Systems and Methods to Treat Cardiac Pacing Conditions

Abstract

The method may include administering to a subject in need thereof an effective amount of an HCN polynucleotide. The HCN polynucleotide includes a nucleotide sequence encoding an HCN polypeptide having channel activity. The amino acid sequence of the HCN polypeptide and the amino acid sequence of a reference polypeptide have at least 80% identity, where the reference polypeptide begins with an amino acid selected from amino acids 92-214 and ends with an amino acid selected from amino acids 723-1188 of SEQ ID NO:8. An example of a reference polypeptide is amino acids 214-723 of SEQ ID NO:8. The HCN polynucleotide may be DNA or RNA.

Description

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 60/351,836 filed Jun. 4, 2010 and cross reference is hereby made to the commonly assigned related U.S. application Ser. No. ______ (Attorney Docket Number P0036355.02), entitled "Compositions and Methods to Treat Cardiac Pacing Conditions" and U.S. application Ser. No. ______ (Attorney Docket Number P0036355.04), entitled "Compositions to Treat Cardiac Pacing Conditions), filed concurrently herewith and incorporated herein by reference in its entirety.

BACKGROUND

[0002] Cardiac contraction in a healthy human heart is initiated by spontaneous excitation of the sinoatrial ("SA") node, which is located in the right atrium. The electric impulse generated by the SA node travels to the atrioventricular ("AV") node where it is transmitted to the bundle of His and to the Purkinje network. The fibers in the Purkinje network branch out in many directions to facilitate coordinated contraction of the left and right ventricles. In some disease states, the heart loses some of its natural capacity to pace properly. Such dysfunction is commonly treated by implanting a pacemaker.

[0003] While effectively improving the lives of many patients, implantable pacemakers have certain technical limitations. For example, implantable pacemakers rely on a self-contained power source such as a battery and consequently have a limited lifetime before the power source is in need of replacement. Implantable pacemakers also require pacing leads, which may fail and result in loss of therapy. Hence, an otherwise healthy patient may require multiple surgeries to replace the power source, leads, or the entire implantable pacemaker. Also, implantable pacemakers may not directly respond to physiological signals similar to the way the SA node responds to such signals.

[0004] Recently, biological methods of influencing a patient's cardiac cells have been developed, some of which include administering biopharmaceutical compositions that affect cardiac pacing. Developments in genetic engineering have produced methods for genetically modifying cardiac cells to modify non-pacemaking cardiac cells to pacemaker-like cardiac cells or regenerate the pacing capabilities of cells in the conduction system of the heart. For example, Johns and Marban (U.S. Pat. No. 6,214,620) describes a method for modulating the excitability of ventricular cells by controlling the regulation of the expression of certain ion channels (e.g. K.sup.+ channels). Marban and Li (PCT Publication No. WO 02/087419) and Sigg et al. (PCT Publication No. WO 05/062890A3) describe methods and systems for modulating electrophysiological behavior of cardiac cells by genetic modification of inwardly rectifying K.sup.+ channels (I_K1) in quiescent ventricular cells.

[0005] Another recent biological approach for modulating cardiac pacing involves implanting into the SA node or other suitable heart regions cells having particular ion channels that are commonly referred to as hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels. For example, see Rosen and Robinson (PCT Publication No. WO 02/098286) and Sigg et al. (PCT Publication No. WO 05/062958A2). Physiologically originating in the SA node, the HCN channels play a prominent role in the control of rhythmic electrical heart activity. Cyclic nucleotides modulate the HCN channel activity, and channel activation occurs upon hyperpolarization rather than depolarization. There are four isoforms of HCN channels (HCN1-4), and each has greater or lesser prevalence in different heart regions. Because the HCN isoforms are directly involved in pacemaker current modulation and activation, implantation of HCN-expressing cells into cardiac tissue that is diseased or experiencing conduction blockage is a viable method for regulating cardiac pacemaker function.

SUMMARY OF THE INVENTION

[0006] The present invention provides methods for treating a cardiac pacing condition. The method may include administering to a subject in need thereof an effective amount of an HCN polynucleotide. The HCN polynucleotide includes a nucleotide sequence encoding an HCN polypeptide having channel activity. The amino acid sequence of the HCN polypeptide and the amino acid sequence of a reference polypeptide have at least 80% identity, where the reference polypeptide begins with an amino acid selected from amino acids 92-214 and ends with an amino acid selected from amino acids 723-1188 of SEQ ID NO:8. An example of a reference polypeptide is amino acids 214-723 of SEQ ID NO:8. The HCN polynucleotide may be DNA or RNA. The HCN polynucleotide may be present in a vector, such as a viral vector (including, for instance, a single strand adeno-associated virus or a self complementary adeno-associated virus), a transposon vector, or a plasmid vector.

[0007] The HCN polynucleotide administered to the subject may be present in a genetically modified cell, and the HCN polynucleotide may be integrated in the genomic DNA of the genetically modified cell, or may be present as part of an extra-chromosomal vector in the cell. The method may further include administering to the subject a second HCN polynucleotide.

[0008] The HCN polynucleotide may be administered by introduction of the HCN polynucleotide into cardiac atrium cells or cardiac ventricle cells. Various methods may be used to introduce the HCN polynucleotide, including, for instance, a syringe or a catheter.

[0009] The present invention also provides a composition that includes an HCN polynucleotide present in a vector. Preferably, the HCN polynucleotide includes a nucleotide sequence encoding an HCN polypeptide having channel activity, wherein the amino acid sequence of the HCN polypeptide and the amino acid sequence of a reference polypeptide have at least 80% identity, and where the reference polypeptide begins with an amino acid selected from amino acids 92-214 and ends with an amino acid selected from amino acids 723-1188 of SEQ ID NO:8. The composition may further include a pharmaceutically acceptable carrier. The vector may be a viral vector (including, for instance, a single strand adeno-associated virus or a self complementary adeno-associated virus), a transposon vector, or a plasmid vector.

[0010] Further provided by the invention is a genetically modified cell that includes an HCN polynucleotide. Preferably, the HCN polynucleotide includes a nucleotide sequence encoding an HCN polypeptide having channel activity, wherein the amino acid sequence of the HCN polypeptide and the amino acid sequence of a reference polypeptide have at least 80% identity, and where the reference polypeptide begins with an amino acid selected from amino acids 92-214 and ends with an amino acid selected from amino acids 723-1188 of SEQ ID NO:8. The HCN polynucleotide may be integrated in the genomic DNA of the genetically modified cell, or may be present as part of an extra-chromosomal vector in the cell. The genetically modified cell may be part of a composition, and the composition may include a pharmaceutically acceptable carrier.

[0011] The present invention is also directed to methods that include identifying a distal end of a catheter at a tissue site of a patient and delivering a fluid or polymer that contains an HCN polynucleotide to the tissue site of the patient via the catheter. The identifying may include an electrically sensing contact between a distal end of a catheter and the tissue site of the patient.

[0012] The method may further include delivering an electrical stimulus to the cardiac tissue site of the patient to enhance transfer of the HCN polynucleotide to the tissue site via electroporation. The electrical stimulus may be delivered to the tissue site via the catheter, for instance, via an electrode coupled to the catheter and an electrode coupled to a distal tip of a probe extending from the catheter. An example of delivering the electrical stimulus to the tissue site via the catheter may include delivering the electrical stimulus to the tissue site via an electrode coupled to the catheter and a distal tip of a probe extending from the catheter, the distal tip of the probe is formed from an electrically conductive material. The electrical stimulus may be delivered to the tissue site via an implanted medical device. The electrical stimulus delivered to the tissue site may include a stimulation pulse, or a series of stimulation pulses. The delivering fluid to the tissue site of the patient via the catheter may include delivering fluid to the tissue site of the patient via one or more exit ports of a distal tip of a probe extending from the catheter. The distal tip of the probe may include a needle or may be a helix shaped distal tip. The distal tip of the probe may extend from a body of the catheter upon sensing contact between the tissue site of the patient and the catheter.

[0013] Also provided by the present invention is a system that includes a fluid supply, a catheter, and a power supply to generate an electrical stimulus that is delivered to the tissue site. The system may include a pump to drive fluid from the fluid supply through the catheter, and/or the power supply may include an implanted medical device that delivers the electrical stimulus to the tissue site. The implanted medical device may include one of an implantable pulse generator, an implantable cardioverter/defibrillator, and an implantable pacemaker/cardioverter/defibrillator. The fluid supply may include an implanted fluid reservoir. The power supply may be coupled to the catheter, and the catheter may deliver the electrical stimulus to the tissue site.

[0014] The catheter of the system may include a catheter body that defines an inner lumen, a probe within the inner lumen that delivers fluid from the fluid supply to a tissue site of a patient, and at least one electrode coupled to the catheter to detect contact between the catheter and the tissue site, wherein the fluid comprises an HCN polynucleotide described herein. The probe may include a distal tip made from an electrically conductive material, and the electrode may be coupled to the catheter body, where the catheter delivers the electrical stimulus to the tissue site via the electrode coupled to the catheter body and the distal tip of the probe. The catheter may include a pair of electrodes, a first electrode coupled to the probe and a second electrode coupled to the catheter body, where the catheter delivers the electrical stimulus to the tissue site via the electrode coupled to the catheter body and the electrode coupled to the probe. The electrical stimulus delivered to the tissue site may include a stimulation pulse or a series of stimulation pulses. The fluid may include a polymer.

[0015] As used herein, an "isolated" substance is one that has been removed from its natural environment, produced using recombinant techniques, or chemically or enzymatically synthesized. For instance, a polypeptide, a polynucleotide, or a cell can be isolated. Preferably, a substance is purified, i.e., is at least 60% free, preferably at least 75% free, and most preferably at least 90% free from other components with which they are naturally associated.

[0016] As used herein, the term "polynucleotide" refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxynucleotides, and includes both double- and single-stranded RNA and DNA. A polynucleotide can be obtained directly from a natural source, or can be prepared with the aid of recombinant, enzymatic, or chemical techniques. A polynucleotide can be linear or circular in topology. A polynucleotide may be, for example, a portion of a vector, such as an expression or cloning vector, or a fragment. A polynucleotide may include nucleotide sequences having different functions, including, for instance, coding regions, and non-coding regions such as regulatory regions.

[0017] As used herein, the terms "coding region" and "coding sequence" are used interchangeably and refer to a nucleotide sequence that encodes a polypeptide and, when placed under the control of appropriate regulatory sequences, expresses the encoded polypeptide. The boundaries of a coding region are generally determined by a translation start codon at its 5' end and a translation stop codon at its 3' end. A "regulatory sequence" is a nucleotide sequence that regulates expression of a coding sequence to which it is operably linked. Non-limiting examples of regulatory sequences include promoters, enhancers, transcription initiation sites, translation start sites, translation stop sites, and transcription terminators. The term "operably linked" refers to a juxtaposition of components such that they are in a relationship permitting them to function in their intended manner. A regulatory sequence is "operably linked" to a coding region when it is joined in such a way that expression of the coding region is achieved under conditions compatible with the regulatory sequence.

[0018] A polynucleotide that includes a coding region may include heterologous nucleotides that flank one or both sides of the coding region. As used herein, "heterologous nucleotides" refer to nucleotides that are not normally present flanking a coding region that is present in a wild-type cell. For instance, a coding region present in a wild-type cell and encoding an HCN polypeptide is flanked by homologous sequences, and any other nucleotide sequence flanking the coding region is considered to be heterologous. Examples of heterologous nucleotides include, but are not limited to regulatory sequences. Typically, heterologous nucleotides are present in a polynucleotide of the present invention through the use of standard genetic and/or recombinant methodologies well known to one skilled in the art. A polynucleotide of the present invention may be included in a suitable vector.

[0019] As used herein, an "exogenous polynucleotide" refers to a polynucleotide that has been introduced into a cell by artificial means. As used herein, the term "endogenous polynucleotide" refers to a polynucleotide that is normally or naturally found in a cell. An "endogenous polynucleotide" is also referred to as a "native polynucleotide."

[0020] The terms "complement" and "complementary" as used herein, refer to the ability of two single stranded polynucleotides to base pair with each other, where an adenine on one strand of a polynucleotide will base pair to a thymine or uracil on a strand of a second polynucleotide, and a cytosine on one strand of a polynucleotide will base pair to a guanine on a strand of a second polynucleotide. Two polynucleotides are complementary to each other when a nucleotide sequence in one polynucleotide can base pair with a nucleotide sequence in a second polynucleotide. For instance, 5'-ATGC and 5'-GCAT are complementary. The term "substantial complement" and cognates thereof as used herein, refer to a polynucleotide that is capable of selectively hybridizing to a specified polynucleotide under stringent hybridization conditions. Stringent hybridization can take place under a number of pH, salt and temperature conditions. The pH can vary from 6 to 9, preferably 6.8 to 8.5. The salt concentration can vary from 0.15 M sodium to 0.9 M sodium, and other cations can be used as long as the ionic strength is equivalent to that specified for sodium. The temperature of the hybridization reaction can vary from 30° C. to 80° C., preferably from 45° C. to 70° C. Additionally, other compounds can be added to a hybridization reaction to promote specific hybridization at lower temperatures, such as at or approaching room temperature. Among the compounds contemplated for lowering the temperature requirements is formamide. Thus, a polynucleotide is typically substantially complementary to a second polynucleotide if hybridization occurs between the polynucleotide and the second polynucleotide. As used herein, "specific hybridization" refers to hybridization between two polynucleotides under stringent hybridization conditions.

[0021] As used herein, the term "polypeptide" refers broadly to a polymer of two or more amino acids joined together by peptide bonds. The term "polypeptide" also includes molecules which contain more than one polypeptide joined by a disulfide bond, or complexes of polypeptides that are joined together, covalently or noncovalently, as multimers (e.g., dimers, tetramers). Thus, the terms peptide, oligopeptide, enzyme, and protein are all included within the definition of polypeptide and these terms are used interchangeably. It should be understood that these terms do not connote a specific length of a polymer of amino acids, nor are they intended to imply or distinguish whether the polypeptide is produced using recombinant techniques, chemical or enzymatic synthesis, or is naturally occurring.

[0022] As used herein, "identity" refers to sequence similarity between two polypeptides or two polynucleotides. The sequence similarity between two polypeptides is determined by aligning the residues of the two polypeptides (e.g., a candidate amino acid sequence and a reference amino acid sequence, such as SEQ ID NO:2, 4, 6, or 8, or a portion thereof) to optimize the number of identical amino acids along the lengths of their sequences; gaps in either or both sequences are permitted in making the alignment in order to optimize the number of shared amino acids, although the amino acids in each sequence must nonetheless remain in their proper order. The sequence similarity is typically at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity. Sequence similarity may be determined, for example, using sequence techniques such as the BESTFIT algorithm in the GCG package (Madison Wis.), or the Blastp program of the BLAST 2 search algorithm, as described by Tatusova, et al. (FEMS Microbiol Lett 1999, 174:247-250), and available through the World Wide Web, for instance at the internet site maintained by the National Center for Biotechnology Information, National Institutes of Health. Preferably, sequence similarity between two amino acid sequences is determined using the Blastp program of the BLAST 2 search algorithm. Preferably, the default values for all BLAST 2 search parameters are used, including matrix=BLOSUM62; open gap penalty=11, extension gap penalty=1, gap x_dropoff=50, expect=10, wordsize=3, and optionally, filter on. In the comparison of two amino acid sequences using the BLAST search algorithm, structural similarity is referred to as "identities."

[0023] The sequence similarity between two polynucleotides is determined by aligning the residues of the two polynucleotides (e.g., a candidate nucleotide sequence and a reference nucleotide sequence, such as SEQ ID NO:1, 3, 5, 7, or a portion thereof) to optimize the number of identical nucleotides along the lengths of their sequences; gaps in either or both sequences are permitted in making the alignment in order to optimize the number of shared nucleotides, although the nucleotides in each sequence must nonetheless remain in their proper order. The sequence similarity is typically at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity. Sequence similarity may be determined, for example, using sequence techniques such as GCG FastA (Genetics Computer Group, Madison, Wis.), MacVector 4.5 (Kodak/IBI software package) or other suitable sequencing programs or methods known in the art. Preferably, sequence similarity between two nucleotide sequences is determined using the Blastn program of the BLAST 2 search algorithm, as described by Tatusova, et al. (1999, FEMS Microbiol Lett., 174:247-250), and available through the World Wide Web, for instance at the internet site maintained by the National Center for Biotechnology Information, National Institutes of Health. Preferably, the default values for all BLAST 2 search parameters are used, including reward for match=1, penalty for mismatch=-2, open gap penalty=5, extension gap penalty=2, gap x_dropoff=50, expect=10, wordsize=11, and optionally, filter on. In the comparison of two nucleotide sequences using the BLAST search algorithm, sequence similarity is referred to as "identities."

[0024] Conditions that "allow" an event to occur or conditions that are "suitable" for an event to occur, such as an enzymatic reaction, or "suitable" conditions are conditions that do not prevent such events from occurring. Thus, these conditions permit, enhance, facilitate, and/or are conducive to the event.

[0025] As used herein, "genetically modified cell" refers to a cell into which has been introduced an exogenous polynucleotide, e.g., an expression vector. For example, a cell is a genetically modified cell by virtue of introduction into a suitable cell of an exogenous polynucleotide that is foreign to the cell, or an exogenous polynucleotide that encodes a polypeptide that is normally present in the cell. "Genetically modified cell" also refers to a cell that has been genetically manipulated such that endogenous nucleotides have been altered. For example, a cell is a genetically modified cell by virtue of introduction into a suitable cell of an alteration of endogenous nucleotides. For instance, an endogenous coding region could be mutagenized. Such mutations may result in a polypeptide having a different amino acid sequence than was encoded by the endogenous polynucleotide. Another example of a genetically modified cell is one having an altered regulatory sequence, such as a promoter, to result in increased or decreased expression of an operably linked endogenous coding region.

[0026] The term "and/or" means one or all of the listed elements or a combination of any two or more of the listed elements.

[0027] The words "preferred" and "preferably" refer to embodiments of the invention that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the invention.

[0028] The terms "comprises" and variations thereof do not have a limiting meaning where these terms appear in the description and claims.

[0029] Unless otherwise specified, "a," "an," "the," and "at least one" are used interchangeably and mean one or more than one.

[0030] Also herein, the recitations of numerical ranges by endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

[0031] The above summary of the present invention is not intended to describe each disclosed embodiment or every implementation of the present invention. The description that follows more particularly exemplifies illustrative embodiments. In several places throughout the application, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list.

BRIEF DESCRIPTION OF THE FIGURES

[0032] FIG. 1. Multiple sequence alignment of a human HCN1 (SEQ ID NO:2), a human HCN2 (SEQ ID NO:4), a human HCN3 (SEQ ID NO:6), and a human HCN4 (SEQ ID NO:8). The six α-helical segments (S1-S6), the pore helix (PoreH), the selectivity filter (SF), and the cyclic-nucleotide binding domain (CNBD) are depicted.

[0033] FIG. 2. Nucleotide sequence encoding a human HCN1 (SEQ ID NO:1), a human HCN2 (SEQ ID NO:3), a human HCN3 (SEQ ID NO:5), and a human HCN4 (SEQ ID NO:7), and the corresponding amino acid sequences of the HCN1 (SEQ ID NO:2), the HCN2 (SEQ ID NO:4), the HCN3 (SEQ ID NO:6), and the HCN4 (SEQ ID NO:8) polypeptides.

[0034] FIG. 3. Biological pacemaker from AV node ablated canine injected with the hHCN4t coding region carried by adenovirus vector. A significant increase in ventricular heart rate was observed following Adv-hHCN4t administration. Biological pacemaker reached a peak on day 2 and maintained a rate at peak level through termination at one week.

[0035] FIG. 4. Sequence homology between canine and human HCN4 coding regions. In comparison with each other, human and canine share high similarity in RNA sequence blocks of I, II and III (92, 95%, and 91% respectively). Other areas (in front of I block, between I to II and II to III blocks, and after block III) display significant distinctive sequences within these two species.

[0036] FIG. 5. Schematic diagram of proposed membrane topology of a HCN channel subunit.

[0037] FIG. 6. Three different super-truncated versions of HCN4 (HCN4st) in comparison with HCN4 wt and HCN4t.

[0038] FIG. 7. Schematic diagram of generating super-truncated HCN4 plasmids for biological pacemaker gene therapy.

[0039] FIG. 8. Confirmation of functionality of HCN4st1 using patch clamp experiments. The panel on the left shows current responses as the cell membrane is pulsed to increasing hyperpolarizing voltages from a positive holding potential at which HCN4 channels are closed. The right panel shows the current-voltage relationship using two different pulse protocols. The electrophysiology of the channel, including reversal potential, is unaltered compared to its wild typed (wtHCN4) and truncated (HCN4t) counterparts.

[0040] FIG. 9. Cell image and recordings obtained using a micro-electrode array (MEA) of non-transduced neo-natal rat venetricular myocytes (NRVMs).

[0041] FIG. 10. Cell image and MEA recordings obtained from NRVM cells transduced with 10¹¹ vg/ml of B-AAV1-HCN4st1 virus.

[0042] FIG. 11. MEA recordings obtained from NRVMs transduced with 10¹⁰-10⁸ vg/ml of B-AAV1-HCN4st1 virus.

[0043] FIG. 12. Time course of NRVMs beating rates following transduction with B-AAV1-HCN4st1 at various doses.

[0044] FIG. 13. Time course of NRVMs beating rates after transduction with B-AAV1-empty vector at various doses.

[0045] FIG. 14. Biological pacemaker from AV node ablated canine injected with hHCN4st1 gene.

[0046] FIG. 15. Nucleotide sequence of HCN4 with MyC fusion.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Polypeptides

[0047] The present invention includes isolated polypeptides having hyperpolarization-activated and cyclic nucleotide-gated channel activity, also referred to herein as channel activity. A polypeptide having channel activity is referred to herein as an HCN polypeptide. An HCN polypeptide includes at least three domains, the transmembrane core, the cytosolic N-terminal domain, and the cytosolic C-terminal domain (Wahl-Schott and Biel, 2009, Coll. Mol. Life Sci., 66:470-494). The transmembrane core includes six α-helical segments (S1-S6) and an ion conducting pore loop between S5 and S6 (see FIG. 6). A highly conserved asparagine residue is typically present in the extracellular loop between S5 and the pore loop (Much et al., 2003, J. Biol. Chem. 278:43781-43786). The pore loop typically includes a glycine-tyrosine-glycine (GYG) motif. The voltage sensor of HCN polypeptides is formed by a charged S4-helix carrying a series of arginine or lysine residues regularly spaced at every third position (Vaca et al., 2000, FEBS Lett., 479:35-40), optionally including a serine residue (Chen et al., 2000, J. Biol. Chem., 275:36465-36471). There is evidence suggesting that the loop connecting the S4 with the S5 segment plays a role in conferring the differential response to voltage (Long et al., 2005, Science, 309:903-908, Decher et al., 2004, J. Biol. Chem., 279:13859-13865, and Prole et al., 2006, J. Gen. Physiol. 128:273-282). The proximal portion of the cytosolic C-terminus mediates the sensitivity of HCN polypeptides to cAMP (Zagotta et al., 2003, Nature, 425:200-205). This part of an HCN polypeptide includes a cyclic nucleotide-binding domain of about 120 amino acids (CNBD) and an 80 amino acid long linker region that connects the CNBD with the S6 segment.

[0048] Examples of HCN polypeptides useful in the methods described herein are depicted at SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8. In some embodiments an HCN polypeptide useful in the methods disclosed herein is truncated at the amino terminal end. Typically, such a truncation may result in the deletion of amino acids up to, but not including, amino acids corresponding to the S1 α-helical segment. For instance, with reference to the HCN polypeptide depicted at SEQ ID NO:8, the truncation may be from amino acid 1 to amino acid 258, 257, 256, 255, 254, 253, 252, 251, 250, 249, 248, 247, 246, 245, 244, 243, 242, 241, 240, 239, 238, 237, 236, 235, 234, 233, 232, 231, 230, 229, 228, 227, 226, 225, 224, 223, 222, 221, 220, 219, 218, 217, 216, 215, 214, 213, 212, 211, or 210, and so on up to the second amino acid of SEQ ID NO:8. In some embodiments an HCN polypeptide is truncated at the carboxy terminal end. Typically, such a truncation may result in the deletion of amino acids from the carboxy terminal end up to the amino acids that make up the CNBD domain. For instance, with reference to the HCN polypeptide depicted at SEQ ID NO:8, the truncation may begin at amino acid 723 (i.e., amino acids 723 to the last amino acid, 1203, are not present), 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, or 741, and so on up to amino acid 1202 of SEQ ID NO:8.

[0049] In some embodiments, an HCN polypeptide useful in the methods described herein includes a truncation at both the amino terminal and carboxy terminal ends. Accordingly, an HCN polypeptide of the present invention may include an amino acid sequence corresponding to a core region selected from the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:8. The core region may be, for instance, amino acids 139-603 of SEQ ID NO:2, 208-672 of SEQ ID NO:4, 80-545 of SEQ ID NO:6, or 214-723 of SEQ ID NO:8, and may optionally include additional amino acids located on the amino terminal end and/or the carboxy terminal end. Such additional amino acids may be selected from the corresponding regions of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:8, or may be other amino acid sequences as discussed herein. For instance, in one aspect an HCN polypeptide may have an amino acid sequence beginning with an amino acid selected from amino acids 92-214 of SEQ ID NO:8 and ending with an amino acid selected from amino acids 723-1188 of SEQ ID NO:8. Specific examples of HCN polypeptides include, but are not limited to, a core region from SEQ ID NO:8 beginning at amino acid 213, 214, 221, or 226, and ending at amino acid 723, 738, or 753.

[0050] Other examples of HCN polypeptides include those having sequence similarity with a region selected from the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:8. An HCN polypeptide having sequence similarity with the region selected from the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:8 has channel activity. An HCN polypeptide may be isolated from a eukaryotic cell, such as a vertebrate cell or an invertebrate cell. Examples of suitable vertebrate cells include, but are not limited to, cells obtained from members of the Family Canidae (such as dogs), members of the Family Suidae (such as pigs), members of the Family Muridae (such as rats and mice), members of the genus Ovis (such as sheep), non-human primates, and human cells. An HCN polypeptide may be produced using recombinant techniques, or chemically or enzymatically synthesized using routine methods.

[0051] HCN polypeptides form channels that are unique among vertebrate voltage-gated ion channels: they have a reverse voltage dependence that leads to activation upon hyperpolarization. In addition, voltage-dependent opening of these channels is directly regulated by the binding of cAMP. HCN polypeptides activate upon hyperpolarization with a characteristic sigmoidal time course. An HCN polypeptide having channel activity will display a reverse voltage dependence that leads to activation upon hyperpolarization under suitable conditions. Suitable conditions typically include the expression of an HCN polypeptide in a cell such that it can form channels, and then assaying for activation upon hyperpolarization. Suitable assays include whole cell patch clamp analysis and microelectrode array. Preferably, whole cell patch clamp analysis is used. Methods for whole cell patch clamp analysis are known and routine in the art (Hamill, 1981, Pflugers Arch., 391(2):85-100). An example of a whole cell patch clamp assay is described in Example 1. A polypeptide that leads to functional HCN4 current under suitable conditions is considered to have channel activity and to be an HCN polypeptide, while a polypeptide that does not lead to functional HCN4 current under suitable conditions is considered to not have channel activity and is not an HCN polypeptide.

[0052] The amino acid sequence of an HCN polypeptide having sequence similarity to a region of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:8 may include conservative substitutions. A conservative substitution is typically the substitution of one amino acid for another that is a member of the same class. For example, it is well known in the art of protein biochemistry that an amino acid belonging to a grouping of amino acids having a particular size or characteristic (such as charge, hydrophobicity, and/or hydrophilicity) may generally be substituted for another amino acid without substantially altering the secondary and/or tertiary structure of a polypeptide. For the purposes of this invention, conservative amino acid substitutions are defined to result from exchange of amino acids residues from within one of the following classes of residues: Class I: Gly, Ala, Val, Leu, and Ile (representing aliphatic side chains); Class II: Gly, Ala, Val, Leu, Ile, Ser, and Thr (representing aliphatic and aliphatic hydroxyl side chains); Class III: Tyr, Ser, and Thr (representing hydroxyl side chains); Class IV: Cys and Met (representing sulfur-containing side chains); Class V: Glu, Asp, Asn and Gln (carboxyl or amide group containing side chains); Class VI: His, Arg and Lys (representing basic side chains); Class VII: Gly, Ala, Pro, Trp, Tyr, Ile, Val, Leu, Phe and Met (representing hydrophobic side chains); Class VIII: Phe, Trp, and Tyr (representing aromatic side chains); and Class IX: Asn and Gln (representing amide side chains). The classes are not limited to naturally occurring amino acids, but also include artificial amino acids, such as beta or gamma amino acids and those containing non-natural side chains, and/or other similar monomers such as hydroxyacids. SEQ ID NOs:2, 4, 6, and 8 are shown in FIG. 1 in a multiple protein alignment. Identical and conserved amino acids are marked. Also depicted are those regions corresponding to the six α-helical segments, the PoreH, the SF, and the CNBD domains. This information, and other information regarding conserved regions of HCN polypeptides described herein, permit the skilled person to predict whether alterations to the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:8 are likely to result in an HCN polypeptide having channel activity.

[0053] Guidance concerning how to make phenotypically silent amino acid substitutions is provided in Bowie et al. (1990, Science, 247:1306-1310), wherein the authors indicate proteins are surprisingly tolerant of amino acid substitutions. For example, Bowie et al. disclose that there are two main approaches for studying the tolerance of a polypeptide sequence to change. The first method relies on the process of evolution, in which mutations are either accepted or rejected by natural selection. The second approach uses genetic engineering to introduce amino acid changes at specific positions of a cloned gene and selects or screens to identify sequences that maintain functionality. As stated by the authors, these studies have revealed that proteins are surprisingly tolerant of amino acid substitutions. The authors further indicate which changes are likely to be permissive at a certain position of the protein. For example, most buried amino acid residues require non-polar side chains, whereas few features of surface side chains are generally conserved. Other such phenotypically silent substitutions are described in Bowie et al, and the references cited therein.

[0054] In some aspects an HCN polypeptide may include specific mutations in the region linking the S3 and S4 segments (e.g., amino acids 360-367 of SEQ ID NO:8). Such mutations are depicted in Table 1 (see Sigg et al., U.S. Patent Application Publication 2009/0099611.

TABLE-US-00001 TABLE 1 S3-S4 linker SEQ ID (amino acids NO: Mutant 360-368) 9 Wild-type HCN4 ETRIDSEVY 10 T360A EARIDSEVY 11 Δ363-367 ETRI 12 T360A, Δ363-367 EARI 13 TRI360-362AGM EAGMDSEVY 14 TRI360-362KGM EKGMDSEVY 15 T360A, I362M EARMDSEVY 16 T360A, Δ365-367 EARIDS 17 E365G ETRIDSGVY 18 E365A ETRIDSAVY 19 R361G ETGIDSEVY 20 TR360-361AA EAAIDSEVY 21 I362C ETRCDSEVY 22 I362S ETRSDSEVY 23 I362T ETRTDSEVY 24 TRI360-362AGM, EAGM Δ363-367

[0055] An HCN polypeptide may be expressed as a fusion polypeptide that includes an HCN polypeptide and an additional amino acid sequence. For instance, the additional amino acid sequence may be useful for purification of the fusion polypeptide by affinity chromatography. Various methods are available for the addition of such affinity purification moieties to proteins. Examples of affinity tags include a polyhistidine-tag and a MyC-tag (see, for instance, Hopp et al. (U.S. Pat. No. 4,703,004), Hopp et al. (U.S. Pat. No. 4,782,137), Sgarlato (U.S. Pat. No. 5,935,824), and Sharma Sgarlato (U.S. Pat. No. 5,594,115)). In another example, the additional amino acid sequence may be useful fortagging the fusion polypeptide to aid in identification of the polypeptide in various conditions, including in a tissue or in a cell.

Polynucleotides

[0056] The present invention also includes isolated polynucleotides encoding an HCN polypeptide described herein. A polynucleotide encoding a polypeptide having channel activity is referred to herein as an HCN polynucleotide. HCN polynucleotides may have a nucleotide sequence encoding a polypeptide having the amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:8, or region of one of those amino acid sequences, as described herein. An example of the class of nucleotide sequences encoding a region of a polypeptide disclosed at SEQ ID NO:2, 4, 6, or 8 is the corresponding region of SEQ ID NO:1, 3, 5, or 7, respectively. It should be understood that a polynucleotide encoding an HCN polypeptide represented by, for instance, SEQ ID NO:8, or a portion thereof, is not limited to the nucleotide sequence disclosed at SEQ ID NO:7, but also includes the class of polynucleotides encoding such polypeptides as a result of the degeneracy of the genetic code. For example, the naturally occurring nucleotide sequence SEQ ID NO:7 is but one member of the class of nucleotide sequences encoding a polypeptide having the amino acid sequence SEQ ID NO:8. The class of nucleotide sequences encoding a selected polypeptide sequence is large but finite, and the nucleotide sequence of each member of the class may be readily determined by one skilled in the art by reference to the standard genetic code, wherein different nucleotide triplets (codons) are known to encode the same amino acid. It should be understood that any nucleotide sequence taught herein also includes the complement thereof, and the corresponding RNA sequences.

[0057] An HCN polynucleotide of the present invention may have sequence similarity with the nucleotide sequence of SEQ ID NO:1, 3, 5, or 7. HCN polynucleotides having sequence similarity with the nucleotide sequence of SEQ ID NO:3 encode an HCN polypeptide. An HCN polynucleotide may be isolated from a vertebrate cell or an invertebrate cell, or may be produced using recombinant techniques, or chemically or enzymatically synthesized. An HCN polynucleotide may further include heterologous nucleotides flanking the open reading frame encoding the HCN polypeptide. Typically, heterologous nucleotides may be at the 5' end of the coding region, at the 3' end of the coding region, or the combination thereof. The number of heterologous nucleotides may be, for instance, at least 10, at least 100, or at least 1000.

[0058] HCN polynucleotides may be obtained from a eukaryotic cell, such as a vertebrate cell or an invertebrate cell. Examples of suitable vertebrate cells include, but are not limited to, cells obtained from members of the Family Canidae, and human cells. HCN polynucleotides may be produced in vitro or in vivo. For instance, methods for in vitro synthesis include, but are not limited to, chemical synthesis with a conventional DNA/RNA synthesizer. Commercial suppliers of synthetic polynucleotides and reagents for such synthesis are well known.

[0059] An HCN polynucleotide may be present in a vector. A vector is a replicating polynucleotide, such as a plasmid, phage, or cosmid, to which another polynucleotide may be attached so as to bring about the replication of the attached polynucleotide. Construction of vectors containing a polynucleotide of the invention employs standard ligation techniques known in the art. See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual., Cold Spring Harbor Laboratory Press (1989). A vector may provide for further cloning (amplification of the polynucleotide), i.e., a cloning vector, or for expression of the polynucleotide, i.e., an expression vector. The term vector includes, but is not limited to, plasmid vectors, viral vectors, cosmid vectors, and artificial chromosome vectors. In some aspects preferred vectors include those useful for gene therapy, e.g., vectors that can be administered to a subject to result in transient or sustained expression of a coding region to result in a beneficial polypeptide. A large variety of such vectors are known in the art and are readily available. Examples of vectors useful in gene therapy include isolated nucleic acid, e.g., plasmid-based vectors which may be extrachromosomally maintained, including minicircle vectors; viral vectors, e.g., recombinant adenovirus, retrovirus, lentivirus, herpesvirus, poxvirus, papilloma virus, or adeno-associated virus; and transposons, e.g., recombinant transposons such as Sleeping Beauty and piggyBac. Preferred examples of vectors include single strand adeno-associated virus (ssAAV) and self complementary AAV (scAAV), a modified adeno-associated virus that bypasses the required second-strand DNA synthesis to achieve transcription of the coding region (McCarty, 2008, Mol Ther., 16:1648-1656). Useful ssAAV vectors and scAAV vectors are commercially available from, for instance, Virovek, Hayward, Calif.

[0060] Selection of a vector depends upon a variety of desired characteristics in the resulting construct, such as a selection marker, vector replication rate, and the like. In some aspects, suitable host cells for cloning or expressing the vectors herein include eukaryotic cells. Suitable eukaryotic cells include fungi and mammalian cells. In other aspects, suitable host cells for cloning or expressing the vectors herein include prokaryotic cells. Suitable prokaryotic cells include eubacteria, such as gram-negative microbes, for example, E. coil. Vectors may be introduced into a host cell using methods that are known and used routinely by the skilled person. For example, calcium phosphate precipitation, electroporation, heat shock, lipofection, microinjection, and viral-mediated nucleic acid transfer are common methods for introducing nucleic acids into host cells.

[0061] An expression vector optionally includes regulatory sequences operably linked to the coding region. An example of a regulatory sequence includes a promoter. The invention is not limited by the use of any particular promoter, and a wide variety of promoters are known. Promoters act as regulatory signals that bind RNA polymerase in a cell to initiate transcription of a downstream (3' direction) coding region. The promoter used may be a constitutive or an inducible promoter. It may be, but need not be, heterologous with respect to the host cell.

[0062] In some aspects, tissue-specific promoters may be used. Tissue-specific expression may enhance the safety of a therapy described herein as expression in non-target tissue becomes less likely. For example, cardiac tissue specific promoters allow cardiac myocyte specific expression of the coding region of interest (including expression in stem cells with cardiac phenotype). Examples of cardiac tissue specific promoters include, but are not limited to, promoters from the following coding regions: an α-myosin heavy chain coding region, e.g., a ventricular α-myosin heavy chain coding region, β-myosin heavy chain coding region, e.g., a ventricular β-myosin heavy chain coding region, myosin light chain 2v coding region, e.g., a ventricular myosin light chain 2 coding region, myosin light chain 2a coding region, e.g., a ventricular myosin light chain 2 coding region, cardiomyocyte-restricted cardiac ankyrin repeat protein (CARP) coding region, cardiac α-actin coding region, cardiac m2 muscarinic acetylcholine coding region, ANP coding region, BNP coding region, cardiac troponin C coding region, cardiac troponin I coding region, cardiac troponin T coding region, cardiac sarcoplasmic reticulum Ca-ATPase coding region, and skeletal α-actin coding region. Further, chamber-specific promoters or enhancers may also be employed, e.g., for atrial-specific expression, the quail slow myosin chain type 3 (MyHC3) or ANP promoter may be used. Examples of ventricular myocyte-specific promoters include a ventricular myosin light chain 2 promoter and a ventricular myosin heavy chain promoter.

[0063] Other useful promoters, for example, would be sensitive to electrical stimulus that could be provided from, for example, an implantable device. Electrical stimulation can promote gene expression (Padua et al., U.S. Patent Application No. 2003/0204206 A1).

[0064] Other regulatory regions include drug-sensitive elements (e.g., a drug-inducible suppressor or promoter). Drug-responsive promoters may induce or suppress expression of an operably linked coding region. For example, a tetracycline responsive element (TRE) that binds doxycycline may present within a promoter construct. When doxycycline is removed, transcription from the TRE is turned off in a dose-dependent manner. Examples of inducible drug-responsive promoters are the ecdysone-inducible promoter (Johns and Marban, U.S. Pat. No. 6,214,620) and rapamycin-dependent expression (Clackson et al., U.S. Pat. No. 6,506,379, see also Discher et al., 1998, J. Biol. Chem., 273:26087-26093; Prentice et al., 1997, Cardiovascular Res., 35: 567-576).

[0065] Further examples of regulatory regions include enhancers, such as cardiac enhancers, to increase the expression of an operably linked coding region in cardiac tissue, such as regions of the cardiac conduction system. Such enhancer elements may include the cardiac specific enhancer elements derived from Csx/Nk×2.5 regulatory regions (Lee and Izumo, U.S. Patent Application 2002/0022259) or the cGATA-6 enhancer.

[0066] An expression vector may optionally include a ribosome binding site and a start site (e.g., the codon ATG) to initiate translation of the transcribed message to produce the polypeptide. It may also include a termination sequence to end translation. A termination sequence is typically a codon for which there exists no corresponding aminoacetyl-tRNA, thus ending polypeptide synthesis. The polynucleotide used to transform the host cell may optionally further include a transcription termination sequence.

[0067] A vector introduced into a host cell optionally includes one or more marker sequences, which typically encode a molecule that inactivates or otherwise detects or is detected by a compound in the growth medium. For example, the inclusion of a marker sequence may render the transformed cell resistant to an antibiotic, or it may confer compound-specific metabolism on the transformed cell. Selectable markers can be positive, negative or bifunctional. Positive selectable markers allow selection for cells carrying the marker, whereas negative selectable markers allow cells carrying the marker to be selectively eliminated. A variety of such marker genes have been described, including bifunctional (i.e., positive/negative) markers (see, e.g., Lupton, PCT Publication Nos. WO 92/08796 and WO 94/28143). Such marker genes can provide an added measure of control that can be advantageous in gene therapy contexts.

[0068] Polypeptides useful in the present invention may be produced using recombinant DNA techniques, such as an expression vector present in a cell. Such methods are routine and known in the art. The polypeptides may also be synthesized in vitro, e.g., by solid phase peptide synthetic methods. The solid phase peptide synthetic methods are routine and known in the art. A polypeptide produced using recombinant techniques or by solid phase peptide synthetic methods may be further purified by routine methods, such as fractionation on immunoaffinity or ion-exchange columns, ethanol precipitation, reverse phase HPLC, chromatography on silica or on an anion-exchange resin such as DEAE, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, gel filtration using, for example, Sephadex G-75, or ligand affinity.

[0069] The present invention also includes genetically modified cells that have an HCN polynucleotide encoding an HCN polypeptide. Compared to a control cell that is not genetically modified according to the present invention, a genetically modified cell may exhibit production of an HCN polypeptide. A polynucleotide encoding an HCN polypeptide may be present in the cell as an extrachromosomal vector or integrated into a chromosome. Examples of cells include, but are not limited to, excitable cells, such as cardiomyocytes and HL-5 cells; and non-excitable cells, such as stem cells, fibroblasts, mesenchymal cells, and HEK293 cells. In some aspects the cells may also be modified to express connexions or gap junctions. The coding regions encoding connexion polypeptides are readily available to the skilled person.

[0070] A genetically modified cell may be ex vivo or in vivo. "Ex vivo" refers to a cell that has been removed from the body of an animal. Ex vivo cells include, for instance, primary cells (e.g., cells that have recently been removed from a animal and are capable of limited growth in tissue culture medium), and cultured cells (e.g., cells that are capable of long term culture in tissue culture medium). "In vivo" refers to cells that are present within the body of an animal.

Compositions

[0071] The present invention is also directed to compositions including an HCN polynucleotide, HCN polypeptide, or genetically modified cell. Such compositions typically include a pharmaceutically acceptable carrier. As used herein "pharmaceutically acceptable carrier" includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Additional active compounds can also be incorporated into the compositions.

[0072] A composition may be prepared by methods well known in the art of pharmacy. In general, a composition can be formulated to be compatible with its intended route of administration. Solutions or suspensions can include the following components: a sterile diluent such as water for administration, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfate; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates; electrolytes, such as sodium ion, chloride ion, potassium ion, calcium ion, and magnesium ion, and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. A composition can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0073] Compositions can include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL® (BASF, Parsippany, N.J.) or phosphate buffered saline. A composition is typically sterile and, when suitable for injectable use, should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0074] Sterile solutions can be prepared by incorporating the active compound (e.g., an HCN polynucleotide, or, in some aspects, an HCN polypeptide or genetically modified cell) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0075] An active compound may be administered by any route including, but not limited to, intramuscular, buccal, rectal, intravenous or intracoronary administration, and transfer to cells may be enhanced using electroporation and/or iontophoresis. Administration may be systemic or local. In some aspects local administration may have advantages for site-specific, targeted disease management. Local therapies may provide high, clinically effective concentrations directly to the treatment site, without causing systemic side effects. Examples of locations to which an active compound can be targeted include, but are not limited to, the right or left atrium, including the sinoatrial node, the right or left ventricle, including the atrioventricular node. For instance, endocardial or myocardial cells of an atrium or a ventricle may be targeted. In some aspects, an HCN polynucleotide can be implanted in, or downstream from, the conduction pathway, in a heart region that is experiencing or may experience poor conduction. For example, if cardiac contraction is not being properly initiated by the SA node but the AV node conduction is intact, an HCN polynucleotide may be implanted in the myocardium of the SA node or the right atrium to cause the targeted region to depolarize and create electric impulses that will travel to the AV node. Alternatively, if cardiac contraction is not being properly conducted by the AV node then an HCN polynucleotide may be implanted downstream in the conduction pathway from the right atrium, i.e. in the bundle of His, the Purkinje network, or one of the ventricles. Other delivery sites include, but are not limited to, left ventricular epicardium.

[0076] Examples of routes of administration include the use of a delivery tool, such as a syringe for direct injection into cardiac tissue (for instance, during open heart surgery) or by catheter. For instance, one type of catheter useful in the methods described herein has electric sensing capabilities, which permits introduction of an active compound directly into the targeted cardiac tissue. The delivery tool may include electrodes for sensing electric activity and delivering pacing stimuli in order to determine the desired location for the biological pacemakers. Once the location is determined, an active compound is delivered to the cardiac tissue. The delivery tool may include an injection device that injects the active compound into cardiac tissue. One suitable method for injecting a genetic construct directly into the myocardium is described by Guzman et al., 1993, Circ. Res., 73:1202-1207. Furthermore, a delivery system for delivering genetic material to a targeted heart region is described in Laske et al. (U.S. Pat. No. 7,103,418) and Stokes et al. (PCT Publication No. WO 98/02150). Systems for myocardial, endocardial, sub-epicardial and epicardial delivery are described in Sullivan and Hezi-Yamit (U.S. Published Patent Application 20100137976), Hiniduma-Lokuge et al. (PCT Publication No. WO/2008/055001), and Sommer et al., (U.S. Pat. No. 7,274,966 and U.S. Pat. No. 7,187,971). Alternatively, genetically engineered cells may be cultured and proliferated on a solid scaffold, and then surgically delivered to the selected heart region together with the scaffold. The scaffold may also be directly injected into cardiac tissue.

[0077] Perfusion protocols that are useful are often sufficiently capable of delivering a genetic construct to at least about 10% of cardiac myocytes. Infusion volumes of between 0.01 ml and 3 ml are useful for direct intramyocardial injection. Also, suitable methods for targeting non-viral vector genetic constructs to the heart are described in Lawrence (U.S. Pat. No. 6,376,471).

[0078] When a polynucleotide is introduced into cardiac cells using any suitable technique, the polynucleotide is delivered into the cells by, for example, transfection or transduction procedures. Transfection and transduction refer to the acquisition by a cell of new genetic material by incorporation of added polynucleotides. Transfection can occur by physical or chemical methods. Many transfection techniques are known to those of ordinary skill in the art including, without limitation, calcium phosphate DNA co-precipitation, DEAE-dextrin DNA transfection, electroporation, naked plasmid adsorption, and cationic liposome-mediated transfection (commonly known as lipofection). Transduction refers to the process of transferring nucleic acid into a cell using a DNA or RNA virus.

[0079] A polynucleotide described herein may be used in combination with other agents assisting the cellular uptake of polynucleotides, or assisting the release of poylnucleotides from endosomes or intracellular compartments into the cytoplasm or cell nuclei by, for instance, conjugation of those to the polynucleotide. The agents may be, but are not limited to, peptides, especially cell-penetrating peptides, protein transduction domains, and/or dsRNA-binding domains which enhance the cellular uptake of polynucleotides (Dowdy et al., US Published Patent Application 2009/0093026, Eguchi et al., 2009, Nature Biotechnology 27:567-571, Lindsay et al., 2002, Curr. Opin. Pharmacol., 2:587-594, Wadia and Dowdy, 2002, Curr. Opin. Biotechnol. 13:52-56. Gait, 2003, Cell. Mol. Life Sci., 60:1-10). The conjugations can be performed at an internal position at the oligonucleotide or at a terminal postions either the 5'-end or the 3'-end.

[0080] A polynucleotide described herein may be present in liposomes, including neutral or cationic liposomes, such as DOSPA/DOPE, DOGS/DOPE or DMRIE/DOPE liposomes, and/or associated with other molecules such as DNA-anti-DNA antibody-cationic lipid (DOTMA/DOPE) complexes or polyethyleneimine (PEI). An active compound may be present in a polymer matrix, for instance, a polymer matrix may be formed of any physiologically compatible material which generally retains a polynucleotide (which is a charged molecule) or optionally other agents including other agents under physiological conditions for a sustained period of time. The polymer matrix may extrude (release) the polynucleotide in response to an external stimulus, such as an electric field created by an electrical signal, or the matrix may provide for passive delivery.

[0081] Toxicity and therapeutic efficacy of such active compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the ED₅₀ (the dose therapeutically effective in 50% of the population). The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For a compound used in the methods of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. In those aspects where a viral vector is used, such as an AAV-based vector, a dosage may be at least 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, or 10¹⁴ viral particles.

[0082] Administration of an HCN polynucleotide, an HCN polypeptide, or a genetically modified cell described herein may be continuous or intermittent, depending, for example, upon the recipient's physiological condition, whether the purpose of the administration is therapeutic or prophylactic, and other factors known to a person skilled in the art. The administration may be essentially continuous over a preselected period of time or may be in a series of spaced doses.

Methods of Use

[0083] The present invention also includes methods of using the HCN polynucleotides, HCN polypeptides, and genetically modified cells described herein. The methods include, for instance, methods of treating a cardiac pacing condition. Examples of cardiac pacing conditions include, but are not limited to, patients with atrioventricular (AV) node dysfunction and/or sinoatrial (SA) node dysfunction. Such patients may have bradyarrhythmia, such as those with the clinical syndrome sick sinus syndrome. Cardiac pacing conditions may also occur in patients undergoing surgery (such as coronary artery bypass surgery or insertion of an artificial heart valve), or having endocarditis. Signs and symptoms associated with cardiac pacing conditions and the evaluation of such signs and symptoms are routine and known in the art. As used herein, the term "symptom" refers to subjective evidence of a cardiac pacing condition experienced by a subject. As used herein, the term "clinical sign" or, simply, "sign" refers to objective evidence of a cardiac pacing condition.

[0084] Treatment may be prophylactic or, alternatively, may be initiated after a cardiac pacing condition is evident. Treatment that is prophylactic, for instance, initiated before an animal manifests symptoms of a cardiac pacing condition, is referred to herein as treatment of a patient that is "at risk" of developing a cardiac pacing condition. Treatment initiated after development of symptoms of a cardiac pacing condition may result in decreasing the severity of the symptoms, or completely removing the symptoms. An "effective amount" is an amount effective to prevent the manifestation of symptoms of a cardiac pacing condition, decrease the severity of the symptoms of cardiac pacing condition, and/or completely remove the symptoms. It is not required that any composition of the present invention completely remove or cure all symptoms of a cardiac pacing condition.

[0085] Treatment may result in increasing the intrinsic pacing rate of cardiac cells, preferably to the normal physiological range for the subject. In some aspects, the intrinsic pacing rate is increased to 60-80 beats per minute (bpm) at rest and 90-115 bpm during moderate exercise.

[0086] The methods may include administering a composition of the present invention to a subject in need thereof. The subject may be human, or an animal, such as an animal typically used as a model in the study and evaluation of treatments for a cardiac pacing condition, such as a dog or pig, sheep, or non-human primate.

[0087] The present invention is illustrated by the following examples. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein.

Example 1

[0088] This example describes the development a super-truncated HCN4 construct in order to be packaged in size limited scAAV vector and to be applicable across species and between human without inducing an immune response. Further, an AAV1 vector carrying this super-truncated HCN4 was to be generated by exploring an alternative AAV generation technology. The pace maker function was confirmed by ex vivo evaluation.

BACKGROUND

[0089] Previously we had demonstrated biological pacemaker function using both human full length HCN4 polypeptide (wtHCN4) and a human C-terminal truncated HCN4 version (HCN4t) having amino acids 1-738 of SEQ ID NO:8, packaged in an adenovirus vector (Adv). We used Adv as a carrier to deliver human hyperpolarization-activated cyclic-nucleotide-gated (HCN) ion channel 4 and established proof of concept that HCN4t, along with the full length HCN4, are capable of generating a ventricular biological pacemaker in an AV blocked canine model (FIG. 3).

[0090] Although Adv has shown remarkable efficacy in gene transfer, we developed a clinically relevant vector, traditional adeno-associated virus vector (AAV), to carry a biological pacemaker coding region, HCN4. In comparison with Adv, traditional AAV, also referred to as single-stranded AAV (ssAAV), is a very attractive vector for cardiac gene therapy because of lack of pathogenicity, very low immunogenicity, and long-term gene transfer potential with decreased risk of malignant transformation in small and large animal models.

[0091] However, the problems associated with ssAAV vector include a latent period from 10 days to 1 month before ssAAV mediated gene expression peaks. While a 3-4 week waiting period required for ssAAV to reach the steady state may be acceptable for one-time life-saving therapy such as a biological pacemaker, a faster expressing system would certainly be most applicable clinically since it would minimize the time a patient is on electronic pacing as a bridge, and would also provide much quicker feedback to the physician if the biological pacemaker were to need any intervention (e.g., to adjust pacing rate). Therefore, it is desirable to determine whether a different type of AAV vector system, the self-complementary AAV vector system (scAAV) will function.

[0092] The traditional AAV system contains single stranded DNA and requires host cell synthesis of the complementary strand. Therefore there is a lag period, when the expression is generally quite slow with the peak function occurring 3-4 weeks after the gene administration. In contrast, scAAV system contains double strand DNA and therefore eliminates the rate-limiting step of second-strand DNA synthesis. Thus, scAAV can attain peak expression in a matter of days. This fast ramp-up feature makes scAAV a very promising vector for clinical applications.

[0093] ssAAV can package a coding region and regulatory sequences (including two ITR regions, promoter, transgene, and poly-A signal) roughly the size of 4.8 kilo-base pairs (kb) and allows packaging of HCN4t (2,214 base pairs (bp) for HCN4t versus 3,612 by for wtHCN4). As for scAAV, it can only package ˜2.5 kb of coding region and regulatory sequences. While much shorter than in its wild-type counterpart, the coding region encoding HCN4t is still longer than the packaging capacity of scAAV. We developed an even shorter version of HCN4 construct, so that the packaging in scAAV vector system is feasible. The shorter version was designated super truncated HCN4 (HCN4st).

[0094] In addition to the limitation related to their large sizes, another limitation of the wtHCN4 and HCN4t genes is that they have regions that show inter-species variability. For example, between canine and human genes, there is a considerable lack of homology in the 5' region of the HCN4 gene (FIG. 4). The HCN4st removes all the regions that exhibit inter-species variability and this would imply that the risk of generating an immune response and loss of pacemaker function would be minimized with an HCN4st gene. There are greater than 98% identical residues between human and canine HCN4st1 sequence.

[0095] HCN4 channels (FIG. 5) consist of 6 transmembrane domains, with a pore region between S5 and S6 and a cyclic nucleotide-binding domain (CNBD) in the cytoplasmic C-terminal region. While HCN4t deleted nucleotides after the CNBD region, HCN4st was designed to delete regions from the amino terminal end, but not extend into the S1 region. It is within block III of FIG. 4.

[0096] In an effort to generate scalable production of AAV vectors, as well as to overcome the toxicity of HCN4 in routine host cells, such as HEK293, a "BAC-to-AAV" technology (Chen U.S. Published Patent Application 20090203071) that uses a baculovirus expression system to produce AAV vectors in insect cells under serum-free condition was used. BAC-to-AAV technology enhances the packaging of much more VP1 proteins into the virions than other systems and therefore greatly increases the performance of AAV vectors.

Methods:

[0097] Generating Super-Truncated Version of HCN4 (HCN4st).

[0098] HCN4t construct was generated by truncating 1,398 base pairs (bp) nucleotides that encode the C-terminus of wild type HCN4. The truncation site was chosen such that a cyclic nucleotide-binding domain (CNBD) was preserved in the C-terminus region of HCN4t. HCN4st constructs included a series of deletions on the N-terminus of the HCN4t, in front of the six transmembrane domains (FIG. 6).

[0099] Each individual HCN4st gene was generated via PCR amplification by using HCN4t as template. Sequence specific primers were designed using the nucleotide sequence available at Genbank accession NM_--005477.2. Upstream primers HCN4st were 5' ata gcg cga att ccc gcc atg cag cgc cag ttc ggg (SEQ ID NO:25), 5' ata gcg cga att ccc gcc atg ctc caa ccc ggg g (SEQ ID NO:26) and 5' ata gcg cga att ccc gcc atg ttc ggc agc cag aaa g (SEQ ID NO:27) respectively for HCN4st1, st2 and st3. The coding sequence encoding HCN4st1 encodes a polypeptide corresponding to amino acids 214-719 of SEQ ID NO:8 (FIG. 1), the coding sequence encoding HCN4st2 encodes a polypeptide corresponding to amino acids 221-719 of SEQ ID NO:8, and the coding sequence encoding HCN4st3 encodes a polypeptide corresponding to amino acids 233-719 of SEQ ID NO:8. The downstream primer for each HCN4st was 5' cggcggatcccctagagatat (SEQ ID NO:28). MyC sequence (AGCAGAAGCTGATCTCAGAGGAGGACCTGCTT, SEQ ID NO:29) was attached at the C-terminal of different HCN4t modifications. Three HCN4st genes were cloned into a pTopo sequence plasmid (Invitrogen, Carlsbad, Calif., USA) and verified by restriction enzyme analysis (New England Biolabs, Ipswich, Mass., USA) and the nucleotide sequence was confirmed (Eurofins MWG Operon, Huntsville, Ala. USA). HCN4st genes were subsequently subcloned into the expression plasmid pIRES-EGFP (Clonetech, Mountain View, Calif., USA) for functional testing by patch clamp. Selected HCN4st genes were further subcloned into an AAV shuttle plasmid (ViroVek, Hayward, Calif., USA) for making B-AAV-HCN4st1 vector (FIG. 7, and described below).

Patch Clamp

[0100] HEK 293 Cell Transfection

[0101] Human embryonic kidney (HEK293, ATCC) cells were split on 12 well plate one day before transfection. Three pHCN4st-IRES-EGFP plasmids were transfected into 70% confluent HEK293 cells with Fugene 6 (Roche) transfection reagents, along with pHCN4t-IRES-EGFP. The transfected cells were replated on gelatin-fibronectin coated glass coverslips 24 hours after transfection.

Whole Cell Patch Clamp

[0102] One day after reculture, the cells plated on a coverslip were transported to a chamber mounted on the stage of a Nikon microscope. The chamber was continuously superfused (˜0.5 ml/min) with the Tyrode solution. The whole-cell configuration of the patch-clamp technique (Hamill et al., 1981, Pflugers Arch. 391(2):85-100) was applied. Briefly, glass electrodes (World Precision Instruments, Sarasota, Fla., USA) with 3 to 5 MΩ resistance were connected via an Ag-AgCl wire to an Axopatch 200A amplifier interfaced with a DigiData-1322 acquisition system. After forming a conventional "gigaohm" seal, electrode capacitance was compensated. Additional suction ruptured the patched membrane and formed the whole-cell configuration. Cell membrane capacitance (C_m) was measured in each patched cells with the pCLAMP program (version 9.2, Axon Instruments, Foster City, Calif., USA).

[0103] The hyperpolarization-activated cyclic nucleotide-gated inward current (I_h) was measured with the modified Tyrode bath solution. I_h was evoked by 5 s hyperpolarizing steps to potentials ranging from 0 to -140 mV from a holding potential of -40 mV. The reversal potential of I_h was evaluated by tail currents recorded by 3 s `tail` steps to membrane potentials ranging from -80 to 20 mV in 10 mV increments followed by a 5 s conditioning potential step to -130 mV every 15 s. The holding potential was set at -40 mV. The activation of I_h was elicited by 3 s `tail` pulses to -130 mV followed 5 s conditioning pulses from 0 mV to -140 mV in 10 mV increments. The pulse rate was every 30 s.

Data Analysis

[0104] Data were collected with the pCLAMP software (version 9.02). I_h was evaluated at a point near the end of each test pulse unless stated otherwise (tail-current measurements). The current amplitudes were normalized with respect to the corresponding values of C_m to minimize the current difference due to cell size. A single-exponential fit (Axon-Clampfit 9.02) of current traces allowed derivation of time constants (τ) of current activation and deactivation. Some data were fitted by a Boltzmann equation {1/[1+exp(V₁/2-V)/k], where V₁/2 is the half-inactivation potential, V is the voltage potential, and k is the slope factor (in mV/e-fold change in current)}. The best-fit procedure was performed with a commercial software program (Origin 7.0, Microcal® Software Inc., Northampton, Ma., USA). All data are presented as mean±standard error of the mean unless otherwise stated. Unpaired Student's t-test was applied for statistical analysis as appropriate. Differences were considered significant if P≦0.05.

B-AAV1-HCN4st1 Generation

[0105] Following patch clamp, functional HCN4st gene was further subcloned and the polynucleotide sequence depicted in FIG. 15 was used as a template for PCR using the primers 5' ata gcg cga att ccc gcc atg cag cgc cag ttc ggg (SEQ ID NO:25) and 5' cggcggatcccctagagatat (SEQ ID NO:28), and the amplification product was used by ViroVek to produce an AAV1 vector that encoded an HCN4st1 polypeptide that included a MyC sequence fused to the amino terminal end of the polypeptide. The methods used by ViroVek include cloning gene of interest into an pFB-AAV shuttle plasmid, generation of Bacmid and purification of Bacmid DNA, transfection of Sf9 cells to generate baculovirus, amplification of baculovirus and titration, production of AAV and CsCl purification, and desalting, filter sterilization, and AAV titration (see Chen, U.S. Published Patent Application 20090203071, U.S. Provisional Patent Application 60/839,761, and International Application PCT/US07/76799, and http://www.virovek.com/AAV_Production.html. Together with AAV rep component and vp component, this shuttle vector were co-transfected into SF9 insect cells for generation of recombinant baculovirus and production of a virus vector containing a coding region encoding HCN4st1, designated B-AAV1-HCN4st1, where the "B" refers to generation using the baculovirus system. Following purification of the virus vector through buffer exchange and sterile filtration, virus titer was determined by qPCR. The resulting B-AAV1-HCN4st1 was dissolved in PBS with 0.001% Pluronic F-68 buffer at concentration of 1.03E+13 vg/mL. Endotoxin level was tested by Biotest Labs with <1EU/ml.

Microelectrode Array (MEA) Measurements

[0106] Primary Cell Isolation

[0107] Neonatal rats were sacrificed by decapitation and hearts were rapidly excised and washed in Ca²+ free balanced salt solution. The ventricles were minced into 1-2 mm³ pieces and dissociated into single cell suspension by repeated digestion with proteolytic enzymes. Each digestion, enhanced with gentle shaking, lasted for 15-20 minutes. Then myocytes were mechanically dispersed by triturating. The undigested masses and first few digestion fractions were discarded after filtering through a cell strainer. Collected cells suspension was mixed with serum for enzyme deactivation, centrifuged, and re-suspended in culturing media. Cells were additionally incubated with 10 μg/ml of DNAse for 10 min at 37° C. For fibroblasts separation and myocytes enrichment two 1.5 hrs pre-plating steps (incubation of cell suspension in 75 cm² flasks) were performed. After pre-plating, slowly attaching myocytes were separated from quickly attaching fibroblasts, collected, counted, re-suspended, and plated in pretreated MEAs (at -500000 cells/ml, -180000 cells/cm²). Each MEA had ˜64 electrodes spaced ˜100 μm and covering a total area of approximately 0.5 mm².

[0108] NRVMs were cultured with Norepinephrine and Bromodeoxyuridine (BrDu) for 2 days, than with BrDu for 2 more days, until synchronized spontaneously beating cell monolayers were formed, than maintained in serum free culture media (contained Insulin, BSA, and Vitamin B₁₂) preconditioned on cultured fibroblasts.

AAV Transduction

[0109] Transfection experiments with serial virus titers were performed twice.

[0110] During the first experiment, transfections of myocytes in MEA chambers were carried out on 5th day in culture at the following titers: 10¹¹, 10¹⁰, 10⁹, 10⁸ vg/ml of B-AAV1-HCN4st1 and non transduction negative control (5 arrays for each condition).

[0111] During the second experiment, in addition to B-AAV1-HCN4st1 and negative control, a control of B-AAV1-empty particles was included.

MEA System

[0112] The day before transduction, and then each following day, electrograms for each culture were recorded with MEA system and microscopic fluorescence and bright field images of each array were taken with Leica inverted microscope at 10× magnification. Cultures were monitored for about 2 weeks, after which MEA recordings were processed using "MC Rack" Multi Channel Systems Software and analyzed in Microsoft Excel; microscopic images were processed with "ImageJ" software.

[0113] NRVM cultures showing signs of degradation were rapidly assessed for apoptosis with Vibrant Apoptosis Assay Kit #5 (Invitrogen, Carlsbbad, Calif., USA) based on fluorescence detection of the compacted state of the chromatin in apoptotic cells.

Results

[0114] HCN4st1, st2 and st3 evaluation by patch clamp.

[0115] We have expressed three HCN4st constructs individually in HEK293 cells by Fugene 6 transfection and performed whole-cell patch clamp experiments to assess their electrophysiology function. Our experiments suggested that only HCN4st1 remained indistinguishable from the HCN4t, thus suggesting that truncation at N-terminus of 213 amino acids does not compromise HCN4 function (FIG. 8). When using HEK293 cells HCN4st2 and HCN4st3 did not lead to functional HCN4 current, indicating that these constructs may not function properly in HEK293 cells, or that further modification on these two deletions may be needed. HCN4st1 gene contains only 1575 by nucleotides, well within the packaging capacity of scAAV vector system, and has both the C-terminus and N-terminus truncated.

B-AAV1-HCN4st1 In Vitro Evaluation by MEA

[0116] Non transduced control NRVM cells demonstrated stable (˜20-40 BPM) beating rates and overall uniform healthy appearance during the two week observation period (FIG. 9).

[0117] NRVM cells transduced with 10¹¹ vg/ml of B-AAV1-HCN4st1 were not beating by the next day after transduction and rapidly degraded showing the signs of apoptosis (FIG. 10).

[0118] For the next lower titer -10¹⁰ vg/ml NRVM cells reached the maximum of beating rate ˜115 BPM by day 3 after transduction, and then degraded by day 7.

[0119] For the 10⁹ vg/ml titer, NRVM cells reached maximum of beating rate ˜130-160 BPM by the day 6 and degraded by the day 10.

[0120] For the lowest tested 10⁸ vg/ml titer, NRVM cells reached beating rate ˜75 BPM by the day 10 and did not show signs of degradation (FIG. 11, FIG. 12).

[0121] NRVM cells transduced with B-AAV1-empty particles did not show any signs of degradation and any noticeable rate increase compared to non transduced controls (FIG. 13).

[0122] Overall, neonatal rat ventricular myocytes transduced with B-AAV1-HCN4st1 showed increased induced beating. At higher titers (10¹¹, 10¹⁰, 10⁹ vg/ml), B-AAV1-HCN4st1 caused cell degradation via apoptosis. At lower titer (10⁸ vg/ml), B-AAV1-HCN4st1 was safe and reached induced beating rate at ˜75 BPM.

Example 2

[0123] AAV1-HCN4-mediated biological pacemaker paces the canine heart with AV block over 7 months and responds well to autonomic challenges

[0124] Implantation of an electronic pacemaker is necessary for a patient with severe bradycardia; however, while effectively improving the lives of many patients, such therapy has several limitations including hardware complications, limited battery life and lack of response to autonomic and physiologic demands on hearts. Compared to its counterpart, a biological pacemaker is a conceptually attractive alternative to electronic pacemakers.

[0125] This study was to test whether using adeno-associate virus-1 (AAV1) with truncated human HCN4 (hHCN4tr) would create a long-term (>6 months) and sustained (nearly 100% biologically induced pacing) biological pacemaker in the left ventricle (LV) of canines with atrioventricular (AV) nodal block.

[0126] Canines (n=3) with neutralizing anti-AAV 1 antibodies (NAb) at a titer ≦1:40 serum dilution were selected. After complete AV block, the AAV1-hHCN4tr vectors were epicardially injected into the LV apex using a side-hole needle as described in Hiniduma-Lokuge et al. (PCT Publication No. WO/2008/055001). An electrical pacemaker (VVI 50 bpm) was implanted for backup pacing and recording of the electrocardiograms every two hours. During the follow-up monitoring, drug and exercise challenges were performed. Twenty-four hour Holter monitoring and pacemaker log record checks were also performed.

[0127] All canines demonstrated biological pacemaker activities 3 days after AAV1-hHCN4tr injection. The biological pacemaker in two canines with NAb at a titer of 1:40 diminished 3 weeks after the gene transfer presumably due to the pre-existing immunity to AAV1. However, the canine with NAb at a titer of 1:20 has exhibited sustained biological pacing activities at a range of 60-150 bpm over 7 months at the time of this abstract submission (FIG. 15). The electrical pacemaker intervened at 50 bpm when spontaneous ventricular rate fell below at that rate, triggering for <2% of the beats. The biological pacemaker responded well to drugs, such as isoproterenol and metoprolol, and also showed physiological diurnal variations and responses to exercise.

CONCLUSION

[0128] LV epicardial injection with AAV1-hHCN4tr creates long-term (over 7 months) and sustained (nearly 100% biologically induced pacing) biological pacemaker activities in a complete AV block canine model. Interestingly, such biological pacemaker responds well to autonomic challenges and physiological exercise.

[0129] The complete disclosure of all patents, patent applications, and publications, and electronically available material (including, for instance, nucleotide sequence submissions in, e.g., GenBank and RefSeq, and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) cited herein are incorporated by reference in their entirety. Supplementary materials referenced in publications (such as supplementary tables, supplementary figures, supplementary materials and methods, and/or supplementary experimental data) are likewise incorporated by reference in their entirety. In the event that any inconsistency exists between the disclosure of the present application and the disclosure(s) of any document incorporated herein by reference, the disclosure of the present application shall govern. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.

[0130] Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0131] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.

[0132] All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.

Sequence CWU 1

3112673DNAhumanCDS(1)..(2673) 1atg gaa gga ggc ggc aag ccc aac tct tcg tct aac agc cgg gac gat 48Met Glu Gly Gly Gly Lys Pro Asn Ser Ser Ser Asn Ser Arg Asp Asp1 5 10 15ggc aac agc gtc ttc ccc gcc aag gcg tcc gcg acg ggc gcg ggg ccg 96Gly Asn Ser Val Phe Pro Ala Lys Ala Ser Ala Thr Gly Ala Gly Pro 20 25 30gcc gcg gcc gag aag cgc ctg ggc acc ccg ccg ggg ggc ggc ggg gcc 144Ala Ala Ala Glu Lys Arg Leu Gly Thr Pro Pro Gly Gly Gly Gly Ala 35 40 45ggc gcg aag gag cac ggc aac tcc gtg tgc ttc aag gtg gac ggc ggt 192Gly Ala Lys Glu His Gly Asn Ser Val Cys Phe Lys Val Asp Gly Gly 50 55 60ggc ggc ggt ggc ggc ggc ggc ggc ggc ggc gag gag ccg gcg ggg ggc 240Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Glu Glu Pro Ala Gly Gly65 70 75 80ttc gaa gac gcc gag ggg ccc cgg cgg cag tac ggc ttc atg cag agg 288Phe Glu Asp Ala Glu Gly Pro Arg Arg Gln Tyr Gly Phe Met Gln Arg 85 90 95cag ttc acc tcc atg ctg cag ccc ggg gtc aac aaa ttc tcc ctc cgc 336Gln Phe Thr Ser Met Leu Gln Pro Gly Val Asn Lys Phe Ser Leu Arg 100 105 110atg ttt ggg agc cag aag gcg gtg gaa aag gag cag gaa agg gtt aaa 384Met Phe Gly Ser Gln Lys Ala Val Glu Lys Glu Gln Glu Arg Val Lys 115 120 125act gca ggc ttc tgg att atc cac cct tac agt gat ttc agg ttt tac 432Thr Ala Gly Phe Trp Ile Ile His Pro Tyr Ser Asp Phe Arg Phe Tyr 130 135 140tgg gat tta ata atg ctt ata atg atg gtt gga aat cta gtc atc ata 480Trp Asp Leu Ile Met Leu Ile Met Met Val Gly Asn Leu Val Ile Ile145 150 155 160cca gtt gga atc aca ttc ttt aca gag caa aca aca aca cca tgg att 528Pro Val Gly Ile Thr Phe Phe Thr Glu Gln Thr Thr Thr Pro Trp Ile 165 170 175att ttc aat gtg gca tca gat aca gtt ttc cta ttg gac ctg atc atg 576Ile Phe Asn Val Ala Ser Asp Thr Val Phe Leu Leu Asp Leu Ile Met 180 185 190aat ttt agg act ggg act gtc aat gaa gac agt tct gaa atc atc ctg 624Asn Phe Arg Thr Gly Thr Val Asn Glu Asp Ser Ser Glu Ile Ile Leu 195 200 205gac ccc aaa gtg atc aag atg aat tat tta aaa agc tgg ttt gtg gtt 672Asp Pro Lys Val Ile Lys Met Asn Tyr Leu Lys Ser Trp Phe Val Val 210 215 220gac ttc atc tca tcc atc cca gtg gat tat atc ttt ctt att gta gaa 720Asp Phe Ile Ser Ser Ile Pro Val Asp Tyr Ile Phe Leu Ile Val Glu225 230 235 240aaa gga atg gat tct gaa gtt tac aag aca gcc agg gca ctt cgc att 768Lys Gly Met Asp Ser Glu Val Tyr Lys Thr Ala Arg Ala Leu Arg Ile 245 250 255gtg agg ttt aca aaa att ctc agt ctc ttg cgt tta tta cga ctt tca 816Val Arg Phe Thr Lys Ile Leu Ser Leu Leu Arg Leu Leu Arg Leu Ser 260 265 270agg tta att aga tac ata cat caa tgg gaa gag ata ttc cac atg aca 864Arg Leu Ile Arg Tyr Ile His Gln Trp Glu Glu Ile Phe His Met Thr 275 280 285tat gat ctc gcc agt gca gtg gtg aga att ttt aat ctc atc ggc atg 912Tyr Asp Leu Ala Ser Ala Val Val Arg Ile Phe Asn Leu Ile Gly Met 290 295 300atg ctg ctc ctg tgc cac tgg gat ggt tgt ctt cag ttc tta gta cca 960Met Leu Leu Leu Cys His Trp Asp Gly Cys Leu Gln Phe Leu Val Pro305 310 315 320cta ctg cag gac ttc cca cca gat tgc tgg gtg tct tta aat gaa atg 1008Leu Leu Gln Asp Phe Pro Pro Asp Cys Trp Val Ser Leu Asn Glu Met 325 330 335gtt aat gat tct tgg gga aag cag tat tca tac gca ctc ttc aaa gct 1056Val Asn Asp Ser Trp Gly Lys Gln Tyr Ser Tyr Ala Leu Phe Lys Ala 340 345 350atg agt cac atg ctg tgc att ggg tat gga gcc caa gcc cca gtc agc 1104Met Ser His Met Leu Cys Ile Gly Tyr Gly Ala Gln Ala Pro Val Ser 355 360 365atg tct gac ctc tgg att acc atg ctg agc atg atc gtc ggg gcc acc 1152Met Ser Asp Leu Trp Ile Thr Met Leu Ser Met Ile Val Gly Ala Thr 370 375 380tgc tat gcc atg ttt gtc ggc cat gcc acc gct tta atc cag tct ctg 1200Cys Tyr Ala Met Phe Val Gly His Ala Thr Ala Leu Ile Gln Ser Leu385 390 395 400gat tct tcg agg cgg cag tat caa gag aag tat aag caa gtg gaa caa 1248Asp Ser Ser Arg Arg Gln Tyr Gln Glu Lys Tyr Lys Gln Val Glu Gln 405 410 415tac atg tca ttc cat aag tta cca gct gat atg cgt cag aag ata cat 1296Tyr Met Ser Phe His Lys Leu Pro Ala Asp Met Arg Gln Lys Ile His 420 425 430gat tac tat gaa cac aga tac caa ggc aaa atc ttt gat gag gaa aat 1344Asp Tyr Tyr Glu His Arg Tyr Gln Gly Lys Ile Phe Asp Glu Glu Asn 435 440 445att ctc aat gaa ctc aat gat cct ctg aga gag gag ata gtc aac ttc 1392Ile Leu Asn Glu Leu Asn Asp Pro Leu Arg Glu Glu Ile Val Asn Phe 450 455 460aac tgt cgg aaa ctg gtg gct aca atg cct tta ttt gct aat gcg gat 1440Asn Cys Arg Lys Leu Val Ala Thr Met Pro Leu Phe Ala Asn Ala Asp465 470 475 480cct aat ttt gtg act gcc atg ctg agc aag ttg aga ttt gag gtg ttt 1488Pro Asn Phe Val Thr Ala Met Leu Ser Lys Leu Arg Phe Glu Val Phe 485 490 495caa cct gga gat tat atc ata cga gaa gga gcc gtg ggt aaa aaa atg 1536Gln Pro Gly Asp Tyr Ile Ile Arg Glu Gly Ala Val Gly Lys Lys Met 500 505 510tat ttc att caa cac ggt gtt gct ggt gtc att aca aaa tcc agt aaa 1584Tyr Phe Ile Gln His Gly Val Ala Gly Val Ile Thr Lys Ser Ser Lys 515 520 525gaa atg aag ctg aca gat ggc tct tac ttt gga gag att tgc ctg ctg 1632Glu Met Lys Leu Thr Asp Gly Ser Tyr Phe Gly Glu Ile Cys Leu Leu 530 535 540acc aaa gga cgt cgt act gcc agt gtt cga gct gat aca tat tgt cgt 1680Thr Lys Gly Arg Arg Thr Ala Ser Val Arg Ala Asp Thr Tyr Cys Arg545 550 555 560ctt tac tca ctt tcc gtg gac aat ttc aac gag gtc ctg gag gaa tat 1728Leu Tyr Ser Leu Ser Val Asp Asn Phe Asn Glu Val Leu Glu Glu Tyr 565 570 575cca atg atg agg aga gcc ttt gag aca gtt gcc att gac cga cta gat 1776Pro Met Met Arg Arg Ala Phe Glu Thr Val Ala Ile Asp Arg Leu Asp 580 585 590cga ata gga aag aaa aat tca att ctt ctg caa aag ttc cag aag gat 1824Arg Ile Gly Lys Lys Asn Ser Ile Leu Leu Gln Lys Phe Gln Lys Asp 595 600 605ctg aac act ggt gtt ttc aac aat cag gag aac gaa atc ctc aag cag 1872Leu Asn Thr Gly Val Phe Asn Asn Gln Glu Asn Glu Ile Leu Lys Gln 610 615 620att gtg aaa cat gac agg gag atg gtg cag gca atc gct ccc atc aat 1920Ile Val Lys His Asp Arg Glu Met Val Gln Ala Ile Ala Pro Ile Asn625 630 635 640tat cct caa atg aca acc ctg aat tcc aca tcg tct act acg acc ccg 1968Tyr Pro Gln Met Thr Thr Leu Asn Ser Thr Ser Ser Thr Thr Thr Pro 645 650 655acc tcc cgc atg agg aca caa tct cca ccg gtg tac aca gcg acc agc 2016Thr Ser Arg Met Arg Thr Gln Ser Pro Pro Val Tyr Thr Ala Thr Ser 660 665 670ctg tct cac agc aac ctg cac tcc ccc agt ccc agc aca cag acc ccc 2064Leu Ser His Ser Asn Leu His Ser Pro Ser Pro Ser Thr Gln Thr Pro 675 680 685cag cca tca gcc atc ctg tca ccc tgc tcc tac acc acc gcg gtc tgc 2112Gln Pro Ser Ala Ile Leu Ser Pro Cys Ser Tyr Thr Thr Ala Val Cys 690 695 700agc cct cct gta cag agc cct ctg gcc gct cga act ttc cac tat gcc 2160Ser Pro Pro Val Gln Ser Pro Leu Ala Ala Arg Thr Phe His Tyr Ala705 710 715 720tcc ccc acc gcc tcc cag ctg tca ctc atg caa cag cag ccg cag cag 2208Ser Pro Thr Ala Ser Gln Leu Ser Leu Met Gln Gln Gln Pro Gln Gln 725 730 735cag gta cag cag tcc cag ccg ccg cag act cag cca cag cag ccg tcc 2256Gln Val Gln Gln Ser Gln Pro Pro Gln Thr Gln Pro Gln Gln Pro Ser 740 745 750ccg cag cca cag aca cct ggc agc tcc acg ccg aaa aat gaa gtg cac 2304Pro Gln Pro Gln Thr Pro Gly Ser Ser Thr Pro Lys Asn Glu Val His 755 760 765aag agc acg cag gcg ctt cac aac acc aac ctg acc cgg gaa gtc agg 2352Lys Ser Thr Gln Ala Leu His Asn Thr Asn Leu Thr Arg Glu Val Arg 770 775 780cca ctc tcc gcc tcg cag ccc tcg ctg ccc cat gag gtg tcc act ctg 2400Pro Leu Ser Ala Ser Gln Pro Ser Leu Pro His Glu Val Ser Thr Leu785 790 795 800att tcc aga cct cat ccc act gtg ggc gag tcc ctg gcc tcc atc cct 2448Ile Ser Arg Pro His Pro Thr Val Gly Glu Ser Leu Ala Ser Ile Pro 805 810 815caa ccc gtg acg gcg gtc ccc gga acg ggc ctt cag gca ggg ggc agg 2496Gln Pro Val Thr Ala Val Pro Gly Thr Gly Leu Gln Ala Gly Gly Arg 820 825 830agc act gtc ccg cag cgc gtc acc ctc ttc cga cag atg tcg tcg gga 2544Ser Thr Val Pro Gln Arg Val Thr Leu Phe Arg Gln Met Ser Ser Gly 835 840 845gcc atc ccc ccg aac cga gga gtc cct cca gca ccc cct cca cca gca 2592Ala Ile Pro Pro Asn Arg Gly Val Pro Pro Ala Pro Pro Pro Pro Ala 850 855 860gct gct ctt cca aga gaa tct tcc tca gtc tta aac aca gac cca gac 2640Ala Ala Leu Pro Arg Glu Ser Ser Ser Val Leu Asn Thr Asp Pro Asp865 870 875 880gca gaa aag cca cga ttt gct tca aat tta tga 2673Ala Glu Lys Pro Arg Phe Ala Ser Asn Leu 885 8902890PRThuman 2Met Glu Gly Gly Gly Lys Pro Asn Ser Ser Ser Asn Ser Arg Asp Asp1 5 10 15Gly Asn Ser Val Phe Pro Ala Lys Ala Ser Ala Thr Gly Ala Gly Pro 20 25 30Ala Ala Ala Glu Lys Arg Leu Gly Thr Pro Pro Gly Gly Gly Gly Ala 35 40 45Gly Ala Lys Glu His Gly Asn Ser Val Cys Phe Lys Val Asp Gly Gly 50 55 60Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Glu Glu Pro Ala Gly Gly65 70 75 80Phe Glu Asp Ala Glu Gly Pro Arg Arg Gln Tyr Gly Phe Met Gln Arg 85 90 95Gln Phe Thr Ser Met Leu Gln Pro Gly Val Asn Lys Phe Ser Leu Arg 100 105 110Met Phe Gly Ser Gln Lys Ala Val Glu Lys Glu Gln Glu Arg Val Lys 115 120 125Thr Ala Gly Phe Trp Ile Ile His Pro Tyr Ser Asp Phe Arg Phe Tyr 130 135 140Trp Asp Leu Ile Met Leu Ile Met Met Val Gly Asn Leu Val Ile Ile145 150 155 160Pro Val Gly Ile Thr Phe Phe Thr Glu Gln Thr Thr Thr Pro Trp Ile 165 170 175Ile Phe Asn Val Ala Ser Asp Thr Val Phe Leu Leu Asp Leu Ile Met 180 185 190Asn Phe Arg Thr Gly Thr Val Asn Glu Asp Ser Ser Glu Ile Ile Leu 195 200 205Asp Pro Lys Val Ile Lys Met Asn Tyr Leu Lys Ser Trp Phe Val Val 210 215 220Asp Phe Ile Ser Ser Ile Pro Val Asp Tyr Ile Phe Leu Ile Val Glu225 230 235 240Lys Gly Met Asp Ser Glu Val Tyr Lys Thr Ala Arg Ala Leu Arg Ile 245 250 255Val Arg Phe Thr Lys Ile Leu Ser Leu Leu Arg Leu Leu Arg Leu Ser 260 265 270Arg Leu Ile Arg Tyr Ile His Gln Trp Glu Glu Ile Phe His Met Thr 275 280 285Tyr Asp Leu Ala Ser Ala Val Val Arg Ile Phe Asn Leu Ile Gly Met 290 295 300Met Leu Leu Leu Cys His Trp Asp Gly Cys Leu Gln Phe Leu Val Pro305 310 315 320Leu Leu Gln Asp Phe Pro Pro Asp Cys Trp Val Ser Leu Asn Glu Met 325 330 335Val Asn Asp Ser Trp Gly Lys Gln Tyr Ser Tyr Ala Leu Phe Lys Ala 340 345 350Met Ser His Met Leu Cys Ile Gly Tyr Gly Ala Gln Ala Pro Val Ser 355 360 365Met Ser Asp Leu Trp Ile Thr Met Leu Ser Met Ile Val Gly Ala Thr 370 375 380Cys Tyr Ala Met Phe Val Gly His Ala Thr Ala Leu Ile Gln Ser Leu385 390 395 400Asp Ser Ser Arg Arg Gln Tyr Gln Glu Lys Tyr Lys Gln Val Glu Gln 405 410 415Tyr Met Ser Phe His Lys Leu Pro Ala Asp Met Arg Gln Lys Ile His 420 425 430Asp Tyr Tyr Glu His Arg Tyr Gln Gly Lys Ile Phe Asp Glu Glu Asn 435 440 445Ile Leu Asn Glu Leu Asn Asp Pro Leu Arg Glu Glu Ile Val Asn Phe 450 455 460Asn Cys Arg Lys Leu Val Ala Thr Met Pro Leu Phe Ala Asn Ala Asp465 470 475 480Pro Asn Phe Val Thr Ala Met Leu Ser Lys Leu Arg Phe Glu Val Phe 485 490 495Gln Pro Gly Asp Tyr Ile Ile Arg Glu Gly Ala Val Gly Lys Lys Met 500 505 510Tyr Phe Ile Gln His Gly Val Ala Gly Val Ile Thr Lys Ser Ser Lys 515 520 525Glu Met Lys Leu Thr Asp Gly Ser Tyr Phe Gly Glu Ile Cys Leu Leu 530 535 540Thr Lys Gly Arg Arg Thr Ala Ser Val Arg Ala Asp Thr Tyr Cys Arg545 550 555 560Leu Tyr Ser Leu Ser Val Asp Asn Phe Asn Glu Val Leu Glu Glu Tyr 565 570 575Pro Met Met Arg Arg Ala Phe Glu Thr Val Ala Ile Asp Arg Leu Asp 580 585 590Arg Ile Gly Lys Lys Asn Ser Ile Leu Leu Gln Lys Phe Gln Lys Asp 595 600 605Leu Asn Thr Gly Val Phe Asn Asn Gln Glu Asn Glu Ile Leu Lys Gln 610 615 620Ile Val Lys His Asp Arg Glu Met Val Gln Ala Ile Ala Pro Ile Asn625 630 635 640Tyr Pro Gln Met Thr Thr Leu Asn Ser Thr Ser Ser Thr Thr Thr Pro 645 650 655Thr Ser Arg Met Arg Thr Gln Ser Pro Pro Val Tyr Thr Ala Thr Ser 660 665 670Leu Ser His Ser Asn Leu His Ser Pro Ser Pro Ser Thr Gln Thr Pro 675 680 685Gln Pro Ser Ala Ile Leu Ser Pro Cys Ser Tyr Thr Thr Ala Val Cys 690 695 700Ser Pro Pro Val Gln Ser Pro Leu Ala Ala Arg Thr Phe His Tyr Ala705 710 715 720Ser Pro Thr Ala Ser Gln Leu Ser Leu Met Gln Gln Gln Pro Gln Gln 725 730 735Gln Val Gln Gln Ser Gln Pro Pro Gln Thr Gln Pro Gln Gln Pro Ser 740 745 750Pro Gln Pro Gln Thr Pro Gly Ser Ser Thr Pro Lys Asn Glu Val His 755 760 765Lys Ser Thr Gln Ala Leu His Asn Thr Asn Leu Thr Arg Glu Val Arg 770 775 780Pro Leu Ser Ala Ser Gln Pro Ser Leu Pro His Glu Val Ser Thr Leu785 790 795 800Ile Ser Arg Pro His Pro Thr Val Gly Glu Ser Leu Ala Ser Ile Pro 805 810 815Gln Pro Val Thr Ala Val Pro Gly Thr Gly Leu Gln Ala Gly Gly Arg 820 825 830Ser Thr Val Pro Gln Arg Val Thr Leu Phe Arg Gln Met Ser Ser Gly 835 840 845Ala Ile Pro Pro Asn Arg Gly Val Pro Pro Ala Pro Pro Pro Pro Ala 850 855 860Ala Ala Leu Pro Arg Glu Ser Ser Ser Val Leu Asn Thr Asp Pro Asp865 870 875 880Ala Glu Lys Pro Arg Phe Ala Ser Asn Leu 885 89032670DNAhumanCDS(1)..(2670) 3atg gac gcg cgc ggg ggc ggc ggg cgg ccc ggg gag agc ccg ggc gcg 48Met Asp Ala Arg Gly Gly Gly Gly Arg Pro Gly Glu Ser Pro Gly Ala1 5 10 15acc ccc gcg ccg ggg ccg ccg ccg ccg ccg ccg ccc gcg ccc ccc caa 96Thr Pro Ala Pro Gly Pro Pro Pro Pro Pro Pro Pro Ala Pro Pro Gln 20 25 30cag cag ccg ccg ccg ccg ccg ccg ccc gcg ccc ccc ccg ggc ccc ggg 144Gln Gln Pro Pro Pro Pro Pro Pro Pro Ala Pro Pro Pro Gly Pro Gly 35 40 45ccc gcg ccc ccc cag cac ccg ccc cgg gcc gag gcg ttg ccc ccg gag 192Pro Ala Pro Pro Gln His Pro Pro Arg Ala Glu Ala Leu Pro Pro Glu 50 55 60gcg gcg gat gag ggc ggc ccg cgg ggc cgg ctc cgc agc cgc gac agc 240Ala Ala Asp Glu Gly Gly Pro Arg Gly Arg Leu Arg Ser Arg Asp Ser65 70 75 80tcg tgc ggc cgc ccc ggc acc ccg ggc gcg gcg agc acg gcc aag ggc 288Ser Cys Gly Arg Pro Gly Thr Pro

Gly Ala Ala Ser Thr Ala Lys Gly 85 90 95agc ccg aac ggc gag tgc ggg cgc ggc gag ccg cag tgc agc ccc gcg 336Ser Pro Asn Gly Glu Cys Gly Arg Gly Glu Pro Gln Cys Ser Pro Ala 100 105 110ggg ccc gag ggc ccg gcg cgg ggg ccc aag gtg tcg ttc tcg tgc cgc 384Gly Pro Glu Gly Pro Ala Arg Gly Pro Lys Val Ser Phe Ser Cys Arg 115 120 125ggg gcg gcc tcg ggg ccc gcg ccg ggg ccg ggg ccg gcg gag gag gcg 432Gly Ala Ala Ser Gly Pro Ala Pro Gly Pro Gly Pro Ala Glu Glu Ala 130 135 140ggc agc gag gag gcg ggc ccg gcg ggg gag ccg cgc ggc agc cag gcc 480Gly Ser Glu Glu Ala Gly Pro Ala Gly Glu Pro Arg Gly Ser Gln Ala145 150 155 160agc ttc atg cag cgc cag ttc ggc gcg ctc ctg cag ccg ggc gtc aac 528Ser Phe Met Gln Arg Gln Phe Gly Ala Leu Leu Gln Pro Gly Val Asn 165 170 175aag ttc tcg ctg cgg atg ttc ggc agc cag aag gcc gtg gag cgc gag 576Lys Phe Ser Leu Arg Met Phe Gly Ser Gln Lys Ala Val Glu Arg Glu 180 185 190cag gag cgc gtc aag tcg gcg ggg gcc tgg atc atc cac ccg tac agc 624Gln Glu Arg Val Lys Ser Ala Gly Ala Trp Ile Ile His Pro Tyr Ser 195 200 205gac ttc agg ttc tac tgg gac ttc acc atg ctg ctg ttc atg gtg gga 672Asp Phe Arg Phe Tyr Trp Asp Phe Thr Met Leu Leu Phe Met Val Gly 210 215 220aac ctc atc atc atc cca gtg ggc atc acc ttc ttc aag gat gag acc 720Asn Leu Ile Ile Ile Pro Val Gly Ile Thr Phe Phe Lys Asp Glu Thr225 230 235 240act gcc ccg tgg atc gtg ttc aac gtg gtc tcg gac acc ttc ttc ctc 768Thr Ala Pro Trp Ile Val Phe Asn Val Val Ser Asp Thr Phe Phe Leu 245 250 255atg gac ctg gtg ttg aac ttc cgc acc ggc att gtg atc gag gac aac 816Met Asp Leu Val Leu Asn Phe Arg Thr Gly Ile Val Ile Glu Asp Asn 260 265 270acg gag atc atc ctg gac ccc gag aag atc aag aag aag tat ctg cgc 864Thr Glu Ile Ile Leu Asp Pro Glu Lys Ile Lys Lys Lys Tyr Leu Arg 275 280 285acg tgg ttc gtg gtg gac ttc gtg tcc tcc atc ccc gtg gac tac atc 912Thr Trp Phe Val Val Asp Phe Val Ser Ser Ile Pro Val Asp Tyr Ile 290 295 300ttc ctt atc gtg gag aag ggc att gac tcc gag gtc tac aag acg gca 960Phe Leu Ile Val Glu Lys Gly Ile Asp Ser Glu Val Tyr Lys Thr Ala305 310 315 320cgc gcc ctg cgc atc gtg cgc ttc acc aag atc ctc agc ctc ctg cgg 1008Arg Ala Leu Arg Ile Val Arg Phe Thr Lys Ile Leu Ser Leu Leu Arg 325 330 335ctg ctg cgc ctc tca cgc ctg atc cgc tac atc cat cag tgg gag gag 1056Leu Leu Arg Leu Ser Arg Leu Ile Arg Tyr Ile His Gln Trp Glu Glu 340 345 350atc ttc cac atg acc tat gac ctg gcc agc gcg gtg atg agg atc tgc 1104Ile Phe His Met Thr Tyr Asp Leu Ala Ser Ala Val Met Arg Ile Cys 355 360 365aat ctc atc agc atg atg ctg ctg ctc tgc cac tgg gac ggc tgc ctg 1152Asn Leu Ile Ser Met Met Leu Leu Leu Cys His Trp Asp Gly Cys Leu 370 375 380cag ttc ctg gtg cct atg ctg cag gac ttc ccg cgc aac tgc tgg gtg 1200Gln Phe Leu Val Pro Met Leu Gln Asp Phe Pro Arg Asn Cys Trp Val385 390 395 400tcc atc aat ggc atg gtg aac cac tcg tgg agt gaa ctg tac tcc ttc 1248Ser Ile Asn Gly Met Val Asn His Ser Trp Ser Glu Leu Tyr Ser Phe 405 410 415gca ctc ttc aag gcc atg agc cac atg ctg tgc atc ggg tac ggc cgg 1296Ala Leu Phe Lys Ala Met Ser His Met Leu Cys Ile Gly Tyr Gly Arg 420 425 430cag gcg ccc gag agc atg acg gac atc tgg ctg acc atg ctc agc atg 1344Gln Ala Pro Glu Ser Met Thr Asp Ile Trp Leu Thr Met Leu Ser Met 435 440 445att gtg ggt gcc acc tgc tac gcc atg ttc atc ggc cac gcc act gcc 1392Ile Val Gly Ala Thr Cys Tyr Ala Met Phe Ile Gly His Ala Thr Ala 450 455 460ctc atc cag tcg ctg gac tcc tcg cgg cgc cag tac cag gag aag tac 1440Leu Ile Gln Ser Leu Asp Ser Ser Arg Arg Gln Tyr Gln Glu Lys Tyr465 470 475 480aag cag gtg gag cag tac atg tcc ttc cac aag ctg cca gct gac ttc 1488Lys Gln Val Glu Gln Tyr Met Ser Phe His Lys Leu Pro Ala Asp Phe 485 490 495cgc cag aag atc cac gac tac tat gag cac cgt tac cag ggc aag atg 1536Arg Gln Lys Ile His Asp Tyr Tyr Glu His Arg Tyr Gln Gly Lys Met 500 505 510ttt gac gag gac agc atc ctg ggc gag ctc aac ggg ccc ctg cgg gag 1584Phe Asp Glu Asp Ser Ile Leu Gly Glu Leu Asn Gly Pro Leu Arg Glu 515 520 525gag atc gtc aac ttc aac tgc cgg aag ctg gtg gcc tcc atg ccg ctg 1632Glu Ile Val Asn Phe Asn Cys Arg Lys Leu Val Ala Ser Met Pro Leu 530 535 540ttc gcc aac gcc gac ccc aac ttc gtc acg gcc atg ctg acc aag ctc 1680Phe Ala Asn Ala Asp Pro Asn Phe Val Thr Ala Met Leu Thr Lys Leu545 550 555 560aag ttc gag gtc ttc cag ccg ggt gac tac atc atc cgc gaa ggc acc 1728Lys Phe Glu Val Phe Gln Pro Gly Asp Tyr Ile Ile Arg Glu Gly Thr 565 570 575atc ggg aag aag atg tac ttc atc cag cac ggc gtg gtc agc gtg ctc 1776Ile Gly Lys Lys Met Tyr Phe Ile Gln His Gly Val Val Ser Val Leu 580 585 590act aag ggc aac aag gag atg aag ctg tcc gat ggc tcc tac ttc ggg 1824Thr Lys Gly Asn Lys Glu Met Lys Leu Ser Asp Gly Ser Tyr Phe Gly 595 600 605gag atc tgc ctg ctc acc cgg ggc cgc cgc acg gcg agc gtg cgg gct 1872Glu Ile Cys Leu Leu Thr Arg Gly Arg Arg Thr Ala Ser Val Arg Ala 610 615 620gac acc tac tgc cgc ctc tat tcg ctg agc gtg gac aac ttc aac gag 1920Asp Thr Tyr Cys Arg Leu Tyr Ser Leu Ser Val Asp Asn Phe Asn Glu625 630 635 640gtg ctg gag gag tac ccc atg atg cgg cgc gcc ttc gag acg gtg gcc 1968Val Leu Glu Glu Tyr Pro Met Met Arg Arg Ala Phe Glu Thr Val Ala 645 650 655atc gac cgc ctg gac cgc atc ggc aag aag aat tcc atc ctc ctg cac 2016Ile Asp Arg Leu Asp Arg Ile Gly Lys Lys Asn Ser Ile Leu Leu His 660 665 670aag gtg cag cat gac ctc aac tcg ggc gta ttc aac aac cag gag aac 2064Lys Val Gln His Asp Leu Asn Ser Gly Val Phe Asn Asn Gln Glu Asn 675 680 685gcc atc atc cag gag atc gtc aag tac gac cgc gag atg gtg cag cag 2112Ala Ile Ile Gln Glu Ile Val Lys Tyr Asp Arg Glu Met Val Gln Gln 690 695 700gcc gag ctg ggt cag cgc gtg ggc ctc ttc ccg ccg ccg ccg ccg ccg 2160Ala Glu Leu Gly Gln Arg Val Gly Leu Phe Pro Pro Pro Pro Pro Pro705 710 715 720ccg cag gtc acc tcg gcc atc gcc acg ctg cag cag gcg gcg gcc atg 2208Pro Gln Val Thr Ser Ala Ile Ala Thr Leu Gln Gln Ala Ala Ala Met 725 730 735agc ttc tgc ccg cag gtg gcg cgg ccg ctc gtg ggg ccg ctg gcg ctc 2256Ser Phe Cys Pro Gln Val Ala Arg Pro Leu Val Gly Pro Leu Ala Leu 740 745 750ggc tcg ccg cgc ctc gtg cgc cgc ccg ccc ccg ggg ccc gca cct gcc 2304Gly Ser Pro Arg Leu Val Arg Arg Pro Pro Pro Gly Pro Ala Pro Ala 755 760 765gcc gcc tca ccc ggg ccc ccg ccc ccc gcc agc ccc ccg ggc gcg ccc 2352Ala Ala Ser Pro Gly Pro Pro Pro Pro Ala Ser Pro Pro Gly Ala Pro 770 775 780gcc agc ccc cgg gca ccg cgg acc tcg ccc tac ggc ggc ctg ccc gcc 2400Ala Ser Pro Arg Ala Pro Arg Thr Ser Pro Tyr Gly Gly Leu Pro Ala785 790 795 800gcc ccc ctt gct ggg ccc gcc ctg ccc gcg cgc cgc ctg agc cgc gcg 2448Ala Pro Leu Ala Gly Pro Ala Leu Pro Ala Arg Arg Leu Ser Arg Ala 805 810 815tcg cgc cca ctg tcc gcc tcg cag ccc tcg ctg cct cac ggc gcc ccc 2496Ser Arg Pro Leu Ser Ala Ser Gln Pro Ser Leu Pro His Gly Ala Pro 820 825 830ggc ccc gcg gcc tcc aca cgc ccg gcc agc agc tcc aca ccg cgc ttg 2544Gly Pro Ala Ala Ser Thr Arg Pro Ala Ser Ser Ser Thr Pro Arg Leu 835 840 845ggg ccc acg ccc gct gcc cgg gcc gcc gcg ccc agc ccg gac cgc agg 2592Gly Pro Thr Pro Ala Ala Arg Ala Ala Ala Pro Ser Pro Asp Arg Arg 850 855 860gac tcg gcc tca ccc ggc gcc gcc ggc ggc ctg gac ccc cag gac tcc 2640Asp Ser Ala Ser Pro Gly Ala Ala Gly Gly Leu Asp Pro Gln Asp Ser865 870 875 880gcg cgc tcg cgc ctc tcg tcc aac ttg tga 2670Ala Arg Ser Arg Leu Ser Ser Asn Leu 8854889PRThuman 4Met Asp Ala Arg Gly Gly Gly Gly Arg Pro Gly Glu Ser Pro Gly Ala1 5 10 15Thr Pro Ala Pro Gly Pro Pro Pro Pro Pro Pro Pro Ala Pro Pro Gln 20 25 30Gln Gln Pro Pro Pro Pro Pro Pro Pro Ala Pro Pro Pro Gly Pro Gly 35 40 45Pro Ala Pro Pro Gln His Pro Pro Arg Ala Glu Ala Leu Pro Pro Glu 50 55 60Ala Ala Asp Glu Gly Gly Pro Arg Gly Arg Leu Arg Ser Arg Asp Ser65 70 75 80Ser Cys Gly Arg Pro Gly Thr Pro Gly Ala Ala Ser Thr Ala Lys Gly 85 90 95Ser Pro Asn Gly Glu Cys Gly Arg Gly Glu Pro Gln Cys Ser Pro Ala 100 105 110Gly Pro Glu Gly Pro Ala Arg Gly Pro Lys Val Ser Phe Ser Cys Arg 115 120 125Gly Ala Ala Ser Gly Pro Ala Pro Gly Pro Gly Pro Ala Glu Glu Ala 130 135 140Gly Ser Glu Glu Ala Gly Pro Ala Gly Glu Pro Arg Gly Ser Gln Ala145 150 155 160Ser Phe Met Gln Arg Gln Phe Gly Ala Leu Leu Gln Pro Gly Val Asn 165 170 175Lys Phe Ser Leu Arg Met Phe Gly Ser Gln Lys Ala Val Glu Arg Glu 180 185 190Gln Glu Arg Val Lys Ser Ala Gly Ala Trp Ile Ile His Pro Tyr Ser 195 200 205Asp Phe Arg Phe Tyr Trp Asp Phe Thr Met Leu Leu Phe Met Val Gly 210 215 220Asn Leu Ile Ile Ile Pro Val Gly Ile Thr Phe Phe Lys Asp Glu Thr225 230 235 240Thr Ala Pro Trp Ile Val Phe Asn Val Val Ser Asp Thr Phe Phe Leu 245 250 255Met Asp Leu Val Leu Asn Phe Arg Thr Gly Ile Val Ile Glu Asp Asn 260 265 270Thr Glu Ile Ile Leu Asp Pro Glu Lys Ile Lys Lys Lys Tyr Leu Arg 275 280 285Thr Trp Phe Val Val Asp Phe Val Ser Ser Ile Pro Val Asp Tyr Ile 290 295 300Phe Leu Ile Val Glu Lys Gly Ile Asp Ser Glu Val Tyr Lys Thr Ala305 310 315 320Arg Ala Leu Arg Ile Val Arg Phe Thr Lys Ile Leu Ser Leu Leu Arg 325 330 335Leu Leu Arg Leu Ser Arg Leu Ile Arg Tyr Ile His Gln Trp Glu Glu 340 345 350Ile Phe His Met Thr Tyr Asp Leu Ala Ser Ala Val Met Arg Ile Cys 355 360 365Asn Leu Ile Ser Met Met Leu Leu Leu Cys His Trp Asp Gly Cys Leu 370 375 380Gln Phe Leu Val Pro Met Leu Gln Asp Phe Pro Arg Asn Cys Trp Val385 390 395 400Ser Ile Asn Gly Met Val Asn His Ser Trp Ser Glu Leu Tyr Ser Phe 405 410 415Ala Leu Phe Lys Ala Met Ser His Met Leu Cys Ile Gly Tyr Gly Arg 420 425 430Gln Ala Pro Glu Ser Met Thr Asp Ile Trp Leu Thr Met Leu Ser Met 435 440 445Ile Val Gly Ala Thr Cys Tyr Ala Met Phe Ile Gly His Ala Thr Ala 450 455 460Leu Ile Gln Ser Leu Asp Ser Ser Arg Arg Gln Tyr Gln Glu Lys Tyr465 470 475 480Lys Gln Val Glu Gln Tyr Met Ser Phe His Lys Leu Pro Ala Asp Phe 485 490 495Arg Gln Lys Ile His Asp Tyr Tyr Glu His Arg Tyr Gln Gly Lys Met 500 505 510Phe Asp Glu Asp Ser Ile Leu Gly Glu Leu Asn Gly Pro Leu Arg Glu 515 520 525Glu Ile Val Asn Phe Asn Cys Arg Lys Leu Val Ala Ser Met Pro Leu 530 535 540Phe Ala Asn Ala Asp Pro Asn Phe Val Thr Ala Met Leu Thr Lys Leu545 550 555 560Lys Phe Glu Val Phe Gln Pro Gly Asp Tyr Ile Ile Arg Glu Gly Thr 565 570 575Ile Gly Lys Lys Met Tyr Phe Ile Gln His Gly Val Val Ser Val Leu 580 585 590Thr Lys Gly Asn Lys Glu Met Lys Leu Ser Asp Gly Ser Tyr Phe Gly 595 600 605Glu Ile Cys Leu Leu Thr Arg Gly Arg Arg Thr Ala Ser Val Arg Ala 610 615 620Asp Thr Tyr Cys Arg Leu Tyr Ser Leu Ser Val Asp Asn Phe Asn Glu625 630 635 640Val Leu Glu Glu Tyr Pro Met Met Arg Arg Ala Phe Glu Thr Val Ala 645 650 655Ile Asp Arg Leu Asp Arg Ile Gly Lys Lys Asn Ser Ile Leu Leu His 660 665 670Lys Val Gln His Asp Leu Asn Ser Gly Val Phe Asn Asn Gln Glu Asn 675 680 685Ala Ile Ile Gln Glu Ile Val Lys Tyr Asp Arg Glu Met Val Gln Gln 690 695 700Ala Glu Leu Gly Gln Arg Val Gly Leu Phe Pro Pro Pro Pro Pro Pro705 710 715 720Pro Gln Val Thr Ser Ala Ile Ala Thr Leu Gln Gln Ala Ala Ala Met 725 730 735Ser Phe Cys Pro Gln Val Ala Arg Pro Leu Val Gly Pro Leu Ala Leu 740 745 750Gly Ser Pro Arg Leu Val Arg Arg Pro Pro Pro Gly Pro Ala Pro Ala 755 760 765Ala Ala Ser Pro Gly Pro Pro Pro Pro Ala Ser Pro Pro Gly Ala Pro 770 775 780Ala Ser Pro Arg Ala Pro Arg Thr Ser Pro Tyr Gly Gly Leu Pro Ala785 790 795 800Ala Pro Leu Ala Gly Pro Ala Leu Pro Ala Arg Arg Leu Ser Arg Ala 805 810 815Ser Arg Pro Leu Ser Ala Ser Gln Pro Ser Leu Pro His Gly Ala Pro 820 825 830Gly Pro Ala Ala Ser Thr Arg Pro Ala Ser Ser Ser Thr Pro Arg Leu 835 840 845Gly Pro Thr Pro Ala Ala Arg Ala Ala Ala Pro Ser Pro Asp Arg Arg 850 855 860Asp Ser Ala Ser Pro Gly Ala Ala Gly Gly Leu Asp Pro Gln Asp Ser865 870 875 880Ala Arg Ser Arg Leu Ser Ser Asn Leu 88552325DNAhumanCDS(1)..(2325) 5atg gag gca gag cag cgg ccg gcg gcg ggg gcc agc gaa ggg gcg acc 48Met Glu Ala Glu Gln Arg Pro Ala Ala Gly Ala Ser Glu Gly Ala Thr1 5 10 15cct gga ctg gag gcg gtg cct ccc gtt gct ccc ccg cct gcg acc gcg 96Pro Gly Leu Glu Ala Val Pro Pro Val Ala Pro Pro Pro Ala Thr Ala 20 25 30gcc tca ggt ccg atc ccc aaa tct ggg cct gag cct aag agg agg cac 144Ala Ser Gly Pro Ile Pro Lys Ser Gly Pro Glu Pro Lys Arg Arg His 35 40 45ctt ggg acg ctg ctc cag cct acg gtc aac aag ttc tcc ctt cgg gtg 192Leu Gly Thr Leu Leu Gln Pro Thr Val Asn Lys Phe Ser Leu Arg Val 50 55 60ttc ggc agc cac aaa gca gtg gaa atc gag cag gag cgg gtg aag tca 240Phe Gly Ser His Lys Ala Val Glu Ile Glu Gln Glu Arg Val Lys Ser65 70 75 80gcg ggg gcc tgg atc atc cac ccc tac agc gac ttc cgg ttt tac tgg 288Ala Gly Ala Trp Ile Ile His Pro Tyr Ser Asp Phe Arg Phe Tyr Trp 85 90 95gac ctg atc atg ctg ctg ctg atg gtg ggg aac ctc atc gtc ctg cct 336Asp Leu Ile Met Leu Leu Leu Met Val Gly Asn Leu Ile Val Leu Pro 100 105 110gtg ggc atc acc ttc ttc aag gag gag aac tcc ccg cct tgg atc gtc 384Val Gly Ile Thr Phe Phe Lys Glu Glu Asn Ser Pro Pro Trp Ile Val 115 120 125ttc aac gta ttg tct gat act ttc ttc cta ctg gat ctg gtg ctc aac 432Phe Asn Val Leu Ser Asp Thr Phe Phe Leu Leu Asp Leu Val Leu Asn 130 135 140ttc cga acg ggc atc gtg gtg gag gag ggt gct gag atc ctg ctg gca 480Phe Arg Thr Gly Ile Val Val Glu Glu Gly Ala Glu Ile Leu Leu Ala145 150 155 160ccg cgg gcc atc cgc acg cgc tac ctg cgc acc tgg ttc ctg gtt gac 528Pro Arg Ala Ile Arg Thr Arg Tyr Leu Arg Thr Trp Phe Leu Val Asp 165 170 175ctc atc tct tct atc cct gtg gat tac atc ttc cta gtg gtg gag ctg

576Leu Ile Ser Ser Ile Pro Val Asp Tyr Ile Phe Leu Val Val Glu Leu 180 185 190gag cca cgg ttg gac gct gag gtc tac aaa acg gca cgg gcc cta cgc 624Glu Pro Arg Leu Asp Ala Glu Val Tyr Lys Thr Ala Arg Ala Leu Arg 195 200 205atc gtt cgc ttc acc aag atc cta agc ctg ctg agg ctg ctc cgc ctc 672Ile Val Arg Phe Thr Lys Ile Leu Ser Leu Leu Arg Leu Leu Arg Leu 210 215 220tcc cgc ctc atc cgc tac ata cac cag tgg gag gag atc ttt cac atg 720Ser Arg Leu Ile Arg Tyr Ile His Gln Trp Glu Glu Ile Phe His Met225 230 235 240acc tat gac ctg gcc agt gct gtg gtt cgc atc ttc aac ctc att ggg 768Thr Tyr Asp Leu Ala Ser Ala Val Val Arg Ile Phe Asn Leu Ile Gly 245 250 255atg atg ctg ctg cta tgt cac tgg gat ggc tgt ctg cag ttc ctg gtg 816Met Met Leu Leu Leu Cys His Trp Asp Gly Cys Leu Gln Phe Leu Val 260 265 270ccc atg ctg cag gac ttc cct ccc gac tgc tgg gtc tcc atc aac cac 864Pro Met Leu Gln Asp Phe Pro Pro Asp Cys Trp Val Ser Ile Asn His 275 280 285atg gtg aac cac tcg tgg ggc cgc cag tat tcc cat gcc ctg ttc aag 912Met Val Asn His Ser Trp Gly Arg Gln Tyr Ser His Ala Leu Phe Lys 290 295 300gcc atg agc cac atg ctg tgc att ggc tat ggg cag cag gca cct gta 960Ala Met Ser His Met Leu Cys Ile Gly Tyr Gly Gln Gln Ala Pro Val305 310 315 320ggc atg ccc gac gtc tgg ctc acc atg ctc agc atg atc gta ggt gcc 1008Gly Met Pro Asp Val Trp Leu Thr Met Leu Ser Met Ile Val Gly Ala 325 330 335aca tgc tac gcc atg ttc atc ggc cat gcc acg gca ctc atc cag tcc 1056Thr Cys Tyr Ala Met Phe Ile Gly His Ala Thr Ala Leu Ile Gln Ser 340 345 350ctg gac tct tcc cgg cgt cag tac cag gag aag tac aag cag gtg gag 1104Leu Asp Ser Ser Arg Arg Gln Tyr Gln Glu Lys Tyr Lys Gln Val Glu 355 360 365cag tac atg tcc ttc cac aag ctg cca gca gac acg cgg cag cgc atc 1152Gln Tyr Met Ser Phe His Lys Leu Pro Ala Asp Thr Arg Gln Arg Ile 370 375 380cac gag tac tat gag cac cgc tac cag ggc aag atg ttc gat gag gaa 1200His Glu Tyr Tyr Glu His Arg Tyr Gln Gly Lys Met Phe Asp Glu Glu385 390 395 400agc atc ctg ggc gag ctg agc gag ccg ctt cgc gag gag atc att aac 1248Ser Ile Leu Gly Glu Leu Ser Glu Pro Leu Arg Glu Glu Ile Ile Asn 405 410 415ttc acc tgt cgg ggc ctg gtg gcc cac atg ccg ctg ttt gcc cat gcc 1296Phe Thr Cys Arg Gly Leu Val Ala His Met Pro Leu Phe Ala His Ala 420 425 430gac ccc agc ttc gtc act gca gtt ctc acc aag ctg cgc ttt gag gtc 1344Asp Pro Ser Phe Val Thr Ala Val Leu Thr Lys Leu Arg Phe Glu Val 435 440 445ttc cag ccg ggg gat ctc gtg gtg cgt gag ggc tcc gtg ggg agg aag 1392Phe Gln Pro Gly Asp Leu Val Val Arg Glu Gly Ser Val Gly Arg Lys 450 455 460atg tac ttc atc cag cat ggg ctg ctc agt gtg ctg gcc cgc ggc gcc 1440Met Tyr Phe Ile Gln His Gly Leu Leu Ser Val Leu Ala Arg Gly Ala465 470 475 480cgg gac aca cgc ctc acc gat gga tcc tac ttt ggg gag atc tgc ctg 1488Arg Asp Thr Arg Leu Thr Asp Gly Ser Tyr Phe Gly Glu Ile Cys Leu 485 490 495cta act agg ggc cgg cgc aca gcc agt gtt cgg gct gac acc tac tgc 1536Leu Thr Arg Gly Arg Arg Thr Ala Ser Val Arg Ala Asp Thr Tyr Cys 500 505 510cgc ctt tac tca ctc agc gtg gac cat ttc aat gct gtg ctt gag gag 1584Arg Leu Tyr Ser Leu Ser Val Asp His Phe Asn Ala Val Leu Glu Glu 515 520 525ttc ccc atg atg cgc cgg gcc ttt gag act gtg gcc atg gat cgg ctg 1632Phe Pro Met Met Arg Arg Ala Phe Glu Thr Val Ala Met Asp Arg Leu 530 535 540ctc cgc atc ggc aag aag aat tcc ata ctg cag cgg aag cgc tcc gag 1680Leu Arg Ile Gly Lys Lys Asn Ser Ile Leu Gln Arg Lys Arg Ser Glu545 550 555 560cca agt cca ggc agc agt ggt ggc atc atg gag cag cac ttg gtg caa 1728Pro Ser Pro Gly Ser Ser Gly Gly Ile Met Glu Gln His Leu Val Gln 565 570 575cat gac aga gac atg gct cgg ggt gtt cgg ggt cgg gcc ccg agc aca 1776His Asp Arg Asp Met Ala Arg Gly Val Arg Gly Arg Ala Pro Ser Thr 580 585 590gga gct cag ctt agt gga aag cca gta ctg tgg gag cca ctg gta cat 1824Gly Ala Gln Leu Ser Gly Lys Pro Val Leu Trp Glu Pro Leu Val His 595 600 605gcg ccc ctt cag gca gct gct gtg acc tcc aat gtg gcc att gcc ctg 1872Ala Pro Leu Gln Ala Ala Ala Val Thr Ser Asn Val Ala Ile Ala Leu 610 615 620act cat cag cgg ggc cct ctg ccc ctc tcc cct gac tct cca gcc acc 1920Thr His Gln Arg Gly Pro Leu Pro Leu Ser Pro Asp Ser Pro Ala Thr625 630 635 640ctc ctt gct cgc tct gct tgg cgc tca gca ggc tct cca gct tcc ccg 1968Leu Leu Ala Arg Ser Ala Trp Arg Ser Ala Gly Ser Pro Ala Ser Pro 645 650 655ctg gtg ccc gtc cga gct ggc cca tgg gca tcc acc tcc cgc ctg ccc 2016Leu Val Pro Val Arg Ala Gly Pro Trp Ala Ser Thr Ser Arg Leu Pro 660 665 670gcc cca cct gcc cga acc ctg cac gcc agc cta tcc cgg gca ggg cgc 2064Ala Pro Pro Ala Arg Thr Leu His Ala Ser Leu Ser Arg Ala Gly Arg 675 680 685tcc cag gtc tcc ctg ctg ggt ccc cct cca gga gga ggt gga cgg cgg 2112Ser Gln Val Ser Leu Leu Gly Pro Pro Pro Gly Gly Gly Gly Arg Arg 690 695 700cta gga cct cgg ggc cgc cca ctc tca gcc tcc caa ccc tct ctg cct 2160Leu Gly Pro Arg Gly Arg Pro Leu Ser Ala Ser Gln Pro Ser Leu Pro705 710 715 720cag cgg gca aca ggc gat ggc tct cct ggg cgt aag gga tca gga agt 2208Gln Arg Ala Thr Gly Asp Gly Ser Pro Gly Arg Lys Gly Ser Gly Ser 725 730 735gag cgg ctg cct ccc tca ggg ctc ctg gcc aaa cct cca agg aca gcc 2256Glu Arg Leu Pro Pro Ser Gly Leu Leu Ala Lys Pro Pro Arg Thr Ala 740 745 750cag ccc ccc agg cca cca gtg cct gag cca gcc aca ccc cgg ggt ctc 2304Gln Pro Pro Arg Pro Pro Val Pro Glu Pro Ala Thr Pro Arg Gly Leu 755 760 765cag ctt tct gcc aac atg taa 2325Gln Leu Ser Ala Asn Met 7706774PRThuman 6Met Glu Ala Glu Gln Arg Pro Ala Ala Gly Ala Ser Glu Gly Ala Thr1 5 10 15Pro Gly Leu Glu Ala Val Pro Pro Val Ala Pro Pro Pro Ala Thr Ala 20 25 30Ala Ser Gly Pro Ile Pro Lys Ser Gly Pro Glu Pro Lys Arg Arg His 35 40 45Leu Gly Thr Leu Leu Gln Pro Thr Val Asn Lys Phe Ser Leu Arg Val 50 55 60Phe Gly Ser His Lys Ala Val Glu Ile Glu Gln Glu Arg Val Lys Ser65 70 75 80Ala Gly Ala Trp Ile Ile His Pro Tyr Ser Asp Phe Arg Phe Tyr Trp 85 90 95Asp Leu Ile Met Leu Leu Leu Met Val Gly Asn Leu Ile Val Leu Pro 100 105 110Val Gly Ile Thr Phe Phe Lys Glu Glu Asn Ser Pro Pro Trp Ile Val 115 120 125Phe Asn Val Leu Ser Asp Thr Phe Phe Leu Leu Asp Leu Val Leu Asn 130 135 140Phe Arg Thr Gly Ile Val Val Glu Glu Gly Ala Glu Ile Leu Leu Ala145 150 155 160Pro Arg Ala Ile Arg Thr Arg Tyr Leu Arg Thr Trp Phe Leu Val Asp 165 170 175Leu Ile Ser Ser Ile Pro Val Asp Tyr Ile Phe Leu Val Val Glu Leu 180 185 190Glu Pro Arg Leu Asp Ala Glu Val Tyr Lys Thr Ala Arg Ala Leu Arg 195 200 205Ile Val Arg Phe Thr Lys Ile Leu Ser Leu Leu Arg Leu Leu Arg Leu 210 215 220Ser Arg Leu Ile Arg Tyr Ile His Gln Trp Glu Glu Ile Phe His Met225 230 235 240Thr Tyr Asp Leu Ala Ser Ala Val Val Arg Ile Phe Asn Leu Ile Gly 245 250 255Met Met Leu Leu Leu Cys His Trp Asp Gly Cys Leu Gln Phe Leu Val 260 265 270Pro Met Leu Gln Asp Phe Pro Pro Asp Cys Trp Val Ser Ile Asn His 275 280 285Met Val Asn His Ser Trp Gly Arg Gln Tyr Ser His Ala Leu Phe Lys 290 295 300Ala Met Ser His Met Leu Cys Ile Gly Tyr Gly Gln Gln Ala Pro Val305 310 315 320Gly Met Pro Asp Val Trp Leu Thr Met Leu Ser Met Ile Val Gly Ala 325 330 335Thr Cys Tyr Ala Met Phe Ile Gly His Ala Thr Ala Leu Ile Gln Ser 340 345 350Leu Asp Ser Ser Arg Arg Gln Tyr Gln Glu Lys Tyr Lys Gln Val Glu 355 360 365Gln Tyr Met Ser Phe His Lys Leu Pro Ala Asp Thr Arg Gln Arg Ile 370 375 380His Glu Tyr Tyr Glu His Arg Tyr Gln Gly Lys Met Phe Asp Glu Glu385 390 395 400Ser Ile Leu Gly Glu Leu Ser Glu Pro Leu Arg Glu Glu Ile Ile Asn 405 410 415Phe Thr Cys Arg Gly Leu Val Ala His Met Pro Leu Phe Ala His Ala 420 425 430Asp Pro Ser Phe Val Thr Ala Val Leu Thr Lys Leu Arg Phe Glu Val 435 440 445Phe Gln Pro Gly Asp Leu Val Val Arg Glu Gly Ser Val Gly Arg Lys 450 455 460Met Tyr Phe Ile Gln His Gly Leu Leu Ser Val Leu Ala Arg Gly Ala465 470 475 480Arg Asp Thr Arg Leu Thr Asp Gly Ser Tyr Phe Gly Glu Ile Cys Leu 485 490 495Leu Thr Arg Gly Arg Arg Thr Ala Ser Val Arg Ala Asp Thr Tyr Cys 500 505 510Arg Leu Tyr Ser Leu Ser Val Asp His Phe Asn Ala Val Leu Glu Glu 515 520 525Phe Pro Met Met Arg Arg Ala Phe Glu Thr Val Ala Met Asp Arg Leu 530 535 540Leu Arg Ile Gly Lys Lys Asn Ser Ile Leu Gln Arg Lys Arg Ser Glu545 550 555 560Pro Ser Pro Gly Ser Ser Gly Gly Ile Met Glu Gln His Leu Val Gln 565 570 575His Asp Arg Asp Met Ala Arg Gly Val Arg Gly Arg Ala Pro Ser Thr 580 585 590Gly Ala Gln Leu Ser Gly Lys Pro Val Leu Trp Glu Pro Leu Val His 595 600 605Ala Pro Leu Gln Ala Ala Ala Val Thr Ser Asn Val Ala Ile Ala Leu 610 615 620Thr His Gln Arg Gly Pro Leu Pro Leu Ser Pro Asp Ser Pro Ala Thr625 630 635 640Leu Leu Ala Arg Ser Ala Trp Arg Ser Ala Gly Ser Pro Ala Ser Pro 645 650 655Leu Val Pro Val Arg Ala Gly Pro Trp Ala Ser Thr Ser Arg Leu Pro 660 665 670Ala Pro Pro Ala Arg Thr Leu His Ala Ser Leu Ser Arg Ala Gly Arg 675 680 685Ser Gln Val Ser Leu Leu Gly Pro Pro Pro Gly Gly Gly Gly Arg Arg 690 695 700Leu Gly Pro Arg Gly Arg Pro Leu Ser Ala Ser Gln Pro Ser Leu Pro705 710 715 720Gln Arg Ala Thr Gly Asp Gly Ser Pro Gly Arg Lys Gly Ser Gly Ser 725 730 735Glu Arg Leu Pro Pro Ser Gly Leu Leu Ala Lys Pro Pro Arg Thr Ala 740 745 750Gln Pro Pro Arg Pro Pro Val Pro Glu Pro Ala Thr Pro Arg Gly Leu 755 760 765Gln Leu Ser Ala Asn Met 77073612DNAhumanCDS(1)..(3612) 7atg gac aag ctg ccg ccg tcc atg cgc aag cgg ctc tac agc ctc ccg 48Met Asp Lys Leu Pro Pro Ser Met Arg Lys Arg Leu Tyr Ser Leu Pro1 5 10 15cag cag gtg ggg gcc aag gcg tgg atc atg gac gag gaa gag gac gcc 96Gln Gln Val Gly Ala Lys Ala Trp Ile Met Asp Glu Glu Glu Asp Ala 20 25 30gag gag gag ggg gcc ggg ggc cgc caa gac ccc agc cgc agg agc atc 144Glu Glu Glu Gly Ala Gly Gly Arg Gln Asp Pro Ser Arg Arg Ser Ile 35 40 45cgg ctg cgg cca ctg ccc tcg ccc tcc ccc tcg gcg gcc gcg ggt ggc 192Arg Leu Arg Pro Leu Pro Ser Pro Ser Pro Ser Ala Ala Ala Gly Gly 50 55 60acg gag tcc cgg agc tcg gcc ctc ggg gca gcg gac agc gaa ggg ccg 240Thr Glu Ser Arg Ser Ser Ala Leu Gly Ala Ala Asp Ser Glu Gly Pro65 70 75 80gcc cgc ggc gcg ggc aag tcc agc acg aac ggc gac tgc agg cgc ttc 288Ala Arg Gly Ala Gly Lys Ser Ser Thr Asn Gly Asp Cys Arg Arg Phe 85 90 95cgc ggg agc ctg gcc tcg ctg ggc agc cgg ggc ggc ggc agc ggc ggc 336Arg Gly Ser Leu Ala Ser Leu Gly Ser Arg Gly Gly Gly Ser Gly Gly 100 105 110acg ggg agc ggc agc agt cac gga cac ctg cat gac tcc gcg gag gag 384Thr Gly Ser Gly Ser Ser His Gly His Leu His Asp Ser Ala Glu Glu 115 120 125cgg cgg ctc atc gcc gag ggc gac gcg tcc ccc ggc gag gac agg acg 432Arg Arg Leu Ile Ala Glu Gly Asp Ala Ser Pro Gly Glu Asp Arg Thr 130 135 140ccc cca ggc ctg gcg gcc gag ccc gag cgc ccc ggc gcc tcg gcg cag 480Pro Pro Gly Leu Ala Ala Glu Pro Glu Arg Pro Gly Ala Ser Ala Gln145 150 155 160ccc gca gcc tcg ccg ccg ccg ccc cag cag cca ccg cag ccg gcc tcc 528Pro Ala Ala Ser Pro Pro Pro Pro Gln Gln Pro Pro Gln Pro Ala Ser 165 170 175gcc tcc tgc gag cag ccc tcg gtg gac acc gct atc aaa gtg gag gga 576Ala Ser Cys Glu Gln Pro Ser Val Asp Thr Ala Ile Lys Val Glu Gly 180 185 190ggc gcg gct gcc ggc gac cag atc ctc ccg gag gcc gag gtg cgc ctg 624Gly Ala Ala Ala Gly Asp Gln Ile Leu Pro Glu Ala Glu Val Arg Leu 195 200 205ggc cag gcc ggc ttc atg cag cgc cag ttc ggg gcc atg ctc caa ccc 672Gly Gln Ala Gly Phe Met Gln Arg Gln Phe Gly Ala Met Leu Gln Pro 210 215 220ggg gtc aac aaa ttc tcc cta agg atg ttc ggc agc cag aaa gcc gtg 720Gly Val Asn Lys Phe Ser Leu Arg Met Phe Gly Ser Gln Lys Ala Val225 230 235 240gag cgc gaa cag gag agg gtc aag tcg gcc gga ttt tgg att atc cac 768Glu Arg Glu Gln Glu Arg Val Lys Ser Ala Gly Phe Trp Ile Ile His 245 250 255ccc tac agt gac ttc aga ttt tac tgg gac ctg acc atg ctg ctg ctg 816Pro Tyr Ser Asp Phe Arg Phe Tyr Trp Asp Leu Thr Met Leu Leu Leu 260 265 270atg gtg gga aac ctg att atc att cct gtg ggc atc acc ttc ttc aag 864Met Val Gly Asn Leu Ile Ile Ile Pro Val Gly Ile Thr Phe Phe Lys 275 280 285gat gag aac acc aca ccc tgg att gtc ttc aat gtg gtg tca gac aca 912Asp Glu Asn Thr Thr Pro Trp Ile Val Phe Asn Val Val Ser Asp Thr 290 295 300ttc ttc ctc atc gac ttg gtc ctc aac ttc cgc aca ggg atc gtg gtg 960Phe Phe Leu Ile Asp Leu Val Leu Asn Phe Arg Thr Gly Ile Val Val305 310 315 320gag gac aac aca gag atc atc ctg gac ccg cag cgg att aaa atg aag 1008Glu Asp Asn Thr Glu Ile Ile Leu Asp Pro Gln Arg Ile Lys Met Lys 325 330 335tac ctg aaa agc tgg ttc atg gta gat ttc att tcc tcc atc ccc gtg 1056Tyr Leu Lys Ser Trp Phe Met Val Asp Phe Ile Ser Ser Ile Pro Val 340 345 350gac tac atc ttc ctc att gtg gag aca cgc atc gac tcg gag gtc tac 1104Asp Tyr Ile Phe Leu Ile Val Glu Thr Arg Ile Asp Ser Glu Val Tyr 355 360 365aag act gcc cgg gcc ctg cgc att gtc cgc ttc acg aag atc ctc agc 1152Lys Thr Ala Arg Ala Leu Arg Ile Val Arg Phe Thr Lys Ile Leu Ser 370 375 380ctc tta cgc ctg tta cgc ctc tcc cgc ctc att cga tat att cac cag 1200Leu Leu Arg Leu Leu Arg Leu Ser Arg Leu Ile Arg Tyr Ile His Gln385 390 395 400tgg gaa gag atc ttc cac atg acc tac gac ctg gcc agc gcc gtg gtg 1248Trp Glu Glu Ile Phe His Met Thr Tyr Asp Leu Ala Ser Ala Val Val 405 410 415cgc atc gtg aac ctc atc ggc atg atg ctc ctg ctc tgc cac tgg gac 1296Arg Ile Val Asn Leu Ile Gly Met Met Leu Leu Leu Cys His Trp Asp 420 425 430ggc tgc ctg cag ttc ctg gta ccc atg cta cag gac ttc cct gac gac 1344Gly Cys Leu Gln Phe Leu Val Pro Met Leu Gln Asp Phe Pro Asp Asp 435 440 445tgc tgg gtg tcc atc aac aac atg gtg aac aac tcc tgg ggg aag cag 1392Cys Trp Val Ser Ile Asn Asn Met Val Asn Asn Ser Trp Gly Lys Gln 450 455 460tac tcc tac gcg ctc

ttc aag gcc atg agc cac atg ctg tgc atc ggc 1440Tyr Ser Tyr Ala Leu Phe Lys Ala Met Ser His Met Leu Cys Ile Gly465 470 475 480tac ggg cgg cag gcg ccc gtg ggc atg tcc gac gtc tgg ctc acc atg 1488Tyr Gly Arg Gln Ala Pro Val Gly Met Ser Asp Val Trp Leu Thr Met 485 490 495ctc agc atg atc gtg ggt gcc acc tgc tac gcc atg ttc att ggc cac 1536Leu Ser Met Ile Val Gly Ala Thr Cys Tyr Ala Met Phe Ile Gly His 500 505 510gcc act gcc ctc atc cag tcc ctg gac tcc tcc cgg cgc cag tac cag 1584Ala Thr Ala Leu Ile Gln Ser Leu Asp Ser Ser Arg Arg Gln Tyr Gln 515 520 525gaa aag tac aag cag gtg gag cag tac atg tcc ttt cac aag ctc ccg 1632Glu Lys Tyr Lys Gln Val Glu Gln Tyr Met Ser Phe His Lys Leu Pro 530 535 540ccc gac acc cgg cag cgc atc cac gac tac tac gag cac cgc tac cag 1680Pro Asp Thr Arg Gln Arg Ile His Asp Tyr Tyr Glu His Arg Tyr Gln545 550 555 560ggc aag atg ttc gac gag gag agc atc ctg ggc gag cta agc gag ccc 1728Gly Lys Met Phe Asp Glu Glu Ser Ile Leu Gly Glu Leu Ser Glu Pro 565 570 575ctg cgg gag gag atc atc aac ttt aac tgt cgg aag ctg gtg gcc tcc 1776Leu Arg Glu Glu Ile Ile Asn Phe Asn Cys Arg Lys Leu Val Ala Ser 580 585 590atg cca ctg ttt gcc aat gcg gac ccc aac ttc gtg acg tcc atg ctg 1824Met Pro Leu Phe Ala Asn Ala Asp Pro Asn Phe Val Thr Ser Met Leu 595 600 605acc aag ctg cgt ttc gag gtc ttc cag cct ggg gac tac atc atc cgg 1872Thr Lys Leu Arg Phe Glu Val Phe Gln Pro Gly Asp Tyr Ile Ile Arg 610 615 620gaa ggc acc att ggc aag aag atg tac ttc atc cag cat ggc gtg gtc 1920Glu Gly Thr Ile Gly Lys Lys Met Tyr Phe Ile Gln His Gly Val Val625 630 635 640agc gtg ctc acc aag ggc aac aag gag acc aag ctg gcc gac ggc tcc 1968Ser Val Leu Thr Lys Gly Asn Lys Glu Thr Lys Leu Ala Asp Gly Ser 645 650 655tac ttt gga gag atc tgc ctg ctg acc cgg ggc cgg cgc aca gcc agc 2016Tyr Phe Gly Glu Ile Cys Leu Leu Thr Arg Gly Arg Arg Thr Ala Ser 660 665 670gtg agg gcc gac acc tac tgc cgc ctc tac tcg ctg agc gtg gac aac 2064Val Arg Ala Asp Thr Tyr Cys Arg Leu Tyr Ser Leu Ser Val Asp Asn 675 680 685ttc aat gag gtg ctg gag gag tac ccc atg atg cga agg gcc ttc gag 2112Phe Asn Glu Val Leu Glu Glu Tyr Pro Met Met Arg Arg Ala Phe Glu 690 695 700acc gtg gcg ctg gac cgc ctg gac cgc att ggc aag aag aac tcc atc 2160Thr Val Ala Leu Asp Arg Leu Asp Arg Ile Gly Lys Lys Asn Ser Ile705 710 715 720ctc ctc cac aaa gtc cag cac gac ctc aac tcc ggc gtc ttc aac tac 2208Leu Leu His Lys Val Gln His Asp Leu Asn Ser Gly Val Phe Asn Tyr 725 730 735cag gag aat gag atc atc cag cag att gtg cag cat gac cgg gag atg 2256Gln Glu Asn Glu Ile Ile Gln Gln Ile Val Gln His Asp Arg Glu Met 740 745 750gcc cac tgc gcg cac cgc gtc cag gct gct gcc tct gcc acc cca acc 2304Ala His Cys Ala His Arg Val Gln Ala Ala Ala Ser Ala Thr Pro Thr 755 760 765ccc acg ccc gtc atc tgg acc ccg ctg atc cag gca cca ctg cag gct 2352Pro Thr Pro Val Ile Trp Thr Pro Leu Ile Gln Ala Pro Leu Gln Ala 770 775 780gcc gct gcc acc act tct gtg gcc ata gcc ctc acc cac cac cct cgc 2400Ala Ala Ala Thr Thr Ser Val Ala Ile Ala Leu Thr His His Pro Arg785 790 795 800ctg cct gct gcc atc ttc cgc cct ccc cca gga tct ggg ctg ggc aac 2448Leu Pro Ala Ala Ile Phe Arg Pro Pro Pro Gly Ser Gly Leu Gly Asn 805 810 815ctc ggt gcc ggg cag acg cca agg cac ctg aaa cgg ctg cag tcc ctg 2496Leu Gly Ala Gly Gln Thr Pro Arg His Leu Lys Arg Leu Gln Ser Leu 820 825 830atc cct tct gcg ctg ggc tcc gcc tcg ccc gcc agc agc ccg tcc cag 2544Ile Pro Ser Ala Leu Gly Ser Ala Ser Pro Ala Ser Ser Pro Ser Gln 835 840 845gtg gac aca ccg tct tca tcc tcc ttc cac atc caa cag ctg gct gga 2592Val Asp Thr Pro Ser Ser Ser Ser Phe His Ile Gln Gln Leu Ala Gly 850 855 860ttc tct gcc ccc gct gga ctg agc cca ctc ctg ccc tca tcc agc tcc 2640Phe Ser Ala Pro Ala Gly Leu Ser Pro Leu Leu Pro Ser Ser Ser Ser865 870 875 880tcc cca ccc ccc ggg gcc tgt ggc tcc ccc tcg gct ccc aca cca tca 2688Ser Pro Pro Pro Gly Ala Cys Gly Ser Pro Ser Ala Pro Thr Pro Ser 885 890 895gct ggc gta gcc gcc acc acc ata gcc ggg ttt ggc cac ttc cac aag 2736Ala Gly Val Ala Ala Thr Thr Ile Ala Gly Phe Gly His Phe His Lys 900 905 910gcg ctg ggt ggc tcc ctg tcc tcc tcc gac tct ccc ctg ctc acc ccg 2784Ala Leu Gly Gly Ser Leu Ser Ser Ser Asp Ser Pro Leu Leu Thr Pro 915 920 925ctg cag cca ggc gcc cgc tcc ccg cag gct gcc cag cca tct ccc gcg 2832Leu Gln Pro Gly Ala Arg Ser Pro Gln Ala Ala Gln Pro Ser Pro Ala 930 935 940cca ccc ggg gcc cgg gga ggc ctg gga ctc ccg gag cac ttc ctg cca 2880Pro Pro Gly Ala Arg Gly Gly Leu Gly Leu Pro Glu His Phe Leu Pro945 950 955 960ccc cca ccc tca tcc aga tcc ccg tca tct agc ccc ggg cag ctg ggc 2928Pro Pro Pro Ser Ser Arg Ser Pro Ser Ser Ser Pro Gly Gln Leu Gly 965 970 975cag cct ccc ggg gag ttg tcc cta ggt ctg gcc act ggc cca ctg agc 2976Gln Pro Pro Gly Glu Leu Ser Leu Gly Leu Ala Thr Gly Pro Leu Ser 980 985 990acg cca gag aca ccc cca cgg cag cct gag ccg ccg tcc ctt gtg gca 3024Thr Pro Glu Thr Pro Pro Arg Gln Pro Glu Pro Pro Ser Leu Val Ala 995 1000 1005ggg gcc tct ggg ggg gct tcc cct gta ggc ttt act ccc cga gga 3069Gly Ala Ser Gly Gly Ala Ser Pro Val Gly Phe Thr Pro Arg Gly 1010 1015 1020ggt ctc agc ccc cct ggc cac agc cca ggc ccc cca aga acc ttc 3114Gly Leu Ser Pro Pro Gly His Ser Pro Gly Pro Pro Arg Thr Phe 1025 1030 1035ccg agt gcc ccg ccc cgg gcc tct ggc tcc cac gga tcc ttg ctc 3159Pro Ser Ala Pro Pro Arg Ala Ser Gly Ser His Gly Ser Leu Leu 1040 1045 1050ctg cca cct gca tcc agc ccc cca cca ccc cag gtc ccc cag cgc 3204Leu Pro Pro Ala Ser Ser Pro Pro Pro Pro Gln Val Pro Gln Arg 1055 1060 1065cgg ggc aca ccc ccg ctc acc ccc ggc cgc ctc acc cag gac ctc 3249Arg Gly Thr Pro Pro Leu Thr Pro Gly Arg Leu Thr Gln Asp Leu 1070 1075 1080aag ctc atc tcc gcg tct cag cca gcc ctg cct cag gac ggg gcg 3294Lys Leu Ile Ser Ala Ser Gln Pro Ala Leu Pro Gln Asp Gly Ala 1085 1090 1095cag act ctc cgc aga gcc tcc ccg cac tcc tca ggg gag tcc atg 3339Gln Thr Leu Arg Arg Ala Ser Pro His Ser Ser Gly Glu Ser Met 1100 1105 1110gct gcc ttc ccg ctc ttc ccc agg gct ggg ggt ggc agc ggg ggc 3384Ala Ala Phe Pro Leu Phe Pro Arg Ala Gly Gly Gly Ser Gly Gly 1115 1120 1125agt ggg agc agc ggg ggc ctc ggt ccc cct ggg agg ccc tat ggt 3429Ser Gly Ser Ser Gly Gly Leu Gly Pro Pro Gly Arg Pro Tyr Gly 1130 1135 1140gcc atc ccc ggc cag cac gtc act ctg cct cgg aag aca tcc tca 3474Ala Ile Pro Gly Gln His Val Thr Leu Pro Arg Lys Thr Ser Ser 1145 1150 1155ggt tct ttg cca ccc cct ctg tct ttg ttt ggg gca aga gcc acc 3519Gly Ser Leu Pro Pro Pro Leu Ser Leu Phe Gly Ala Arg Ala Thr 1160 1165 1170tct tct ggg ggg ccc cct ctg act gct gga ccc cag agg gaa cct 3564Ser Ser Gly Gly Pro Pro Leu Thr Ala Gly Pro Gln Arg Glu Pro 1175 1180 1185ggg gcc agg cct gag cca gtg cgc tcc aaa ctg cca tcc aat cta 3609Gly Ala Arg Pro Glu Pro Val Arg Ser Lys Leu Pro Ser Asn Leu 1190 1195 1200tga 361281203PRThuman 8Met Asp Lys Leu Pro Pro Ser Met Arg Lys Arg Leu Tyr Ser Leu Pro1 5 10 15Gln Gln Val Gly Ala Lys Ala Trp Ile Met Asp Glu Glu Glu Asp Ala 20 25 30Glu Glu Glu Gly Ala Gly Gly Arg Gln Asp Pro Ser Arg Arg Ser Ile 35 40 45Arg Leu Arg Pro Leu Pro Ser Pro Ser Pro Ser Ala Ala Ala Gly Gly 50 55 60Thr Glu Ser Arg Ser Ser Ala Leu Gly Ala Ala Asp Ser Glu Gly Pro65 70 75 80Ala Arg Gly Ala Gly Lys Ser Ser Thr Asn Gly Asp Cys Arg Arg Phe 85 90 95Arg Gly Ser Leu Ala Ser Leu Gly Ser Arg Gly Gly Gly Ser Gly Gly 100 105 110Thr Gly Ser Gly Ser Ser His Gly His Leu His Asp Ser Ala Glu Glu 115 120 125Arg Arg Leu Ile Ala Glu Gly Asp Ala Ser Pro Gly Glu Asp Arg Thr 130 135 140Pro Pro Gly Leu Ala Ala Glu Pro Glu Arg Pro Gly Ala Ser Ala Gln145 150 155 160Pro Ala Ala Ser Pro Pro Pro Pro Gln Gln Pro Pro Gln Pro Ala Ser 165 170 175Ala Ser Cys Glu Gln Pro Ser Val Asp Thr Ala Ile Lys Val Glu Gly 180 185 190Gly Ala Ala Ala Gly Asp Gln Ile Leu Pro Glu Ala Glu Val Arg Leu 195 200 205Gly Gln Ala Gly Phe Met Gln Arg Gln Phe Gly Ala Met Leu Gln Pro 210 215 220Gly Val Asn Lys Phe Ser Leu Arg Met Phe Gly Ser Gln Lys Ala Val225 230 235 240Glu Arg Glu Gln Glu Arg Val Lys Ser Ala Gly Phe Trp Ile Ile His 245 250 255Pro Tyr Ser Asp Phe Arg Phe Tyr Trp Asp Leu Thr Met Leu Leu Leu 260 265 270Met Val Gly Asn Leu Ile Ile Ile Pro Val Gly Ile Thr Phe Phe Lys 275 280 285Asp Glu Asn Thr Thr Pro Trp Ile Val Phe Asn Val Val Ser Asp Thr 290 295 300Phe Phe Leu Ile Asp Leu Val Leu Asn Phe Arg Thr Gly Ile Val Val305 310 315 320Glu Asp Asn Thr Glu Ile Ile Leu Asp Pro Gln Arg Ile Lys Met Lys 325 330 335Tyr Leu Lys Ser Trp Phe Met Val Asp Phe Ile Ser Ser Ile Pro Val 340 345 350Asp Tyr Ile Phe Leu Ile Val Glu Thr Arg Ile Asp Ser Glu Val Tyr 355 360 365Lys Thr Ala Arg Ala Leu Arg Ile Val Arg Phe Thr Lys Ile Leu Ser 370 375 380Leu Leu Arg Leu Leu Arg Leu Ser Arg Leu Ile Arg Tyr Ile His Gln385 390 395 400Trp Glu Glu Ile Phe His Met Thr Tyr Asp Leu Ala Ser Ala Val Val 405 410 415Arg Ile Val Asn Leu Ile Gly Met Met Leu Leu Leu Cys His Trp Asp 420 425 430Gly Cys Leu Gln Phe Leu Val Pro Met Leu Gln Asp Phe Pro Asp Asp 435 440 445Cys Trp Val Ser Ile Asn Asn Met Val Asn Asn Ser Trp Gly Lys Gln 450 455 460Tyr Ser Tyr Ala Leu Phe Lys Ala Met Ser His Met Leu Cys Ile Gly465 470 475 480Tyr Gly Arg Gln Ala Pro Val Gly Met Ser Asp Val Trp Leu Thr Met 485 490 495Leu Ser Met Ile Val Gly Ala Thr Cys Tyr Ala Met Phe Ile Gly His 500 505 510Ala Thr Ala Leu Ile Gln Ser Leu Asp Ser Ser Arg Arg Gln Tyr Gln 515 520 525Glu Lys Tyr Lys Gln Val Glu Gln Tyr Met Ser Phe His Lys Leu Pro 530 535 540Pro Asp Thr Arg Gln Arg Ile His Asp Tyr Tyr Glu His Arg Tyr Gln545 550 555 560Gly Lys Met Phe Asp Glu Glu Ser Ile Leu Gly Glu Leu Ser Glu Pro 565 570 575Leu Arg Glu Glu Ile Ile Asn Phe Asn Cys Arg Lys Leu Val Ala Ser 580 585 590Met Pro Leu Phe Ala Asn Ala Asp Pro Asn Phe Val Thr Ser Met Leu 595 600 605Thr Lys Leu Arg Phe Glu Val Phe Gln Pro Gly Asp Tyr Ile Ile Arg 610 615 620Glu Gly Thr Ile Gly Lys Lys Met Tyr Phe Ile Gln His Gly Val Val625 630 635 640Ser Val Leu Thr Lys Gly Asn Lys Glu Thr Lys Leu Ala Asp Gly Ser 645 650 655Tyr Phe Gly Glu Ile Cys Leu Leu Thr Arg Gly Arg Arg Thr Ala Ser 660 665 670Val Arg Ala Asp Thr Tyr Cys Arg Leu Tyr Ser Leu Ser Val Asp Asn 675 680 685Phe Asn Glu Val Leu Glu Glu Tyr Pro Met Met Arg Arg Ala Phe Glu 690 695 700Thr Val Ala Leu Asp Arg Leu Asp Arg Ile Gly Lys Lys Asn Ser Ile705 710 715 720Leu Leu His Lys Val Gln His Asp Leu Asn Ser Gly Val Phe Asn Tyr 725 730 735Gln Glu Asn Glu Ile Ile Gln Gln Ile Val Gln His Asp Arg Glu Met 740 745 750Ala His Cys Ala His Arg Val Gln Ala Ala Ala Ser Ala Thr Pro Thr 755 760 765Pro Thr Pro Val Ile Trp Thr Pro Leu Ile Gln Ala Pro Leu Gln Ala 770 775 780Ala Ala Ala Thr Thr Ser Val Ala Ile Ala Leu Thr His His Pro Arg785 790 795 800Leu Pro Ala Ala Ile Phe Arg Pro Pro Pro Gly Ser Gly Leu Gly Asn 805 810 815Leu Gly Ala Gly Gln Thr Pro Arg His Leu Lys Arg Leu Gln Ser Leu 820 825 830Ile Pro Ser Ala Leu Gly Ser Ala Ser Pro Ala Ser Ser Pro Ser Gln 835 840 845Val Asp Thr Pro Ser Ser Ser Ser Phe His Ile Gln Gln Leu Ala Gly 850 855 860Phe Ser Ala Pro Ala Gly Leu Ser Pro Leu Leu Pro Ser Ser Ser Ser865 870 875 880Ser Pro Pro Pro Gly Ala Cys Gly Ser Pro Ser Ala Pro Thr Pro Ser 885 890 895Ala Gly Val Ala Ala Thr Thr Ile Ala Gly Phe Gly His Phe His Lys 900 905 910Ala Leu Gly Gly Ser Leu Ser Ser Ser Asp Ser Pro Leu Leu Thr Pro 915 920 925Leu Gln Pro Gly Ala Arg Ser Pro Gln Ala Ala Gln Pro Ser Pro Ala 930 935 940Pro Pro Gly Ala Arg Gly Gly Leu Gly Leu Pro Glu His Phe Leu Pro945 950 955 960Pro Pro Pro Ser Ser Arg Ser Pro Ser Ser Ser Pro Gly Gln Leu Gly 965 970 975Gln Pro Pro Gly Glu Leu Ser Leu Gly Leu Ala Thr Gly Pro Leu Ser 980 985 990Thr Pro Glu Thr Pro Pro Arg Gln Pro Glu Pro Pro Ser Leu Val Ala 995 1000 1005Gly Ala Ser Gly Gly Ala Ser Pro Val Gly Phe Thr Pro Arg Gly 1010 1015 1020Gly Leu Ser Pro Pro Gly His Ser Pro Gly Pro Pro Arg Thr Phe 1025 1030 1035Pro Ser Ala Pro Pro Arg Ala Ser Gly Ser His Gly Ser Leu Leu 1040 1045 1050Leu Pro Pro Ala Ser Ser Pro Pro Pro Pro Gln Val Pro Gln Arg 1055 1060 1065Arg Gly Thr Pro Pro Leu Thr Pro Gly Arg Leu Thr Gln Asp Leu 1070 1075 1080Lys Leu Ile Ser Ala Ser Gln Pro Ala Leu Pro Gln Asp Gly Ala 1085 1090 1095Gln Thr Leu Arg Arg Ala Ser Pro His Ser Ser Gly Glu Ser Met 1100 1105 1110Ala Ala Phe Pro Leu Phe Pro Arg Ala Gly Gly Gly Ser Gly Gly 1115 1120 1125Ser Gly Ser Ser Gly Gly Leu Gly Pro Pro Gly Arg Pro Tyr Gly 1130 1135 1140Ala Ile Pro Gly Gln His Val Thr Leu Pro Arg Lys Thr Ser Ser 1145 1150 1155Gly Ser Leu Pro Pro Pro Leu Ser Leu Phe Gly Ala Arg Ala Thr 1160 1165 1170Ser Ser Gly Gly Pro Pro Leu Thr Ala Gly Pro Gln Arg Glu Pro 1175 1180 1185Gly Ala Arg Pro Glu Pro Val Arg Ser Lys Leu Pro Ser Asn Leu 1190 1195 120099PRTartificialregion linking the S3 and S4 segment of HCN 9Glu Thr Arg Ile Asp Ser Glu Val Tyr1 5109PRTartificialregion linking the S3 and S4 segment of HCN 10Glu Ala Arg Ile Asp Ser Glu Val Tyr1 5114PRTartificialregion linking the S3 and S4 segment of HCN 11Glu Thr Arg Ile1124PRTartificialregion linking the S3 and S4 segment of HCN 12Glu Ala Arg Ile1139PRTartificialregion linking the S3

and S4 segment of HCN 13Glu Ala Gly Met Asp Ser Glu Val Tyr1 5149PRTartificialregion linking the S3 and S4 segment of HCN 14Glu Lys Gly Met Asp Ser Glu Val Tyr1 5159PRTartificialregion linking the S3 and S4 segment of HCN 15Glu Ala Arg Met Asp Ser Glu Val Tyr1 5166PRTartificialregion linking the S3 and S4 segment of HCN 16Glu Ala Arg Ile Asp Ser1 5179PRTartificialregion linking the S3 and S4 segment of HCN 17Glu Thr Arg Ile Asp Ser Gly Val Tyr1 5189PRTartificialregion linking the S3 and S4 segment of HCN 18Glu Thr Arg Ile Asp Ser Ala Val Tyr1 5199PRTartificialregion linking the S3 and S4 segment of HCN 19Glu Thr Gly Ile Asp Ser Glu Val Tyr1 5209PRTartificialregion linking the S3 and S4 segment of HCN 20Glu Ala Ala Ile Asp Ser Glu Val Tyr1 5219PRTartificialregion linking the S3 and S4 segment of HCN 21Glu Thr Arg Cys Asp Ser Glu Val Tyr1 5229PRTartificialregion linking the S3 and S4 segment of HCN 22Glu Thr Arg Ser Asp Ser Glu Val Tyr1 5239PRTartificialregion linking the S3 and S4 segment of HCN 23Glu Thr Arg Thr Asp Ser Glu Val Tyr1 5244PRTartificialregion linking the S3 and S4 segment of HCN 24Glu Ala Gly Met12536DNAartificialsynthetic oligonucleotide primer 25atagcgcgaa ttcccgccat gcagcgccag ttcggg 362634DNAartificialsynthetic oligonucleotide primer 26atagcgcgaa ttcccgccat gctccaaccc gggg 342737DNAartificialsynthetic oligonucleotide primer 27atagcgcgaa ttcccgccat gttcggcagc cagaaag 372821DNAartificialsynthetic oligonucleotide primer 28cggcggatcc cctagagata t 212932DNAartificialsynthetic oligonucleotide primer 29agcagaagct gatctcagag gaggacctgc tt 32302358DNAartificialHCN4 - MyC fusion 30agtcgacggt accgcgggcc cagcttggta ccgagctcgg atccactagt aacggccgcc 60agtgtgctgg aattccgcct cgccatggac aagctgccgc cgtccatgcg caagcggctc 120tacagcctcc cgcagcaggt gggggccaag gcgtggatca tggacgagga agaggacgcc 180gaggaggagg gggccggggg ccgccaagac cccagccgca ggagcatccg gctgcggcca 240ctgccctcgc cctccccctc ggcggccgcg ggtggcacgg agtcccggag ctcggccctc 300ggggcagcgg acagcgaagg gccggcccgc ggcgcgggca agtccagcac gaacggcgac 360tgcaggcgct tccgcgggag cctggcctcg ctgggcagcc ggggcggcgg cagcggcggc 420acggggagcg gcagcagtca cggacacctg catgactccg cggaggagcg gcggctcatc 480gccgagggcg acgcgtcccc cggcgaggac aggacgcccc caggcctggc ggccgagccc 540gagcgccccg gcgcctcggc gcagcccgca gcctcgccgc cgccgcccca gcagccaccg 600cagccggcct ccgcctcctg cgagcagccc tcggtggaca ccgctatcaa agtggaggga 660ggcgcggctg ccggcgacca gatcctcccg gaggccgagg tgcgcctggg ccaggccggc 720ttcatgcagc gccagttcgg ggccatgctc caacccgggg tcaacaaatt ctccctaagg 780atgttcggca gccagaaagc cgtggagcgc gaacaggaga gggtcaagtc ggccggattt 840tggattatcc acccctacag tgacttcaga ttttactggg acctgaccat gctgctgctg 900atggtgggaa acctgattat cattcctgtg ggcatcacct tcttcaagga tgagaacacc 960acaccctgga ttgtcttcaa tgtggtgtca gacacattct tcctcatcga cttggtcctc 1020aacttccgca cagggatcgt ggtggaggac aacacagaga tcatcctgga cccgcagcgg 1080attaaaatga agtacctgaa aagctggttc atggtagatt tcatttcctc catccccgtg 1140gactacatct tcctcattgt ggagacacgc atcgactcgg aggtctacaa gactgcccgg 1200gccctgcgca ttgtccgctt cacgaagatc ctcagcctct tacgcctgtt acgcctctcc 1260cgcctcattc gatatattca ccagtgggaa gagatcttcc acatgaccta cgacctggcc 1320agcgccgtgg tgcgcatcgt gaacctcatc ggcatgatgc tcctgctctg ccactgggac 1380ggctgcctgc agttcctggt acccatgcta caggacttcc ctgacgactg ctgggtgtcc 1440atcaacaaca tggtgaacaa ctcctggggg aagcagtact cctacgcgct cttcaaggcc 1500atgagccaca tgctgtgcat cggctacggg cggcaggcgc ccgtgggcat gtccgacgtc 1560tggctcacca tgctcagcat gatcgtgggt gccacctgct acgccatgtt cattggccac 1620gccactgccc tcatccagtc cctggactcc tcccggcgcc agtaccagga aaagtacaag 1680caggtggagc agtacatgtc ctttcacaag ctcccgcccg acacccggca gcgcatccac 1740gactactacg agcaccgcta ccagggcaag atgttcgacg aggagagcat cctgggcgag 1800ctaagcgagc ccctgcggga ggagatcatc aactttaact gtcggaagct ggtggcctcc 1860atgccactgt ttgccaatgc ggaccccaac ttcgtgacgt ccatgctgac caagctgcgt 1920ttcgaggtct tccagcctgg ggactacatc atccgggaag gcaccattgg caagaagatg 1980tacttcatcc agcatggcgt ggtcagcgtg ctcaccaagg gcaacaagga gaccaagctg 2040gccgacggct cctactttgg agagatctgc ctgctgaccc ggggccggcg cacagccagc 2100gtgagggccg acacctactg ccgcctctac tcgctgagcg tggacaactt caatgaggtg 2160ctggaggagt accccatgat gcgaagggcc ttcgagaccg tggcgctgga ccgcctggac 2220cgcattggca agaagaactc catcctcctc cacaaagtcc agcacgacct caactccggc 2280gtcttcaact accaggagca gaagctgatc tcagaggagg acctgctttg agcctgatat 2340ctctagggga tccgcccc 235831783PRTartificialHCN4 - MyC fusion 31Ser Arg Arg Tyr Arg Gly Pro Ser Leu Val Pro Ser Ser Asp Pro Leu1 5 10 15Val Thr Ala Ala Ser Val Leu Glu Phe Arg Leu Ala Met Asp Lys Leu 20 25 30Pro Pro Ser Met Arg Lys Arg Leu Tyr Ser Leu Pro Gln Gln Val Gly 35 40 45Ala Lys Ala Trp Ile Met Asp Glu Glu Glu Asp Ala Glu Glu Glu Gly 50 55 60Ala Gly Gly Arg Gln Asp Pro Ser Arg Arg Ser Ile Arg Leu Arg Pro65 70 75 80Leu Pro Ser Pro Ser Pro Ser Ala Ala Ala Gly Gly Thr Glu Ser Arg 85 90 95Ser Ser Ala Leu Gly Ala Ala Asp Ser Glu Gly Pro Ala Arg Gly Ala 100 105 110Gly Lys Ser Ser Thr Asn Gly Asp Cys Arg Arg Phe Arg Gly Ser Leu 115 120 125Ala Ser Leu Gly Ser Arg Gly Gly Gly Ser Gly Gly Thr Gly Ser Gly 130 135 140Ser Ser His Gly His Leu His Asp Ser Ala Glu Glu Arg Arg Leu Ile145 150 155 160Ala Glu Gly Asp Ala Ser Pro Gly Glu Asp Arg Thr Pro Pro Gly Leu 165 170 175Ala Ala Glu Pro Glu Arg Pro Gly Ala Ser Ala Gln Pro Ala Ala Ser 180 185 190Pro Pro Pro Pro Gln Gln Pro Pro Gln Pro Ala Ser Ala Ser Cys Glu 195 200 205Gln Pro Ser Val Asp Thr Ala Ile Lys Val Glu Gly Gly Ala Ala Ala 210 215 220Gly Asp Gln Ile Leu Pro Glu Ala Glu Val Arg Leu Gly Gln Ala Gly225 230 235 240Phe Met Gln Arg Gln Phe Gly Ala Met Leu Gln Pro Gly Val Asn Lys 245 250 255Phe Ser Leu Arg Met Phe Gly Ser Gln Lys Ala Val Glu Arg Glu Gln 260 265 270Glu Arg Val Lys Ser Ala Gly Phe Trp Ile Ile His Pro Tyr Ser Asp 275 280 285Phe Arg Phe Tyr Trp Asp Leu Thr Met Leu Leu Leu Met Val Gly Asn 290 295 300Leu Ile Ile Ile Pro Val Gly Ile Thr Phe Phe Lys Asp Glu Asn Thr305 310 315 320Thr Pro Trp Ile Val Phe Asn Val Val Ser Asp Thr Phe Phe Leu Ile 325 330 335Asp Leu Val Leu Asn Phe Arg Thr Gly Ile Val Val Glu Asp Asn Thr 340 345 350Glu Ile Ile Leu Asp Pro Gln Arg Ile Lys Met Lys Tyr Leu Lys Ser 355 360 365Trp Phe Met Val Asp Phe Ile Ser Ser Ile Pro Val Asp Tyr Ile Phe 370 375 380Leu Ile Val Glu Thr Arg Ile Asp Ser Glu Val Tyr Lys Thr Ala Arg385 390 395 400Ala Leu Arg Ile Val Arg Phe Thr Lys Ile Leu Ser Leu Leu Arg Leu 405 410 415Leu Arg Leu Ser Arg Leu Ile Arg Tyr Ile His Gln Trp Glu Glu Ile 420 425 430Phe His Met Thr Tyr Asp Leu Ala Ser Ala Val Val Arg Ile Val Asn 435 440 445Leu Ile Gly Met Met Leu Leu Leu Cys His Trp Asp Gly Cys Leu Gln 450 455 460Phe Leu Val Pro Met Leu Gln Asp Phe Pro Asp Asp Cys Trp Val Ser465 470 475 480Ile Asn Asn Met Val Asn Asn Ser Trp Gly Lys Gln Tyr Ser Tyr Ala 485 490 495Leu Phe Lys Ala Met Ser His Met Leu Cys Ile Gly Tyr Gly Arg Gln 500 505 510Ala Pro Val Gly Met Ser Asp Val Trp Leu Thr Met Leu Ser Met Ile 515 520 525Val Gly Ala Thr Cys Tyr Ala Met Phe Ile Gly His Ala Thr Ala Leu 530 535 540Ile Gln Ser Leu Asp Ser Ser Arg Arg Gln Tyr Gln Glu Lys Tyr Lys545 550 555 560Gln Val Glu Gln Tyr Met Ser Phe His Lys Leu Pro Pro Asp Thr Arg 565 570 575Gln Arg Ile His Asp Tyr Tyr Glu His Arg Tyr Gln Gly Lys Met Phe 580 585 590Asp Glu Glu Ser Ile Leu Gly Glu Leu Ser Glu Pro Leu Arg Glu Glu 595 600 605Ile Ile Asn Phe Asn Cys Arg Lys Leu Val Ala Ser Met Pro Leu Phe 610 615 620Ala Asn Ala Asp Pro Asn Phe Val Thr Ser Met Leu Thr Lys Leu Arg625 630 635 640Phe Glu Val Phe Gln Pro Gly Asp Tyr Ile Ile Arg Glu Gly Thr Ile 645 650 655Gly Lys Lys Met Tyr Phe Ile Gln His Gly Val Val Ser Val Leu Thr 660 665 670Lys Gly Asn Lys Glu Thr Lys Leu Ala Asp Gly Ser Tyr Phe Gly Glu 675 680 685Ile Cys Leu Leu Thr Arg Gly Arg Arg Thr Ala Ser Val Arg Ala Asp 690 695 700Thr Tyr Cys Arg Leu Tyr Ser Leu Ser Val Asp Asn Phe Asn Glu Val705 710 715 720Leu Glu Glu Tyr Pro Met Met Arg Arg Ala Phe Glu Thr Val Ala Leu 725 730 735Asp Arg Leu Asp Arg Ile Gly Lys Lys Asn Ser Ile Leu Leu His Lys 740 745 750Val Gln His Asp Leu Asn Ser Gly Val Phe Asn Tyr Gln Glu Gln Lys 755 760 765Leu Ile Ser Glu Glu Asp Leu Leu Ala Tyr Leu Gly Ile Arg Pro 770 775 780

Claims

1. A method comprising: identifying a distal end of a catheter at a tissue site of a patient; delivering a fluid or polymer that contains an HCN polynucleotide comprising a nucleotide sequence encoding an HCN polypeptide having channel activity, wherein the amino acid sequence of the HCN polypeptide and the amino acid sequence of a reference polypeptide have at least 80% identity, the reference polypeptide beginning with an amino acid selected from amino acids 92-214 and ending with an amino acid selected from amino acids 723-1188 of SEQ ID NO:8 to the tissue site of the patient via the catheter.

2. The method of claim 1 wherein the identifying comprises electrically sensing contact between a distal end of a catheter and the tissue site of the patient, and the method further comprises delivering an electrical stimulus to the cardiac tissue site of the patient to enhance transfer HCN polynucleotide to the tissue site via electroporation.

3. The method of claim 2 wherein delivering the electrical stimulus to the tissue site includes delivering the electrical stimulus to the tissue site via the catheter.

4. The method of claim 2 wherein delivering the electrical stimulus to the tissue site via the catheter comprises delivering the electrical stimulus to the tissue site via an electrode coupled to the catheter and an electrode coupled to a distal tip of a probe extending from the catheter.

5. The method of claim 2 wherein delivering the electrical stimulus to the tissue site via the catheter comprises delivering the electrical stimulus to the tissue site via an electrode coupled to the catheter and a distal tip of a probe extending from the catheter, the distal tip of the probe being formed from an electrically conductive material.

6. The method of claim 2 wherein delivering the electrical stimulus to the tissue site includes delivering the electrical stimulus to the tissue site via an implanted medical device.

7. The method of claim 2 wherein the electrical stimulus delivered to the tissue site comprises a stimulation pulse.

8. The method of claim 2 wherein the electrical stimulus delivered to the tissue site comprises a series of stimulation pulses.

9. The method of claim 2 wherein delivering fluid to the tissue site of the patient via the catheter includes delivering fluid to the tissue site of the patient via one or more exit ports of a distal tip of a probe extending from the catheter.

10. The method of claim 9 wherein the distal tip of the probe comprises a needle.

11. The method of claim 9 wherein the distal tip of the probe comprises a helix shaped distal tip.

12. The method of claim 9 wherein the distal tip of the probe extends from a body of the catheter upon sensing contact between the tissue site of the patient and the catheter.

13. The method of claim 1 wherein the HCN polynucleotide is present in a vector.

14. The method of claim 13 wherein the vector is a viral vector, a transposon vector, or a plasmid vector.

15. The method of claim 14 wherein the viral vector is a single strand adeno-associated virus or a self complementary adeno-associated virus.

16. A system comprising: a fluid supply; a catheter that includes a catheter body that defines an inner lumen, a probe within the inner lumen that delivers fluid from the fluid supply to a tissue site of a patient, and at least one electrode coupled to the catheter to detect contact between the catheter and the tissue site, wherein the fluid comprises an HCN polynucleotide of claim 1; and a power supply to generate an electrical stimulus that is delivered to the tissue site.

17. The system of claim 16 further comprising a pump to drive fluid from the fluid supply through the catheter.

18. The system of claim 16 wherein the power supply comprises an implanted medical device that delivers the electrical stimulus to the tissue site.

19. The system of claim 18 wherein the implanted medical device comprises one of an implantable pulse generator, an implantable cardioverter/defibrillator, and an implantable pacemaker/cardioverter/defibrillator.

20. The system of claim 16 wherein the fluid supply comprises an implanted fluid reservoir.

21. The system of claim 16 wherein the power supply is coupled to the catheter, and the catheter delivers the electrical stimulus to the tissue site.

22. The system of claim 21 wherein the probe includes a distal tip made from an electrically conductive material and the electrode is coupled to the catheter body, and the catheter delivers the electrical stimulus to the tissue site via the electrode coupled to the catheter body and the distal tip of the probe.

23. The system of claim 21 wherein the catheter includes a pair of electrodes, a first electrode coupled to the probe and a second electrode coupled to the catheter body, and the catheter delivers the electrical stimulus to the tissue site via the electrode coupled to the catheter body and the electrode coupled to the probe.

24. The system of claim 16 wherein the electrical stimulus delivered to the tissue site includes a stimulation pulse.

25. The system of claim 16 wherein the electrical stimulus delivered to the tissue site includes a series of stimulation pulses.

26. The system of claim 16 wherein the fluid comprises a polymer.

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