severely damaged Such ducted to determine heart function and presence of visible 60 that huge gaps appear within the untreated heart muscle in the heart structure anomalies prior to treatment. left ventricle to the interventricular septum. In contrast, there Nell1 protein and functional derivatives thereof containing is a consistent and dramatic reduction in the amount of break EGF like domains and/or the von Willebrand like domain of down or damage observed in the heart muscle of treated mice. Nell are administered by intraperitoneal injection to young These data from an in vivo MI mouse model illustrates that Nov (Ccm3) mutant mice and corresponding controls during 65 clinical approaches that will enable delivery of Nell1 protein the first two months of life. Various dosages and timing regi directly onto damaged heart muscle will be effective in reduc mens are tested. After treatment, heart function parameters ing the effects of an MI event. US 8,722,625 B2 19 20 TABLE 4

GENES IN NELL1 PATHWAYASSOCIATED WITHKNOWN CARDIOVASCULARDISORDERS UP (I) OR DOWN () REGULATION ASSOCIATION WITH HEART DISORDERS AND GENE and DESCRIPTION p value <0.001) DISEASES REFERENCES Tpm2; tropomyosin 2, beta 43 Cardiac-specific myofibrillogenesis; Cardiomyopathy Denz et al., 2004 Dmn; desmuslin transcript 9.4 Hypertrophic Cardiomyopathy; heart failure Mizuno et al., 2001 variant 1 Acta1; skeletal muscle actin 2.8 Hypertrophic cardiomyopathy; heart failure Lim et al., 2001 alpha 1 Tpm1 tropomyosin alpha 1 4.8 Hypertrophic cardiomyopathy; heart failure Wernicke et al., 2007; Kostin et al., 2007 Lgals3; lectin, 2.6 Acute heart failure biomarker; excellent predictor of mortality Van Kimmenade et al., Galactose binding, soluble 3 within 60 days; increases in failure prone hypertrophied 2006: Sharma et al., hearts; aortic stenosis; induces cardiac fibroblast proliferation, 2004 collagen deposition Spp1 2.3 Heart contractility via control of ECM proteins Okamoto, 2007 Secreted phosphoprotein 1 inflammation control in hypertrophy, myocardial infarction Singh et al., 2007 (osteopontin) and heart failure, valvular stenosis F1 1.3 Atrial fibrillation in cardiac arrhythmia; Chen et al., 2007 Four and a half limb domains B-adrenergic induced cardiomypathy and heart failure (3- Lim et al., 2001 blocker pathway); cardiac remodeling by transcriptional regulation and myofilament assembly Aqpl; aquaporin 1 1.3 Myocardial edema Egan et al., 2006 I6st 1.5 Cardiac hypertrophy Terrell et al., 2006 Interleukin 6 signal Coles et al., 2007 transducer Tnc 1.5 Inflammation induced tissue remodeling in acute myocardial Terasaki et al., 2007 Tenascin c infarction, acute myocarditis and cardiomyopathy, left ventricular remodeling Tnxb 1.8 Cardiac nerve sprouting after MI contributing to arrhythmia Lai et al., 2000 Tenascinxb and Sudden cardiac death 9. bp5 1.3 Atrophy; Adaptive cardiac hypertrophy Baurand et al., 2007 l Sulin growth factor binding protein 5 Fgl2 1.4 Acute congestive heart failure without structural Mu et al., 2007 brinogen-like protein abnormalities; contractile dysfunction and rhythm abnormalities gf; connective tissue 1.3 Excessive myocardial fibrosis and diastolic heart failure Koitabashi et al., 2007 growth factor D pt; dermatopontin 1.5 ECM remodeling in myocardial infarction Takemoto et al., 2002 Ldlr; low density lipoprotein 1.5 Heart failure Weiss et al., 2006 re ceptor ppb 1.3 Cardiac fibrosis Tamura et al., 2000 atriuretic peptide precursor Congestive heart failure and myocardial infarction Heimdal et al., 2007 be b Biomarker for heart failure Seferian et al., 2007 Doust et al., 2004 ppa 1.5 Cardiac fibrosis Tamura et al., 2000 atriuretic peptide precursor Congestive heart failure and myocardial infarction Heimdal et al., 2007 (8. Biomarker for heart failure Seferian et al., 2007 Doust et al., 2004 Ttn 1.4 Cardiac muscle dystrophies (contractility) Fougerousse et al., 1998; Tit Koatin et al., 2000 Cyró1 1.7 inflammatory cardiomyopathy Wittchen et al., 2007: Cysteine rich protein 61 Mo and Lau, 2006 Sgcb Cardiac muscle dystrophies Fougerousse et al., 1998 Sarcoglycan

TABLE 5 Results of Nell1 Protein Treatment of Damaged Heart Tissue in a Mouse Model with Myocardial Infarction

Heart Heart Weight Width Depth Change Heart length Left- Front- Estimated Infarct Size Mouse 17 day Top-Bottom Right Back 17 days post-MI Number period (mm) (mm) (mm) (% left ventricle) Controls (PBS) m2589 O 8.32 5.84 4.91 75% m2588 +1.2 8.55 6.2O S.11 SO% 2733 -0.9 8.78 7.01 S.69 60-70% US 8,722,625 B2 21 22 TABLE 5-continued Results of Nell1 Protein Treatment of Damaged Heart Tissue in a Mouse Model with Myocardial Infarction

Heart Heart Weight Width Depth Change Heart length Left- Front- Estimated Infarct Size Mouse 17 day Top-Bottom Right Back 17 days post-MI Number period (mm) (mm) (mm) (% left ventricle) m2764 +1.1 8.52 6.09 5.57 90% Average --O.35 8.54 6.28 5.32 -70% Nell1 Protein Dose I (312 ng) In 2SSO -3.2 8.42 7.41 6.19 Infarct hardly visible until fixation; ~16% faint area 2597 -2.3 8.04 6.12 6.17 Infarct barely visible until fixation; 30% faint area In 2SS3 -2.3 9.21 6.44 5.52 Infarct hardly visible until fixation; 30% faint area Average -2.6 8.56 6.66 S.96 -25.3% Nell1 Protein Dose II (624 ng) m2668 +0.1 8.51 6.55 5.65 Infarct hardly visible until fixation; 25% faint area 2732 -0.1 8.94 6.44 5.73 Infarct hardly visible until fixation; 10% very small faint 808 m2726 -2.7 8. SO 6.90 5.94 Infarct hardly visible until fixation; very faint layer difficult to estimate 2727 -0.3 8.42 6.95 6.26 Visible infarctat -30% Average -O.75 8.59 6.71 S.90 -16.3%

REFERENCES as Mizuno Tet al. BMC Genet 2001; 2:8. Mizuno Y et al. Proc Natl AcadSci U.S.A. 2001; 98: 6156 Aghaloo Tet al. Am. J of Path 2006; 169:903-915. 6161. Ahn Detal. Am J Physiol Heart Circ Physiol 2004,286:1201 Mo F E et al. Circ. Res. 2006: 99: 961-969. 1207. Mu Jet al. Physiol Genomics Jun. 5, 2007 (Epub). Baurand A et al, Circ Res 2007: 100:1353-1362. 40 Okamoto H. Mol Cell Biochem 2007: 300:1-7. Chen C L et al. Biochim Biophy's Acta 2007: 1772:317-329. Orlic D et al. Ann NY AcadSci 2001; 938:221-229. Coles Bet al. Am J Pathol 2007; May 3 Epub. Orlic D et al. Nature 2001: 410: 701-705. Cundy Tet al. Hum Mol Genet 2002: 11:21 19-2127. Ott et al. Expert Opin Biol Ther 2006; 6(9): 867-78. Denz C R et al. Biochem Biophy's Res Commun 2004; 320: Patten R D et al. Am J Physiol Heart Circ Physiol. 1998: 1291-1297. 45 274:1812-1820. Desai Jet al. Hum Mol Genet 2006; 15:1329-1341. Rosenthal et al., Cell Transplant 2006; 15 Suppl 1: S41-5. Doust J A et al. Arch Intern Med 2004; 164: 1978-1984. Rubart et al., Ann NY AcadSci 2006: 1080: 34-48 Egan J Retal. Biochim Biophy's Acta 2006: 1758: 1043-1052. Seferian K R et al. Clin Chem 2007:53:866-873. Fougerousse F et al. Genomics 1998; 48:145-156. Sharma U C et al. Circulation 2004; 110:3121-3128. Grahame R et al. Ann Rheum Dis 1981; 40:541-546. 50 Helmjdal A et al. J Card Fail 2007: 13:184-188. Singh Metal. Front Biosci 2007: 12:214-221. Jackson G C et al. J Med Genet 2005:41:52-59. Stem cell repair in ischemic heart disease: an experimental Kostin Set al. Heart Fail Rev. 2005:271-280. model. Int J Hematol. 2002: 76 Suppl 1:144-145. Koitabashi Net al. Hypertension 2007:49:1120-1127. Sussman, Nature 2001; 410: 640-641. Kuroda et al., Biochemical Biophysical Research Comm. 265: 55 Takemoto Set al. Basic Res Cardiol 2002; 97: 461-468. 79-86 (1999a). Tamura et al. Proc Natl AcadSci USA 2000:97:4239-4244. Kuroda et al., Biochemical Biophysical Research Comm. 265: Tarnavski Oetal. Physiol Genomics 2004; 16:349-360. 752-757 (1999b). Terrell et al. Shock 2006; 26:226-234. Lai AC, et al. J Cardiovasc Electrophysiol 2000: 11: 1345 Terasaki Fet al. Circ J 2007: 71:327-330. 1351. 60 Ting K. etal. Jof Bone and Mineral Research 1999; 14:80-88. Leier CV et al. Ann Intern Med 1980, 92:171-178. van Kimmenade et al. J. Am. Coll. Cardiol. 2006: 48: 1217 Lim D S et al. JAm Coll Cardiol 2001: 38:1175-1180 1224 Liu L et al. Journal of Undergraduate Research (Vol. 7). Weiss R Metal. Circulation 2006: 114: 2065-2069. 2007. Wernicke D et al. Biomed Tech (Berl) 2007: 52:50-55 Lu et al. The Spine Journal 2007; 7: 50-60. 65 Wittchen F et al. J Mol Med 2007: 85:253-267. Mao J R et al..J Clin Invest 2001; 107: 1063-1069. Zhang X et al. J Bone Miner Res 2003; 18:2126-2134. Mao J R et al. Nat Genet 2002:30:421-425. Zhang X et al...J Clin Invest 2002: 110:861-870.

US 8,722,625 B2 25 26 - Continued tctgct cotg Caaggatggc aagatatt ct gcc.gacggac agcttgttgat tccagaatc 21OO

Caagtgctga cct attctgt tgcc.cagaat gtgacaccag agt cacaagt caatgtttag 216 O accaaaatgg t cacaa.gctg tat caagtg gagacaattg gacccatagc tgtcagcagt 222 O gtcggtgtct ggaaggaga.g gtagattgct ggccact cac ttgc.cccaac ttgagctgtg 228O agtata cagc tat cittagaa ggggaatgtt gtc.ccc.gctg tgtcagtgac c cctd.cc tag 234 O citgata acat caccitatgac atcagaaaaa Cttgcctgga Cagctatggit gttt Cacggc 24 OO ttagtggctic agtgttggacg atggctggat ctic cctdcac aacct gtaaa tgcaagaatg 246 O gaagagtctg ttgttctgtg gattittgagt gtc.ttcaaaa taattgaagt atttacagtg 252O gact caacgc agaagaatgg acgaaatgac catccaacgt. gattalaggat aggaatcggit 2580 agtttggttt ttttgtttgt tttgttttitt taaccacaga taattgccaa agttt coacc 264 O tgaggacggit gtttggaggit tgc ctitttgg acctaccact ttgct catt c ttgctaacct 27 OO agtictaggtg acctacagtg cc.gtgcattt aagtcaatgg ttgttaaaag aagtttic cc.g 276 O tgttgtaaat catgtttic cc titat cagatc atttgcaaat acatttalaat gat ct catgg 282O taaatgttga tgtattttitt ggtttattitt gtgtactaac ataatagaga gag acticago 288O t cottt tatt tattttgttg att tatggat Calaattictaa aataaagttg Cctgttgttga 294 O aaaaaaaaaa. aaaaaaaaaa. aaaaaa. 2966

<210s, SEQ ID NO 2 &211s LENGTH: 810 212. TYPE : PRT &213s ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 2 Met Pro Met Asp Lieu. Ile Leu Val Val Trp Phe Wall Thir Ala 1. 1O 15

Arg Thir Wall Val Gly Phe Gly Met Asp Pro Asp Lell Glin Met Asp Ile 25

Wall Thir Glu Lieu. Asp Lieu Val Asn Thir Thir Lieu. Gly Wall Ala Glin Wall 35 4 O 45

Ser Gly Met His Asn Ala Ser Lys Ala Phe Lieu. Phe Glin Asp Ile Glu SO 55 6 O

Arg Glu Ile His Ala Ala Pro His Wall Ser Glu Lell Ile Gln Lieu. 65 70 7s 8O

Phe Arg Asn Lys Ser Glu Phe Thr Ile Lieu. Ala Thir Wall Glin Gln Lys 85 90 95

Pro Ser Thir Ser Gly Val Ile Lieu Ser Ile Arg Glu Lell Glu His Ser 105 11 O

Phe Glu Lieu. Glu Ser Ser Gly Lieu. Arg Asp Glu Ile Arg Tyr His 115 12 O 125

Ile His Asin Gly Llys Pro Arg Thr Glu Ala Lell Pro Arg Met 13 O 135 14 O

Ala Asp Gly Gln Trp His Llys Val Ala Lieu. Ser Wall Ser Ala Ser His 145 150 155 160

Lell Luell Luell His Val Asp Cys Asn Arg Ile Tyr Glu Arg Wall Ile Asp 1.65 17O 17s

Pro Pro Asp Thir Asn. Leu Pro Pro Gly Ile Asn Lell Trp Luell Gly Glin 18O 185 19 O

Arg Asn Glin Llys His Gly Lieu. Phe Lys Gly Ile Ile Glin Asp Gly Lys 195 2O5

Ile Ile Phe Met Pro Asn Gly Tyr Ile Thr Glin Pro Asn Luell Asn US 8,722,625 B2 27 28 - Continued

21 O 215 22O

His Thir Pro Thir Cys Ser Asp Phe Luell Ser Lell Wall Glin Gly Ile 225 23 O 235 24 O

Met Asp Luell Glin Glu Lell Lell Ala Met Thir Ala Luell Asn Tyr 245 250 255

Ala Glu Thir Arg Lell Ser Glin Luell Glu Asn Cys His Glu Thir 26 O 265 27 O

Glin Wall Ser Gly Lell Lell Tyr Arg Asp Glin Asp Ser Trp Wall Asp 28O 285

Gly Asp His Asn Cys Thir Ser Gly Ala Wall Glu 29 O 295 3 OO

Arg Arg Met Ser Pro Pro Luell Asn Ser Pro Asp Ser Luell Pro 3. OS 310 315

Wall His Ile Ala Gly Glin Wall Arg Pro Cys Ile 3.25 330 335

Gly Gly Lys Wall Lell Ala Glu Gly Glin Ile Lell Thir Ser 34 O 345 35. O

Arg Glu Gly Gly Wall Luell Wall Ile Thir Glu Met Cys 355 360 365

Pro Pro Luell Asn Ser Glu Asp His Ile Lell Pro Glu Asn Glin 37 O 375

Cys Arg Wall Cys Arg Gly His Asn Phe Cys Ala Glu Gly Pro Lys 385 390 395 4 OO

Glu Asn Ser Glu Asn Trp ASn Thir Ala Thir 4 OS 415

Glu Ser Gly Tyr Ile Ser Wall Glin Gly Asp Ser Ala 425 43 O

Glu Asp Ile Asp Glu Ala Ala Met His Cys His Ala Asn 435 44 O 445

Thir Wall Wall Asn Lell Pro Gly Luell Tyr Arg Cys Asp Wall Pro 450 45.5 460

Gly Tyr Arg Wall Asp Asp Phe Ser Thir Glu His Asp Glu Cys 465 470

Gly Ser Glin His Asn Asp Glu Asn Ala Ile Thir Asn Thir 485 490 495

Wall Glin His Ser Thir Lys Pro Gly Tyr Wall Gly Asn Gly SOO 505

Thir Ile Arg Ala Phe Glu Glu Gly Cys Arg Tyr Gly Gly Thir 525

Wall Pro Asn Cys Wall Pro Ser Gly Phe Thir Gly Ser 53 O 535 54 O

His Asp Ile Asp Glu Ser Glu Gly Ile Ile Glu Cys 5.45 550 555 560

His Asn His Ser Arg Wall Asn Luell Pro Gly Trp His Cys Glu 565 st O sts

Arg Ser Gly Phe His Asp Asp Gly Thir Ser Lell Ser Gly Glu 58O 585 59 O

Ser Ile Asp Ile Asp Glu Cys Ala Luell Arg Thir His Thir Trp 595 605

Asn Asp Ser Ala Ile Asn Luell Ala Gly Gly Phe Asp Luell 610 615

Pro Ser Gly Pro Ser Cys Ser Gly Asp Pro His Glu Gly Gly Luell 625 630 635 64 O

US 8,722,625 B2 33 34 - Continued

105 11 O

Phe Glu Luell Glu Ser Ser Gly Pro Arg Glu Glu Ile Arg His 115 12 O 125

Ile His Gly Gly Pro Arg Thir Glu Ala Lell Pro Tyr Arg Met 13 O 135 14 O

Ala Asp Gly Glin Trp His Wall Ala Luell Ser Wall Ser Ala Ser His 145 150 155 160

Lell Luell Luell His Wall Asp Asn Arg Ile Tyr Glu Arg Wall Ile Asp 1.65 17O 17s

Pro Pro Glu Thir Asn Lell Pro Pro Gly Ser ASn Lell Trp Luell Gly Glin 18O 185 19 O

Arg Asn Glin His Gly Phe Phe Gly Ile Ile Glin Asp Gly 195

Ile Ile Phe Met Pro Asn Gly Phe Ile Thir Glin Cys Pro Asn Luell Asn 21 O 215 22O

Arg Thir Pro Thir Cys Ser Asp Phe Luell Ser Lell Wall Glin Gly Ile 225 23 O 235 24 O

Met Asp Luell Glin Glu Lell Lell Ala Met Thir Ala Luell Asn Tyr 245 250 255

Ala Glu Thir Arg Lell Gly Glin Luell Glu Asn Cys His Glu Thir 26 O 265 27 O

Glin Wall Ser Gly Lell Lell Tyr Arg Asp Glin Asp Ser Trp Wall Asp 285

Gly Asp Asn Asn Cys Thir Ser Gly Ala Wall Glu 29 O 295 3 OO

Arg Arg Met Ser Pro Pro Luell Asn Ser Pro Asp Ser Luell Pro 3. OS 310 315 32O

Wall His Ile Ser Gly Glin Wall Arg Pro Cys Ile 3.25 330 335

Gly Gly Lys Wall Lell Ala Glu Gly Glin Ile Lell Thir Thir 34 O 345 35. O

Arg Glu Gly Gly Wall Luell Wall Ile Thir Glu Ala 355 360 365

Pro Pro Luell Asn Ser Glu Asp His Ile Lell Pro Glu Asn Glin 37 O 375

Cys Arg Wall Cys Arg Gly His Asn Phe Cys Ala Glu Ala Pro Lys 385 390 395 4 OO

Glu Asn Ser Glu Asn Trp ASn Thir Ala Thir 4 OS 415

Glu Asn Gly Tyr Ile Ser Wall Glin Gly Asn Ser Ala 425 43 O

Glu Asp Ile Asp Glu Ala Ala Met His Cys His Ala Asn 435 44 O 445

Thir Wall Wall Asn Lell Pro Gly Luell Tyr Arg Cys Asp Ile Pro 450 45.5 460

Gly Tyr Ile Arg Wall Asp Asp Phe Ser Thir Glu His Asp Asp Cys 465 470

Gly Ser Gly Glin His Asn Asp Asn Ala Ile Thir Asn Thir 485 490 495

Wall Glin Gly His Ser Thir Glin Pro Gly Tyr Wall Gly Asn Gly SOO 505 51O

Thir Wall Cys Ala Phe Glu Glu Gly Cys Arg Tyr Gly Gly Thir 515 52O 525 US 8,722,625 B2 35 36 - Continued Wall Ala Pro Asn Lys Cys Val Cys Pro Ser Gly Phe Thir Gly Ser 53 O 535 54 O

His Glu Lys Asp Ile Asp Glu Cys Ala Glu Gly Phe Wall Glu Cys 5.45 550 555 560

His Asn His Ser Arg Cys Val Asn Leu Pro Gly Trp His Cys Glu 565 st O sts

Arg Ser Gly Phe His Asp Asp Gly Thr Tyr Ser Lell Ser Gly Glu 585 59 O

Ser Ile Asp Ile Asp Glu. Cys Ala Lieu. Arg Thir His Thir 595 605

Asn Asp Ser Ala Cys Ile Asn Lieu. Ala Gly Gly Phe Asp Lieu. Cys 610 615

Pro Ser Gly Pro Ser Cys Ser Gly Asp Cys Pro His Glu Gly Lieu. 625 630 635 64 O

His Asn Gly Glin Val Trp Ile Lieu. Arg Glu Asp Arg Ser Wall 645 650 655

Ser Lys Asp Gly Lys Ile Phe Cys Arg Arg Thir Ala 660 665 67 O

Glin Asn Pro Asn Val Asp Lieu Phe Cys Cys Pro Glu Asp Thr 675 685

Arg Wall Thir Ser Glin Cys Lieu. Asp Glin Ser Gly Glin Luell 69 O. 695 7 OO

Ser Gly Asp Asn Trp Thr His Ser Cys Glin Gln Arg Lieu. Glu 7 Os 71O 71s 72O

Gly Glu Ala Asp Cys Trp Pro Lieu. Ala Cys Pro Ser Lell Ser Cys Glu. 72 73 O 73

Thir Ala Ile Phe Glu Gly Glu Cys Cys Pro Arg Wall Ser Asp 740 74. 7 O

Pro Luell Ala Asp Asn. Ile Ala Tyr Asp Ile Arg Lys Thir Cys Lieu. 760 765

Asp Ser Ser Gly Ile Ser Arg Lieu. Ser Gly Ala Wall Trp Thir Met Ala 770 775

Gly Ser Pro Cys Thr Thr Cys Glin Cys Lys Asn Gly Arg Wall 79 O 79.

Ser Wall Asp Lieu Val Cys Lieu. Glu Asn. Asn 805 810

SEO ID NO 5 LENGTH: 2915 TYPE: DNA ORGANISM: Rattus norvegicus

< 4 OOs SEQUENCE: 5 aag cactggit ttcttgttag CCCtgcttgg cgggggttct ccggagcgat 6 O gcc.gatggat gtgattittag ttttgtggitt Ctgtgtatgc accgc.cagga Cagtgttggg 12 O

Ctttgggatg gaccct gacc ttcagctgga CatCatctoa gagct cacc tggtgaacac 18O

CaccCtggga gtcacgcagg tggctggact gcacaacgc.c agtaaag cat t to tatt to a 24 O agatgtacag agagagat CC att cqgcc cc t cacgtgagt gagaa.gctga tccagctatt 3OO ccggaataag agcgagttca c ctittittggc tacagtgcag cagaaac cat ccacct cagg 360 ggtgat actg tccatc.cggg agctggagca cagctattitt gaactggaga gcagtggccC aagaga agag atacgctacc attacataca tggtggaaag CCC aggactg aggcc ct tcc 48O ctaccgcatg gCagacggac aatggcacaa t cagtgagcg c ct cit cacct 54 O

US 8,722,625 B2 39 40 - Continued

<210s, SEQ ID NO 6 &211s LENGTH: 212. TYPE : PRT <213> ORGANISM: Rattus norvegicus

<4 OOs, SEQUENCE: 6

Met Pro Met Asp Wall Ile Lell Wall Luell Trp Phe Wall Thir Ala 1. 5 15

Arg Thir Wall Luell Gly Phe Gly Met Asp Pro Asp Lell Glin Luell Asp Ile 2O 25

Ile Ser Glu Luell Asp Lell Wall Asn Thir Thir Luell Gly Wall Thir Glin Wall 35 4 O 45

Ala Gly Luell His Asn Ala Ser Ala Phe Luell Phe Glin Asp Wall Glin SO 55 6 O

Arg Glu Ile His Ser Ala Pro His Wall Ser Glu Lell Ile Glin Luell 65 70

Phe Arg Asn Ser Glu Phe Thir Phe Luell Ala Thir Wall Glin Glin 85 90 95

Pro Ser Thir Ser Gly Wall Ile Luell Ser Ile Arg Glu Lell Glu His Ser 105 11 O

Phe Glu Luell Glu Ser Ser Gly Pro Arg Glu Glu Ile Arg His 115 12 O 125

Ile His Gly Gly Pro Arg Thir Glu Ala Lell Pro Arg Met 13 O 135 14 O

Ala Asp Gly Glin Trp His Wall Ala Luell Ser Wall Ser Ala Ser His 145 150 155 160

Lell Luell Luell His Ile Asp Asn Arg Ile Glu Arg Wall Ile Asp 1.65 17O 17s

Pro Pro Glu Thir Asn Lell Pro Pro Gly Ser ASn Lell Trp Luell Gly Glin 18O 185 19 O

Arg Asn Glin His Gly Phe Phe Gly Ile Ile Glin Asp Gly 195

Ile Ile Phe Met Pro Asn Gly Phe Ile Thir Glin Cys Pro Asn Luell Asn 21 O 215 22O

Arg Thir Pro Thir Cys Ser Asp Phe Luell Ser Lell Wall Glin Gly Ile 225 23 O 235 24 O

Met Asp Luell Glin Glu Lell Lell Ala Met Thir Ala Luell Asn 245 250 255

Ala Glu Thir Arg Lell Gly Glin Luell Glu Asn His Glu Thir 26 O 265 27 O

Glin Wall Ser Gly Lell Lell Tyr Arg Asp Glin Asp Ser Trp Wall Asp 285

Gly Asp Asn Gly Asn Cys Thir Ser Gly Ala Wall Glu 29 O 295 3 OO

Arg Arg Met Ser Cys Pro Pro Luell Asn Ser Pro Asp Ser Luell Pro 3. OS 310 315 32O

Wall His Ile Ser Gly Glin Wall Cys Arg Pro Cys Ile 3.25 330 335

Gly Gly Lys Wall Lell Ala Glu Gly Glin Arg Ile Lell Thir Thir 34 O 345 35. O

Arg Glu Gly Gly Wall Luell Wall Lys Ile Thir Glu Ala 355 360 365

Pro Pro Luell Asn Cys Ser Ala Asp His Ile Lell Pro Glu Asn Glin 37 O 375 38O US 8,722,625 B2 41 42 - Continued

Cys Arg Wall Cys Pro Gly His Asn Phe Cys Ala Glu Ala Pro Lys 385 390 395 4 OO

Glu Asn Ser Glu Asn Trp ASn Thir Lys Ala Thir 4 OS 41O 415

Glu Asn Gly Ile Ser Wall Glin Gly Asn Ser Ala 425 43 O

Glu Asp Ile Asp Glu Ala Ala Met His Cys His Ala Asn 435 44 O 445

Thir Wall Wall Asn Lell Pro Gly Luell Arg Cys Asp Wall Pro 450 45.5 460

Gly Arg Wall Asp Asp Phe Ser Thir Glu His Asp Asp Cys 465 470

Gly Ser Glin His Asn Asp Asn Ala Ile Thir Asn Thir 485 490 495

Wall Glin His Ser Thir Glin Pro Gly Wall Gly Asn Gly SOO 505

Thir Ile Ala Phe Glu Glu Gly Arg Tyr Gly Gly Thir 525

Wall Pro Asn Cys Wall Pro Ser Gly Phe Thir Gly Ser 53 O 535 54 O

His Asp Ile Asp Glu Ala Glu Gly Phe Wall Glu Cys 5.45 550 555 560

His Asn Ser Arg Wall Asn Luell Pro Gly Trp His Cys Glu 565 st O sts

Arg Ser Gly Phe His Asp Asp Gly Thir Ser Lell Ser Gly Glu 585 59 O

Ser Ile Asp Ile Asp Glu Cys Ala Luell Arg Thir His Thir Trp 595 605

Asn Asp Ser Ala Cys Ile Asn Luell Ala Gly Gly Phe Asp Luell 610 615

Pro Ser Gly Pro Ser Cys Ser Gly Asp Pro His Glu Gly Luell 625 630 635 64 O

His Asn Gly Glin Wall Trp Ile Luell Arg Glu Asp Arg Ser Wall 645 650 655

Ser Lys Asp Gly Ile Phe Arg Arg Thir Ala Asp 660 665 67 O

Glin Asn Pro Asn Wall Asp Luell Phe Pro Glu Thir 675 685

Arg Wall Thir Ser Glin Lell Asp Glin Ser Gly Glin Luell 69 O. 695 7 OO

Ser Gly Asp Asn Trp Thir His Ser Glin Glin Arg Luell Glu 7 Os 71O

Gly Glu Ala Asp Cys Trp Pro Luell Ala Cys Pro Ser Lell Gly Cys Glu 72 73 O 73

Thir Ala Met Phe Glu Gly Glu Cys Pro Arg Wall Ser Asp 740 74. 7 O

Pro Luell Ala Gly Asn Ile Ala Asp Ile Arg Lys Thir Luell 760 765

Asp Ser Phe Gly Wall Ser Arg Luell Ser Gly Ala Wall Trp Thir Met Ala 770 775

Gly Ser Pro Thir Thir Lys ASn Gly Arg Wall 79 O 79.

Ser Wall US 8,722,625 B2 43 44 What is claimed is: 6. The method of claim 5, wherein said Nell1 protein or 1. A method of retarding progression or ameliorating said nucleic acid is administered by ingestion, injection or Symptoms of a cardiovascular disorder in a subject in need implantation. thereof comprising administering a Nelll protein to said sub 7. The method of claim 1 or 2, wherein said Nell1 protein ject, wherein said cardiovascular disorder is myocardial inf 5 or said nucleic acid is administered locally. arction, and wherein said Nell1 protein has an amino acid 8. The method of claim 7, wherein said Nell1 protein or sequence having at least 85% sequence identity to SEQ ID said nucleic acid is administered by injection or implantation NO: 2, 4, or 6 and stimulates differentiation of cardiomyocyte at or near the site of cardiac muscle damage. precursor cells. 9. The method of claim 1 or 2, wherein said Nell1 protein 2. A method of retarding progression or ameliorating 10 Symptoms of a cardiovascular disorder in a subject in need or said nucleic acid is administered via catheter to or near the thereof comprising administering a nucleic acid coding for a site of cardiac muscle damage. 10. The method of claim 1 or 2, wherein said Nell1 protein Nell protein to said subject, wherein said cardiovascular or said nucleic acid is administered in conjunction with cells disorder is myocardial infarction, and wherein said nucleic for the repair and regeneration of damaged cardiac muscles acid is selected from the group consisting of: 15 a) a nucleic acid coding for a Nelll protein that has an and blood vessels. amino acid sequence having at least 85% sequence iden 11. The method of claim 10, wherein said cells are cardi tity to SEQID NO: 2, 4, or 6 and stimulates differentia omyocytes. tion of cardiomyocyte precursor cells; and 12. The method of claim 10, wherein said cells are stem b) a nucleic acid having the sequence set forth in SEQID cells. NO: 1, 3, or 5; and 13. The method of claim 1, wherein said Nell1 protein has wherein said nucleic acid is in an expression vector to an amino acid sequence having at least 95% sequence identity effect expression of Nell1 in said subject. to SEQ ID NO: 2, 4, or 6 and stimulates differentiation of 3. The method of claim 1 or 2, wherein said Nell1 protein cardiomyocyte precursor cells. comprises an amino acid sequence as set forth in any one of 25 14. The method of claim 2, wherein said a nucleic acid SEQID NO: 2, SEQID NO. 4 or SEQID NO: 6. codes for a Nelll protein that has an amino acid sequence 4. The method of claim 2, wherein said expression vectoris having at least 95% sequence identity to SEQID NO: 2, 4, or a viral or non-viral vector. 6 and stimulates differentiation of cardiomyocyte precursor 5. The method of claim 1 or 2, wherein said Nell1 protein cells. or said nucleic acid is administered systemically.