US008859273B2 (12) United States Patent (10) Patent No.: US 8,859,273 B2 Sigg et al. (45) Date of Patent: *Oct. 14, 2014 (54) METHODS OF USING HCNGENESTO 2003/0204206 A1 10/2003 Padua et al. TREAT CARDACARRHYTHMAS 2004.0068299 A1 4/2004 Laske et al. 2004.0068312 A1 4/2004 Sigg et al. (75) Inventors: Daniel Sigg, St. Paul, MN (US); James FOREIGN PATENT DOCUMENTS A. Coles, Jr., Columbia, MD (US); Erica TenBroek, Roseville, MN (US) GB WOO2/O2630 * 1?2OO2 WO WO99,36538 7, 1999 WO WO O2, 19966 A 3, 2002 (73) Assignee: Medtronic, Inc., Minneapolis, MN (US) WO WO O2/50300 6, 2002 WO WO?O2/O87419 * 11 2002 (*) Notice: Subject to any disclaimer, the term of this WO WO O2/O87419 11, 2002 patent is extended or adjusted under 35 WO WO O2/O87419 A 11, 2002 U.S.C. 154(b) by 474 days. WO WO O2/O98286 12/2002 This patent is Subject to a terminal dis OTHER PUBLICATIONS claimer. Zhang etal Circulation, 2002: 106(10): 1294-9.* (21) Appl. No.: 11/022,172 Mocini et al, Ital Heart J. 2005; 6(3): 267-71.* Ecke et al Goodman & Gilman's The Pharmacological basis of (22) Filed: Dec. 22, 2004 Therapeutics, McGraw-Hill, New York, NY. pp. 77-101.* Verma and Somia (1997) Nature 389: 239-242.* (65) Prior Publication Data Verma et al Annu Rev Biochem. (2005), 74:711-38.* Opalinska et al. Nature Reviews Drug Discovery 2002.* US 2007/0218O34 A1 Sep. 20, 2007 Ngo et al., 1994. The protein Folding Problem and Tertiary Structure Prediction, pp. 491-495.* Gepstein Expert Opinion Biol Ther, 5(12): 1531-1537, 2005.* Related U.S. Application Data Menasché et al Thromb Haemost. Oct. 2005:94(4):697-701.* Ishii et al Journal of Physiology, 2001, 537, 93-100.* (60) Provisional application No. 60/532,764, filed on Dec. BrondykW Promega Notes Magazine No. 51, 1995, p. 10.* 24, 2003. Rosen, M.R., Recreating the Biological Pacemaker, The Anatomical Record Part A. 2004, vol. 180A, pp. 1046-1052. (51) Int. Cl. Rosen, M.R., Biolotical Pacemaking: In Our Lifetime?. Heart CI2N IS/00 (2006.01) Rhythm, 2005, vol. 2, No. 4, pp. 418-428. A6 IK 4.8/00 (2006.01) Benson, D.A. et al. Nucl. Acids. Res. 25: 1-6 (1997). C7H 2L/04 (2006.01) Biel, M. et al. Trends Cardiovasc. Med. 12:206-213 (2002). A6 IK38/17 (2006.01) Discher, D.J. et al., J. Biol. Chem. 273:26087-26093 (1998). Edelberg, J., Heart 86:559-562 (2001). (52) U.S. Cl. Fozzard, H. A. et al. eds. The Heart and Cardiovascular System. CPC ......... A6IK38/177 (2013.01); C12N 284.0/203 Scientific Foundations Raven Press, NY (1986). (2013.01); A61K 48/005 (2013.01); C12N Guzman, R.J. et al., Circ. Res. 73:1202-1207 (1993). 2750/14143 (2013.01) Han, X. et al., Proc. Natl. Acad. Sci. 92.9747-9751 (1995). USPC ..................... 435/320.1; 424/93.21; 536/23.1 Hardy, S. et al., J. of Virology 71:1842-1849 (1997). (58) Field of Classification Search Jiang, A. et al., J. Virol. 72: 10148-10156 (1998). CPC .................. A60K 48/005; C12N 2750/14143; Klugherz, B.D., et al., Hum. Gene Ther. 13(3):443-54 (2002). C12N 15/86, C12N 284.0/203; C12N Kurata, Y. et al., Am. J. Physiol. Heart Circ. Physiol. 283:H2074 2799/022; C12N 2502/1329; C12N H2101 (2002). 2502/1358; C12N 2510/00; C12N 5/065; Lapointe, M.C. et al., Am. J. Physiol. Heart Circ. Physiol. CO7K 14/4705 283:H1439-45 (2002). USPC ...................... 435/320.1; 424/93.2: 514/44 R Lesso, H. and Li, R.A., J.of Biol. Chem. 278 (25): 22290-22297 See application file for complete search history. (2003). Much, B. et al., J. Biol. Chem. 278: 43781-43786 (2003). (56) References Cited Prentice, H. et al., Cardiovascular Res. 35:567-574 (1997). Sakhuja, K. et al., Human Gene Therapy 14:243-254 (2003). U.S. PATENT DOCUMENTS (Continued) 5,580,777 A * 12/1996 Bernard et al. ............... 435/456 6,162,796 A 12/2000 Kaplitt et al. Primary Examiner — Anoop Singh 6,214,620 B1 4/2001 Johns et al. 6,376.471 B1 4/2002 Lawrence et al. (74) Attorney, Agent, or Firm — Mueting, Raasch & 6,451,594 B1 9, 2002 Chien et al. Gebhardt, PA. 6,506,379 B1 1/2003 Clackson et al. 6,551,821 B1 4/2003 Kandel et al. 6,979,532 B2 * 12/2005 Jansen et al. ...................... 435/4 (57) ABSTRACT 2002/0022259 A1 2/2002 Lee et al. 2002fOO25577 A1 2/2002 Goldspink The subject invention is directed to methods of treating car 2002fO155101 A1 10, 2002 Donahue diac pacing dysfunction by administering HCN genes, alone 2002/0187948 A1 12/2002 Rosen et al. or in combination with other genes. 2003, OO82153 A1 5/2003 Epstein 2003.0118988 A1 6, 2003 Kandel et al. 2003. O166593 A1 9, 2003 Chien et al. 2 Claims, 24 Drawing Sheets US 8,859.273 B2 Page 2 (56) References Cited Ulens, C. and Tytgat, J., J. Biol. Chem. 276(9):6069-6072 (2001). Weitz, D. et al., Neuron 36: 881-889 (2002). OTHER PUBLICATIONS Xu, S.Z. and Beech. D.J., Circ. Res. 88:84-87 (2001). Zhang, H. et al., Am. J. Physiol. Heart Circ. Physiol. 279:H397-H421 Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, Sec- (2000). tion 1. Cold Spring Harbor Laboratory Press (1989). Zheng, J. et al., Neuron 36:891-896 (2002). Sartiani, L. et al., Journal of Physiology 545(1):81-92 (2002). Zhong, H. et al., Nature 420:193-196 (2002). Silva, J. and Rudy, Y. Circ. Res. 92:261-263 (2003). Stieber, J., et al., J. Biol. Chem. 278 (36):33672-33680 (2003). * cited by examiner U.S. Patent Oct. 14, 2014 Sheet 1 of 24 US 8,859.273 B2 Ø U.S. Patent Oct. 14, 2014 Sheet 2 of 24 US 8,859.273 B2 U.S. Patent Oct. 14, 2014 Sheet 3 of 24 US 8,859.273 B2 U.S. Patent Oct. 14, 2014 Sheet 4 of 24 US 8,859.273 B2 U.S. Patent Oct. 14, 2014 Sheet 5 of 24 US 8,859.273 B2 500 CURRENT(pA) -500 --- nV -1500-1000 “ow. -140 mV. O 1 2 TIME (SECONDS) FIG.5 U.S. Patent Oct. 14, 2014 Sheet 7 of 24 US 8,859.273 B2 CD CD 2 O n U.S. Patent Oct. 14, 2014 Sheet 8 of 24 US 8,859.273 B2 ?qz?g?TEN?EI | dqÇIÇZVNOHOBIWONNI U.S. Patent Oct. 14, 2014 Sheet 10 of 24 US 8,859.273 B2 U.S. Patent Oct. 14, 2014 Sheet 11 of 24 US 8,859.273 B2 U.S. Patent Oct. 14, 2014 Sheet 12 of 24 US 8,859.273 B2 -130 "YSmV F.G. 12A 0 pA -> FIG. 1 2G -300 --es----1 s - -600 / 3S2 -900 /O -o- CONTROL / -O- HCN4-TRANSFECTED -1200 -140 - 120 -100 -80 -60 -40 MEMBRANE VOLTAGE, m V FIG. 1 2D U.S. Patent 000Z009|000||009 Sul‘BW|| U.S. Patent Oct. 14, 2014 Sheet 15 of 24 US 8,859.273 B2 (Wu) N3 no U.S. Patent Oct. 14, 2014 Sheet 16 of 24 US 8,859.273 B2 O s 5 3Co n s is a Sos Co so ea (Nul) 38WION (Wu) N3&n) U.S. Patent Oct. 14, 2014 Sheet 18 of 24 US 8,859.273 B2 -40 mV. F -140 mV ELCITED CURRENT TRACES FITTED TO GET TIME CONSTANT OF ACTIVATION TAL CURRENT AT THIS POINT USED FOR VOLTAGE-DEPENDENT ACTIVATION CURVES FIG. 18 U.S. Patent Oct. 14, 2014 Sheet 19 of 24 US 8,859.273 B2 FIG. 19 U.S. Patent Oct. 14, 2014 Sheet 20 of 24 US 8,859.273 B2 40 mV -40 mV -100 mV -140 mV CURRENT AT THIS POINT USED FOR DETERMINATION OF Erey FIG.2O U.S. Patent Oct. 14, 2014 Sheet 21 of 24 US 8,859.273 B2 FIG.21 U.S. Patent Oct. 14, 2014 Sheet 22 of 24 US 8,859.273 B2 0.0 hCN4trunc hHCN4trunc + cAMP hCN4 hCN4 - CAMP -140 -120 -100 -80 -60 -40 ACTIVATION VOLTAGE (mV) FIG.22 U.S. Patent Oct. 14, 2014 Sheet 23 of 24 US 8,859.273 B2 -o- hCN4trunc -o- hCN4trunc + CAMP -- hCN4 -O- hCN4 - CAMP s2 10 *p<0.05 (ONLY AT -90 mV) -140 -130 - 120 -110 -100 -90 ACTIVATION VOLTAGE (mV) FIG.23 U.S. Patent Oct. 14, 2014 Sheet 24 of 24 US 8,859.273 B2 VOLTAGE (mV) -o- hCN4trunc -- hCN4 FIG.24 US 8,859,273 B2 1. 2 METHODS OF USING HCN GENESTO different isoforms have been identified in vertebrates (HCN1, TREAT CARDACARRHYTHMAS HCN2. HCN3 and HCN4) and all except HCN3 have been found in the heart. HCN3 is specifically expressed in neurons. CROSS-REFERENCES TO RELATED HCN channels directly interact with intracellular cAMP so APPLICATIONS that an increase in cAMP levels results in increased If and more positive activation potentials. This increase thereby The present application claims priority under Title 35, accelerates the heart rate (HR) in response to sympathetic United States Code, S119 to provisional application U.S. stimulation. In contrast, muscarinic stimulation slows the Patent application Ser. No. 60/532,764 filed Dec. 24, 2003. heart rate in part due to a decrease in cAMP levels and a 10 resulting reduction of I, and more negative activation poten FIELD OF INVENTION tials.