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(12) United States Patent (10) Patent No.: US 9,109,232 B2 Schwartz Et Al

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(12) United States Patent (10) Patent No.: US 9,109,232 B2 Schwartz Et Al US009 109232B2 (12) United States Patent (10) Patent No.: US 9,109,232 B2 Schwartz et al. (45) Date of Patent: Aug. 18, 2015 (54) ETS2 AND MESP1 GENERATE CARDIAC Islas et al., Transcription factors ETS2 and MESP1 transdifferentiate PROGENITORS FROM FIBROBLASTS human dermal fibroblasts into cardiac progenitors; PNAS, Published online before print Jul. 23, 2012, doi: 10.1073/pnas. 11202991.09, 2012.* (71) Applicants: University of Houston, Houston, TX Melotti et al., Ets-2 and c-Myb Act Independently in Regulating (US); Texas Heart Institute, Houston, Expression of the Hematopoietic StemCell Antigen CD34:JBC, vol. TX (US); The Texas A&M University 269, No. 41, pp. 25303-25309, 1994.* System, College Station, TX (US) Takahashi et al., Induction of pluripotent stem cells from adulthuman fibroblasts by defined factors; Cell, vol. 131, pp. 861-872, 2007.* Naldini et al. In vivo gene delivery and stable transduction of (72) Inventors: Robert J. Schwartz, Houston, TX (US); nondividing cells by a lentiviral vector; Science, vol. 272, pp. 263 Vladimir N. Potaman, Houston, TX 267, 1996.* (US); Jose Francisco Islas, Houston, TX European Patent Office; Office Action; European Application No. (US) 117114082; Jul 22, 2013. European Patent Office; Response to Office Action; European Appli (73) Assignees: University of Houston, Houston, TX cation No. 11711408.2; Aug. 30, 2013. Chinese Patent Office; Office Action; Chinese Patent Application No. (US); Texas Heart Institute, Houston, 2011800 19561.0; Sep. 18, 2013. TX (US); The Texas A&M University Chinese Patent Office; Office Action (English translation); Chinese System, College Station, TX (US) Patent Application No. 2011800 19561.0; Sep. 18, 2013. European Patent Office; International Search Report and Written (*) Notice: Subject to any disclaimer, the term of this Opinion; PCT Application No. PCT/US2011/027160; Jul. 6, 2011. patent is extended or adjusted under 35 Bondue A., et al.; Mesplacts as a Master Regulator of Multipotent Cardiovascular Progenitor Specification; Cell Stem Cell 3, 69-84, U.S.C. 154(b) by 0 days. Jul. 2008. Lindsley, R.C.. etal; Mespl Coordinately Regulates Cardiovascular (21) Appl. No.: 13/915,677 Fate Restriction and Epithelial-Mesenchymal Transition in Differen tiating ESCs: Cell Stem Cell 3, 55-68, Jul. 2008. (22) Filed: Jun. 12, 2013 Lie-Venema, H., et al.; Ets-1 and Ets-2 Transcription Factors are Essential for Normal Coronary and Myocardial Development in (65) Prior Publication Data Chicken Embryos; Circulaton Research, vol.92, No. 7,749-756, Apr. 18, 2003. US 2014/0004604 A1 Jan. 2, 2014 Bergmann, O. etal. Evidence for cardiomyocyte renewal in humans. Science 324: 5923, 98-102, 2009. Beh, J. et al. FoxF is essential for FGF-induced migration of heart progenitor cells in the ascidian Ciona intestinalis. Development 134: Related U.S. Application Data 3297-3305, 2007. (62) Division of application No. 13/040,611, filed on Mar. Davidson, B. Ciona intestinalis as a model for cardiac development. Semin. Cell Dev. Biol. 18:16-26, 2007. il. 4, 2011, now Pat. No. 8,486,701. Yu, J. Induced pluripotent stem cell lines derived from human (60) Provisional application No. 61/339,509, filed on Mar. somatic cells. Science 3 18:1917-1920, 2007. Davidson, B. and Levine, M. Evolutionary origins of the vertebrate 5, 2010. heart: Specification of the cardiac lineage in Ciona intestinalis. Proc. Natl. Acad. Sci. USA 100: 11469-11473. (51) Int. Cl. Imai, K.S., Satoh, N. and Satou, Y. A Twist-like bHLH gene is a CI2N 15/85 (2006.01) downstream factor of an endogenous FGF and determines CI2N 5/077 (2010.01) mesenchymal fate in the ascidian embryos. Development 130:4461 CI2N 5/074 (2010.01) 4472, 2003. Imai, K.S., Hino, K., Yagi, K., Satoh, N. and Satou, Y. Gene expres (52) U.S. Cl. sion profiles of transcription factors and signaling molecules in the CPC .............. CI2N 15/85 (2013.01); C12N5/0657 ascidian embryo: towards a comprehensive understanding of gene (2013.01); C12N5/0696 (2013.01); C12N networks. Development 131: 4047-4058, 2004. 2501/40 (2013.01); C12N 2501/60 (2013.01); Kitajima, S. et al. Mesp1 and Mesp2 are essential for the develop CI2N2506/1307 (2013.01); C12N 2510/00 ment of cardiac mesoderm. Development 127: 3215-3226, 2000. (2013.01) Moretti, A. et al. Multipotent embryonic isl+ progenitor cells lead to (58) Field of Classification Search cardiac, Smooth muscle and endothelial cell diversification. Cell 127: None 1151-1165, 2006. See application file for complete search history. (Continued) (56) References Cited Primary Examiner — Jennifer Dunston Assistant Examiner — Addison D Ault U.S. PATENT DOCUMENTS (74) Attorney, Agent, or Firm — Jackson Walker L.L.P. 2009/0227032 A1 9/2009 Yamanaka et al. (57) ABSTRACT A method for modulating cell differentiation capabilities FOREIGN PATENT DOCUMENTS using heterologous gene expression. Some embodiments of WO 2010017562 A2 2, 2010 the invention relate to a method for inducing a cardiac pro genitor cell by delivering a reprogramming factor to the cell, OTHER PUBLICATIONS wherein the reprogramming factor comprises ETS2 oracom Steinhauser et al., Regeneration of the heart; EMBO Molecular bination of ETS2 and Mesp1. Medicine, vol. 3,701-712, 2011.* 10 Claims, 13 Drawing Sheets US 9,109,232 B2 Page 2 (56) References Cited Chinese Patent Office; Response to Office Action; Chinese Patent Application No. 2011800 195610; Jan. 27, 2014. OTHER PUBLICATIONS European Patent Office; Response to Office Action; European Appli cation No. 11711408.2: Jun. 25, 2014. Park, I.H. et al. Reprogramming of human Somatic cells to Chinese Patent Office; Second Office Action (in Chinese); Chinese pluripotency with defined factors. Nature 451: 141-146, 2008. Patent Application No. 2011800 19561.0; Jun. 3, 2014. Saga, Y. et al. Mespl is expressed in the heart precursor cells and Chinese Patent Office; Second Office Action (in English); Chinese required for the formation of a single heart tube. Development Patent Application No. 2011800 19561.0; Jun. 3, 2014. 126:3437-3447, 1999. Chinese Patent Office; Response to Second Office Action; Chinese Saga, Y. Kitajima, S. and Miyagawa-Tomita, SMespl expression is Patent Application No. 2011800 19561.0; Aug. 15, 2014. the earliest sign of cardiovascular development. Trends Cardiovasc Chinese Patent Office; Third Office Action (in Chinese); Chinese Med. 10:345-352, 2000. Patent Application No. 2011800 19561.0; Dec. 8, 2014. Takahashi, K. et al. Induction of pluripotent stem cells from adult Chinese Patent Office; Third Office Action (in English); Chinese human fibroblasts by defined factors. Cell 131:861-872, 2007. Patent Application No. 2011800 19561.0; Dec. 8, 2014. European Patent Office; Written Opinion, PCT Application No. PCT/ Chinese Patent Office; Response to Third Office Action; Chinese US2011/027160; Feb. 7, 2012. Patent Application No. 2011800 19561.0; Feb. 11, 2015. European Patent Office; Office Action; European Application No. 11711408.2: Mar. 28, 2014. * cited by examiner U.S. Patent Aug. 18, 2015 Sheet 1 of 13 US 9,109,232 B2 CN V aL . U.S. Patent Aug. 18, 2015 Sheet 2 of 13 US 9,109,232 B2 U.S. Patent Aug. 18, 2015 Sheet 3 of 13 US 9,109,232 B2 i s Y A. (f) ad s H O U.S. Patent Aug. 18, 2015 Sheet 4 of 13 US 9,109,232 B2 t IX L Xe 2 XOS % O SoueN pdeo NA W Od-O 398HWN SOONW/N U.S. Patent Aug. 18, 2015 Sheet 5 of 13 US 9,109,232 B2 9090n-longº-e-vass %,zº OzT 0 „01golz01t01001 0 08 09 Oty 001 09 U.S. Patent Aug. 18, 2015 Sheet 6 of 13 US 9,109,232 B2 |0 09.T. 0 8 OZ 08 09 U.S. Patent Aug. 18, 2015 Sheet 7 of 13 US 9,109,232 B2 U.S. Patent Aug. 18, 2015 Sheet 8 of 13 US 9,109,232 B2 W/3TdS3WZSLEA itviv? G“ZXXIN ITSI 0:2-HEUN SX81 £OHWN ZOTW St7XO ZSIB TdS3W U.S. Patent Aug. 18, 2015 Sheet 9 of 13 US 9,109,232 B2 £OHWN ZOTIVN U.S. Patent Aug. 18, 2015 Sheet 10 of 13 US 9,109,232 B2 U.S. Patent Aug. 18, 2015 Sheet 11 of 13 US 9,109,232 B2 XIT00800900t?00Z 8Post XIT00800900?700Z OZI 06 09 08 U.S. Patent Aug. 18, 2015 Sheet 12 of 13 US 9,109,232 B2 V U.S. Patent Aug. 18, 2015 Sheet 13 of 13 US 9,109,232 B2 zgi requinulº,seosvoºzoszowoezo 3. anuu red Saeeg US 9,109,232 B2 1. 2 ETS2 AND MESP1. GENERATE CARDAC ited number of transcriptional factors important for maintain PROGENITORS FROM FIBROBLASTS ing self renewal and pluripotency has been reported by Yamanaka's, Thomson's and Daley's groups (Takahashi et This application claims priority to U.S. Provisional Patent al., 2007;Yu et al., 2007: Parket al., 2008). One aspect of the Application Ser. No. 61/339,509, filed on Mar. 5, 2010, 5 present invention provides a means of reprogramming the entitled ETS2 AND MESP1. GENERATE CARDIAC PRO Somatic cells and directed differentiation into cardiac pro GENITORS FROM FIBROBLASTS, the entire content of genitor cells. Therefore, one embodiment of this application which is hereby incorporated by reference. provides a way to test a unique regulatory paradigm that ETS2 and Mesp1 are transformative, and unlike NKX2.5 and FIELD OF THE INVENTION 10 ISL1, convert non-embryonic normal human dermal fibro blasts (NHDFs) into primary cardiac progenitors. Another One aspect of the present invention relates generally to the aspect of the present application was to elucidate the role of field of cell differentiation, and more specifically to a strategy Mesp1 in the regulatory hierarchy directing the appearance of for cardiovascular tissue regeneration via the isolation, cardiac progenitors.
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