Patent Publication Number: US-2023151372-A1

Title: Composition and Method of Treatment for Heart Protection and Regeneration

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     This application claims the benefit of U.S. Provisional Application No. 63/253,526, filed Oct. 7, 2021 which is herein incorporated in its entirety. 
    
    
     REFERENCE TO AN ELECTRONIC SEQUENCE LISTING 
     The contents of the electronic sequence listing (Composition and Method of Treatment for Heart Protection and Regeneration.xml; Size: 57,880 bytes; and Date of Creation: Oct. 6, 2022) is herein incorporated by reference in its entirety. 
     BACKGROUND OF THE INVENTION 
     Metabolic flexibility is essential for the heart to adapt to various changes in the microenvironment (Karwi et al., 2018), and changes in metabolism and substrate utilization are well-demonstrated in cardiomyocytes (CMs) during development and following injury. Proliferative fetal CMs favor glycolysis to generate ATP during cardiac development; however, soon after birth, CMs begin to utilize primarily aerobic fatty acid (FA) metabolism. During the same time period, neonatal human CMs rapidly lose their proliferative ability (Bergmann et al., 2015). As the heart enlarges through childhood, rod-shaped CMs undergo hypertrophy, rather than hyperplasia. When injured by hypoxic stress, CMs enlarge due to pathological hypertrophy and their sarcomeric structures become disorganized. During this process, they also regain a small amount of proliferative ability along with a metabolic switch to glycolysis (Neubauer., 2007). This suggests that CM metabolism, dedifferentiation, and proliferation are intrinsically linked. Yet, in adult mammals this adaptive response is not strong enough for complete or even adequate cardiac regeneration after injury. Therefore, there is a need to amplify the metabolic switch or reprogramming to induce substantially higher level of adult CM dedifferentiation and proliferation following injury to provide higher level of CM regeneration. 
     SUMMARY OF THE INVENTION 
     The present invention provides a gene delivery composition comprising a gene delivery vehicle and a heterologous genome wherein the gene delivery vehicle houses or encapsulates the heterologous genome and wherein the heterologous genome comprises nucleic acid sequence at least 80%, 90% or 95% identical to SEQ. ID NO.:1. In an embodiment, the heterologous genome encodes human 3-hydroxy-3-methylglutaryl-CoA synthase 2 (mitochondrial) (HMGCS2) or its various isoforms. In an embodiment, the heterologous genome further comprises a 5′ primer site and a 3′ primer site flanking the nucleic acid sequence. In another embodiment, the heterologous genome encodes HMGCS2 enzyme or any of its functionally homologous forms. In an embodiment, the 5′ primer site comprises nucleotide sequence at least 80%, 90% or 95% identical to the nucleotide sequence of SEQ ID NO:2 and the 3′ primer site comprises nucleotide sequence at least 80%, 90% or 95% identical to the nucleotide sequence of SEQ ID NO:3. In another embodiment, the gene delivery vehicle comprises a nanoparticle. In an embodiment, the gene delivery vehicle comprises a recombinant adeno-associated virus (rAAV). In an embodiment, the rAAV comprises an AAV9 capsid. 
     The present invention also provides a method of treatment for cardiac ischemia comprising the step of providing a therapeutically effective amount of HMGCS2 to a patient. In an embodiment, the step of providing a therapeutically effective amount of HMGCS2 to the patient comprises the step of upregulating the expression of HMGCS2 in the patient&#39;s CM. In another embodiment, the step of upregulating the expression of HMGCS2 in the patient&#39;s CM comprises the step of administration of a therapeutically effective amount of the composition of claim  1  to the patient&#39;s heart. In an embodiment, step of administration of a therapeutically effective amount of the composition to the heart comprises administration of between about 10 7 -10 18 , about 10 11 -10 17  or about 10 12 -10 13  of the rAAV particles. In an embodiment, the step of providing a therapeutic effective amount of HMGCS2 to the patient is performed before the cardiac ischemia. In another embodiment, the step of providing a therapeutic effective amount of HMGCS2 to the patient is performed after the cardiac ischemia. In an embodiment, the step of providing a therapeutic effective amount of HMGCS2 to the patient is performed 1 day, 2 days, 5, days, 10 days, 20 days or 30 after the cardiac ischemia. 
     The present invention also provides a method of treatment for cardiac ischemia comprising the step inducing a metabolic switch of adult cardiomyocyte (CM) using HMGCS2. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIGS.  1 A- 1 S  show that in vivo CM-reprogramming induces metabolic switch, CM dedifferentiation and increased CM proliferation.  FIG.  1 A  illustrates the experimental design for investigating adult CM reprogramming in vivo.  FIG.  1 B  illustrates OSKM expression level and induction level in adult CMs after inducing OSKM reprogramming for 2 days.  FIG.  1 C  depicts the flow cytometry analysis of isolated proliferative CMs through BrdU tracking in CM-reprogramming mice after OSKM induction.  FIG.  1 D  depicts the percentage of proliferative CMs at each CM-reprogramming day determined by flow cytometry.  FIG.  1 E  depicts the schematic diagram of intravital imaging protocol used for live investigating CM-reprogramming hearts after PBS or OSKM induction in vivo for 2 days.  FIG.  1 F  depicts an investigation of CM alignment in the whole CM-reprogramming hearts by intravital microscopy after PBS or OSKM induction in vivo for 2 days.  FIG.  1 G  depicts the morphology of CMs in CM-reprogramming hearts determined by length and width in intravital imaging data after PBS or OSKM induction in vivo for 2 days. Each dot represents one CM in one Ctrl or reprogramming heart.  FIG.  1 H  depicts the aspect ratio determined by length-to-width ratio of each adult CMs of one control or CM-reprogramming mouse in intravital imaging data after PBS or OSKM induction in vivo for 2 days.  FIG.  1 I  depicts the aspect ratio determined by length-to-width ratio of each CM-reprogramming mouse in intravital imaging data after PBS or OSKM induction specifically in CMs in vivo for 2 days. Each dot represents one mouse sample.  FIG.  1 J  shows immunofluorescence staining of heart tissue sections showing morphology of proliferative CMs through H3P and WGA staining on CM-reprogramming hearts after PBS or OSKM induction for 2 days. Arrow heads represented H3P+ proliferative CMs. Scale bars were 50 μm.  FIG.  1 K  shows the percentage of proliferative CM percentage (H3P+%) in the heart tissue sections of CM-reprogramming hearts after PBS or OSKM induction for 2 days.  FIG.  1 L  depicts the morphology of H3P+ CMs in three CM-reprogramming hearts determined by length, width, and aspect ratio in heart tissue sections after OSKM induction in vivo for 2 days. Each dot represents one CM in one Ctrl or reprogramming heart.  FIG.  1 M  shows immunofluorescence of heart tissue sections showing morphology of proliferative CMs through Aurora B Kinase (AURKB) and cardiac Troponin T (cTnT) staining on control or CM-reprogramming hearts after PBS or OSKM induction for 2 days. Arrow heads represented AURKB+/cTnT+ proliferative CMs. Scale bars were 25 μm.  FIG.  1 N  shows the statistics of proliferative CM percentage (AURKB+%) in the heart tissue sections of CM-reprogramming hearts after PBS or OSKM induction for 2 days.  FIG.  1 O  depicts the experimental design for discovering the detail mechanism for adult CM reprogramming at day 2 by microarray analysis.  FIG.  1 P  depicts gene ontology analysis of gene expressional changes in adult CMs after PBS or OSKM induction for 2 days in vivo.  FIG.  1 Q  is a heat map showing metabolism-related gene expressional changes in adult CMs after PBS or OSKM induction for 2 days in vivo.  FIG.  1 R and  1 S  show live imaging of CM-specific OSKM mice, related to  FIG.  1 A to  1 Q .  FIG.  1 R  shows OSKM RNA expression measured by real-time PCR in several tissues isolated from control or CM-reprogramming mice after doxycycline treatment for 2 days.  FIG.  1 S  shows intravital live imaging of one construction in control or CM-reprogramming hearts after doxycycline treatment for 2 days. 
         FIGS.  2 A to  2 V  show how cardiac-specific ketogenesis creates a systemic and specific metabolic switch along with mitochondrial changes, inducing CM dedifferentiation at CM-reprogramming day 2.  FIG.  2 A  depicts the experimental design for metabolic profiling using LC-MS analysis.  FIG.  2 B  shows hits detected by LC-MS analysis especially in both control and CM-reprogramming hearts.  FIG.  2 C  depicts grouping of metabolic hits detected by LC-MS analysis in control or CM-reprogramming hearts.  FIG.  2 D  shows the experimental design for metabolic profiling using a working heart system perfused with 13C-metabolites, detected by NMR.  FIG.  2 E  depicts the oxidation percentage of control and CM-reprogramming hearts measured by 13C-glutamate level derived from different 13C-metabolic substrates through NMR analysis.  FIG.  2 F  depicts ratio (CM-reprogramming to control hearts) of specific 13C-metabolites of control and CM-reprogramming hearts detected by NMR.  FIG.  2 G  depicts the experimental design for measuring ketogenesis in the control or CM-reprogramming hearts.  FIG.  2 H  depicts the HMG-CoA level detected by HPLC in the isolated mitochondria from control or CM-reprogramming hearts.  FIG.  2 I  depicts the OHB level measured by OHB colorimetric assay in the isolated CMs from control or CM-reprogramming mice.  FIG.  2 J  depicts the OCR detected by Seahorse analysis in the isolated CMs from control or CM-reprogramming mice.  FIG.  2 K  shows the quantification of basal and maximal OCRs in the control or reprogramming CMs isolated from PBS or OSKM-treated hearts.  FIG.  2 L  depicts the RNA expression of Hmgcs2 normalized by GAPDH in CMs isolated from control or OSKM-treated mice.  FIG.  2 M  depicts protein expression of HMGCS2 in CMs isolated from control or OSKM-treated mice.  FIG.  2 N  depicts a schematic diagram showing metabolic switch in adult CMs after OSKM induction for 2 days.  FIG.  2 O  shows mitochondrial copy numbers detected by mtDNA through real-time PCR in control or reprogramming CMs isolated from PBS or OSKM-treated hearts.  FIG.  2 P  shows mitochondrial RNA expression detected by real-time PCR in control or reprogramming CMs isolated from PBS or OSKM treated hearts. These RNA expressions were normalized by GAPDH.  FIG.  2 Q  shows mitochondrial structure examined by TEM in isolated control or CM-reprogramming hearts.  FIG.  2 R  shows mitochondrial size in isolated control or CM-reprogramming hearts, determined by TEM.  FIG.  2 S  shows the aspect ratio of mitochondrial length-to-width in isolated control or CM-reprogramming, determined by TEM.  FIG.  2 T to  2 V  show cardiac function of control or CM-OSKM mice, related to  FIG.  2 A to  2 S .  FIG.  2 T  shows NMR peaks for measuring oxidation % of different metabolic substrates in control or CM-reprogramming hearts.  FIG.  2 U  depicts cardiac function measured by echocardiography in control or CM-reprogramming hearts.  FIG.  2 V  shows Western-blotting of phosphorylated DRP-1 on Ser616 or DRP-1 protein expression in control or CM-reprogramming CMs. 
         FIGS.  3 A to  3 S  show that forced HMGCS2 overexpression increases adult CM dedifferentiation and proliferation for heart function improvement after myocardial infarction or under hypoxia when the forced HMGCS2 overexpression is effected before the myocardial infarction or imposition of the hypoxia environment.  FIG.  3 A  depicts the experimental design for performing myocardial infarction (MI) in AAV9-EGFP or AAV9-HMGCS2 mice.  FIG.  3 B  depicts heart function measured by echocardiography in AAV9-EGFP or AAV9-HMGCS2 mice.  FIG.  3 C  depicts heart function measured by catheterization in AAV9-EGFP or AAV9-HMGCS2 mice.  FIG.  3 D  depicts the fibrotic area in AAV9-EGFP or AAV9-HMGCS2 hearts shown by Masson Tri-chrome staining of heart tissue sections at post-MI day 21.  FIG.  3 E  shows quantification of fibrotic percentage in AAV9-EGFP or AAV9-HMGCS2 hearts at post-MI day 21 measured by Masson Trichrome Staining.  FIG.  3 F  shows immunofluorescence staining of heart tissue sections showing morphology of proliferative CMs through H3P and cTnT staining at the border zone of AAV9-EGFP or AAV9-HMGCS2 mice at post-MI day 3. Arrow heads represented H3P+/cTnT+ proliferative CMs. Scale bars were 50 μm.  FIG.  3 G  shows quantification of proliferative CMs (H3P+%) in the heart tissue sections of at the border zone of AAV9-EGFP or AAV9-HMGCS2 mice at post-MI day 3.  FIG.  3 H  shows immunofluorescence staining of heart tissue sections showing morphology of proliferative CMs through AURKB and cTnT staining at the border zone of AAV9-EGFP or AAV9-HMGCS2 mice at post-MI day 3. Arrow heads represented AURKB+/cTnT+ proliferative CMs. Scale bars were 25 μm.  FIG.  3 I  shows quantification of proliferative CM percentage (AURKB+%) in the heart tissue sections at the border zone of AAV9-EGFP or AAV9-HMGCS2 mice at post-MI day 3.  FIG.  3 J  shows experimental design for examining effects on forced HMGCS2 expression in hiPSC-CMs after Lenti-EGFP or Lenti-HMGCS2 infection.  FIG.  3 K  shows protein expression of HMGCS2 measured by western-blot in Ctrl or HMGCS2 overexpressed hiPSC-CM under hypoxia.  FIG.  3 L  shows OHB levels detected by OHB colorimetric assay in Ctrl or HMGCS2 overexpressed hiPSC-CM under hypoxia.  FIG.  3 M  shows the morphology of control or HMGCS2 overexpressed hiPSC-CM under hypoxia.  FIG.  3 N  shows the length of each control or HMGCS2 overexpressed hiPSC-CM under hypoxia.  FIG.  3 O  shows the width of each control or HMGCS2 overexpressed hiPSC-CM under hypoxia.  FIG.  3 P  shows the aspect ratio determined by length-to-width ratio of each control or HMGCS2 overexpressed hiPSC-CM under hypoxia.  FIG.  3 Q  shows the proliferative ability determined by calculation of CM numbers of control or HMGCS2 overexpressed hiPSC-CM after culturing in hypoxia chamber for 24 hours.  FIGS.  3 R and  3 S  show Lentiviral infection efficiency in hiPSC-CMs, related to  FIG.  3 A to  3 Q .  FIG.  3 R  shows the morphology of BF in hiPSC-CMs.  FIG.  3 S  shows the morphology of Lentiviral infection efficiency in hiPSC-CMs. 
         FIGS.  4 A to  4 I  show that forced HMGCS2 overexpression increases adult CM dedifferentiation and proliferation for heart function improvement after myocardial infarction or under hypoxia when the forced HMGCS2 overexpression is effected after the myocardial infarction or imposition of the hypoxia environment.  FIG.  4 A  depicts the experimental design for performing myocardial infarction (MI) in AAV9-EGFP or AAV9-HMGCS2 mice.  FIG.  4 B  depicts heart function measured by echocardiography in AAV9-EGFP or AAV9-HMGCS2 mice.  FIG.  4 C  depicts heart function measured by catheterization in AAV9-EGFP or AAV9-HMGCS2 mice.  FIG.  4 D  shows the fibrotic area in AAV9-EGFP or AAV9-HMGCS2 mice hearts shown by Masson Tri-chrome staining of heart tissue sections at post-cI/R day 21.  FIG.  4 E  shows quantification of infarct area % in heart sections of AAV9-EGFP or AAV9-HMGCS2 mice 21 day after cI/R. IS: infarct size; AAR: area at risk; LV: left ventricle.  FIG.  4 F  depicts the fibrotic area in AAV9-EGFP or AAV9-HMGCS2 hearts shown by Masson Tri-chrome staining of heart tissue sections at post-MI day 21.  FIG.  4 G  shows quantification of fibrotic percentage in AAV9-EGFP or AAV9-HMGCS2 hearts at post-MI day 21 measured by Masson Trichrome Staining.  FIG.  4 H  shows immunofluorescence staining of heart tissue sections showing morphology of proliferative CMs through H3P and cTnT staining at the border zone of AAV9-EGFP or AAV9-HMGCS2 mice at post-MI day 3. Arrow heads represented H3P+/cTnT+ proliferative CMs. Scale bars were 50 μm.  FIG.  4 I  shows quantification of proliferative CMs (H3P+%) in the heart tissue sections of at the border zone of AAV9-EGFP or AAV9-HMGCS2 mice at post-MI day 3. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The compositions of the present invention can comprise, consist of, or consist essentially of the essential elements and limitations of the invention described herein, as well as any of the additional or optional ingredients, components, or limitations described herein. 
     As used in the specification and claims, the singular form “a” “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a” cell includes a plurality of cells, including mixtures thereof. 
     “About” in the context of amount values refers to an average deviation of maximum ±20%, ±10% or ±5% based on the indicated value. For example, an amount of about 30 mg refers to 30 mg±6 mg, 30 mg±3 mg or 30 mg±1.5 mg. 
     A “therapeutically effective amount” is an amount sufficient to effect beneficial or desired results. A therapeutically effective amount can be administered in one or more administrations, applications or dosages. 
     A “subject,” “individual” or “patient” is used interchangeably herein, which refers to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets, By “AAV virion” is meant a complete virus particle, such as a wild-type (wt) AAV virus particle (comprising a linear, single-stranded AAV nucleic acid genome associated with an AAV capsid protein coat). In this regard, single-stranded AAV nucleic acid molecules of either complementary sense, i.e., “sense” or “antisense” strands, can be packaged into any one AAV virion and both strands are equally infectious. The term “adeno-associated virus” (AAV) in the context of the present invention includes without limitation AAV type 1, AAV type 2, AAV type 3 (including types 3A and  3 B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, avian AAV, bovine AAV, canine AAV, equine AAV, and ovine AAV and any other AAV now known or later discovered. 
     By “recombinant virus” is meant a virus that has been genetically altered, e.g., by the deletion of endogenous nucleic acid and/or addition or insertion of a heterologous nucleic acid construct into the particle. 
     A “nucleic acid” or “nucleotide sequence” is a sequence of nucleotide bases, and may be RNA, DNA or DNA-RNA hybrid sequences (including both naturally occurring and non-naturally occurring nucleotide), but is preferably either single or double stranded DNA sequences. The term should also be understood to include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs, and, as applicable to the embodiment being described, single (sense or antisense) and double-stranded polynucleotides. The terms “polynucleotide sequence” and “nucleotide sequence” are also used interchangeably herein. 
     A “coding sequence” or a sequence which “encodes” a particular protein, is a nucleic acid sequence which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5′ (amino) terminus and a translation stop codon at the 3′ (carboxy) terminus. A coding sequence can include, but is not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and even synthetic DNA sequences. A transcription termination sequence will usually be located 3′ to the coding sequence. 
     As used herein, the term “gene” or “recombinant gene” refers to a nucleic acid comprising an open reading frame encoding a polypeptide, including both exon and (optionally) intron sequences. 
     The term “heterologous” as it relates to nucleic acid sequences such as coding sequences and control sequences, denotes sequences that do not occur in nature or are not normally joined together in nature, and/or are not associated with a particular cell in nature. Thus, a “heterologous” region of a nucleic acid construct or a vector is a segment of nucleic acid within or attached to another nucleic acid molecule that is not found in association with the other molecule in nature. For example, a heterologous region of a nucleic acid construct could include a coding sequence flanked by sequences not found in association with the coding sequence in nature. Another example of a heterologous coding sequence is a construct where the coding sequence itself is not found in nature (e.g., synthetic sequences having codons different from the native gene). Similarly, a cell transformed with a construct which is not normally present in the cell would be considered heterologous for purposes of this invention. Allelic variation or naturally occurring mutational events do not give rise to heterologous DNA, as used herein. 
     A “recombinant AAV virion,” or “rAAV virion” is defined herein as an infectious, replication-defective virus comprising an AAV protein shell encapsulating one or more heterologous nucleotide sequence that may be flanked on both sides by AAV ITRs. A rAAV virion may be produced in a suitable host cell comprising an AAV vector, AAV helper functions, and accessory functions. In this manner, the host cell may be rendered capable of encoding AAV polypeptides that are required for packaging the AAV vector containing a recombinant nucleotide sequence of interest into infectious recombinant virion particles for subsequent gene delivery. 
     “Homology” refers to the percent of identity between two polynucleotide or two polypeptide moieties. The correspondence between the sequence from one moiety to another can be determined by techniques known in the art. For example, homology can be determined by a direct comparison of the sequence information between two polypeptide molecules by aligning the sequence information and using readily available computer programs. Alternatively, homology can be determined by hybridization of polynucleotides under conditions which allow for the formation of stable duplexes between homologous regions, followed by digestion with single stranded-specific nuclease(s), and size determination of the digested fragments. Two DNA, or two polypeptide sequences are “substantially homologous” to each other when at least about 80%, at least about 90% or at least about 95% of the nucleotides or amino acids match over a defined length of the molecules, as determined using the methods above. 
     A “functional homologue” or a “functional equivalent” of a given polypeptide may be molecules derived from the native polypeptide sequence, as well as recombinantly produced or chemically synthesized polypeptides that function in a manner similar to the reference molecule to achieve a desired result. Thus, a functional homologue of AAV Rep68 or Rep78 encompasses derivatives and analogues of those polypeptides, including any single or multiple amino acid additions, substitutions and/or deletions occurring internally or at the amino or carboxy termini thereof—so long as integration activity remains. 
     A “functional homologue” or a “functional equivalent” of a given adenoviral nucleotide region may be similar regions derived from a heterologous adenovirus serotype, nucleotide regions derived from another virus or from a cellular source, and recombinantly produced or chemically synthesized polynucleotides which function in a manner similar to the reference nucleotide region to achieve a desired result. Thus, a functional homologue of an adenoviral VA RNA gene region or an adenoviral E2A gene region encompasses derivatives and analogues of such gene regions-including any single or multiple nucleotide base additions, substitutions and/or deletions occurring within the regions, so long as the homologue retains the ability to provide its inherent accessory function to support AAV virion production at levels detectable above background. 
     A “gene delivery vehicle” comprises any method or composition capable of fully or partially encapsulating or housing genome to be carried or delivered to a desired target in a human body such as a cardiomyocyte. The gene delivery vehicle may be biological, chemical or physical in nature or a combination thereof and provides protection for the genome while being carried to be delivered to the desired target. Biological gene delivery vehicle may be bacterial or viral such as rAAV. Chemical gene delivery vehicle may be polymeric particles, liposomes, polymer-lipid hybrid nanoparticles, other biocompatible materials, or combinations thereof. Physical gene delivery vehicle may comprise microinjection, electroporation, ultrasound, gene dun, hydrodynamic applications, or combinations thereof. 
     The present invention provides a cardiac protection and/or regeneration composition and method of treatment based on 3-hydroxy-3-methylglutaryl-CoA synthase 2 (mitochondrial) (HMGCS2). 
     HMGCS2 is an enzyme in humans that is encoded by the HMGCS2 gene. A complete human HMGCS2 sequence hereby defined as SEQ ID NO. 1 is listed in the sequence listing section below as well as in Rojnueangnit et al. Eur J Med Genet. 2020 December; 63(12):104086 which is hereby incorporated in its entirety. The HMGCS2, belonging to the HMG-CoA synthase family, is known to be a mitochondrial enzyme that catalyzes the second and rate-limiting reaction of ketogenesis, a metabolic pathway that provides lipid-derived energy for various organs during times of carbohydrate deprivation, such as fasting, by addition of a third acetyl group to acetoacetyl-CoA, producing HMG-CoA. Mutations in this gene are associated with HMG-CoA synthase deficiency. Alternatively spliced transcript variants encoding different isoforms have been found for this gene such as those published by Puisac et al., Mol Biol Rep. 2012. 39:4777-4785 which is hereby incorporated in its entirety. 
     Cardiac regeneration after injury in adult mammals including adult humans is limited by the low proliferative capacity of cardiomyocytes (CMs). However, certain animals such as zebrafish, newts, and neonatal mice readily regenerate lost myocardium via a process involving dedifferentiation, which unlocks their proliferative capacities. Inspired by this concept, in Example 1 detailed below, we created an experimental model comprising mice with inducible, CM-specific expression of the Yamanaka factors, enabling adult CM reprogramming in vivo. Specifically, two days following induction by doxycycline, adult CMs presented a dedifferentiated phenotype and increased proliferation of CM in vivo indicating cardiac regeneration. Moreover, in Example 2 detailed below, microarray analysis revealed that metabolic changes were central to this process. In particular, metabolic switch from fatty acid to ketone utilization as indicated by increase in ketogenic enzyme HMGCS2. 
     Furthermore, Examples 3 and 4 showed that HMGCS2 overexpression by exogenous means is capable of rescuing cardiac function following ischemic injury when HMGCS2 overexpression is effect before (Example 3) as well as after (Example 4) the ischemic injury. Thus, experiments disclosed in the Examples below reveal that HMGCS2-induced ketogenesis leads to metabolic switch in adult CMs during early reprogramming, and this metabolic adaptation substantially increases adult CM dedifferentiation, facilitating cardiac regeneration after injury. 
     Therefore, embodiments of the present invention encompass various compositions capable of providing a therapeutically effective amount of HMGCS2, variants thereof disclosed herein or functional homologues to a patient capable of effecting cardiac protection and/or regeneration in infarcted or injured areas of the heart of the patient. The composition of the present invention may also encompass various compositions which when administered to the patient effects expression of a therapeutically effective amount of HMGCS2, variants thereof disclosed herein or functional homologues in cells of the patient such as cardiomyocyte capable of effecting cardiac protection and/or regeneration in infarcted or injured areas of the heart, including but not limited to compositions capable of effecting viral-mediated gene delivery, naked DNA delivery, mRNA delivery, transfection methods etc. . . . The composition of the present invention may also encompass various compositions which when administered to the patient effects expression of a therapeutically effective amount of HMGCS2, variants thereof disclosed herein or functional homologues in cells of the patient capable of effecting cardiac protection and/or regeneration in infarcted or injured areas of the heart, including but not limited to compositions comprising gene delivery vehicles housing or fully or partially encapsulating the HMGCS2 genome capable of effecting viral-mediated gene delivery, naked DNA delivery, mRNA delivery, transfection methods etc . . . . 
     In an embodiment, the composition of the present invention comprises rAAV comprising heterologous nucleic acids encoding HMGCS2, variants thereof disclosed herein or functional homologues capable of effecting cells of the patient to express HMGCS2, variants thereof disclosed herein or functional homologues at a substantially higher level than without the rAAV. AAV is a parvovirus belonging to the genus Dependovirus. Although it can infect human cells, AAV has not been associated with any human or animal disease and is stable at a wide range of physical and chemical conditions. In addition, making AAV a desirable gene delivery vehicle. 
     The wild type AAV genome is a linear, single-stranded DNA molecule containing 4681 nucleotides. It comprises an internal non-repeating genome flanked on each end by inverted terminal repeats (ITRs) which are approximately 145 base pairs (bp) in length. The ITRs have multiple functions, including originals of DNA replication and as packaging signals for the viral genome. 
     The internal non-repeated portion of the wild type AAV genome includes two large open reading frames, known as the AAV replication (rep) and capsid (cap) genes. The rep and cap genes code for viral proteins that allow the virus to replicate and package the viral genome into a virion. In particular, a family of at least four viral proteins an expressed from the AAV rep region, Rep 78, Rep 69, Rep 52 and Rep 40, named according to their apparent molecular weight, the AAV cap region encodes at least three proteins, VP1, VP2 and VP3. 
     AAV can be engineered to deliver genes of interest as rAAV by deleting at least some of the internal non-repeating portion of the AAV genome such as rep and cap and inserting one or more heterologous gene between the ITRs. In an embodiment, the rAAV of the present invention comprises AAV type 1, AAV type 2, AAV type 3 (including types 3A and  3 B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, avian AAV, bovine AAV, canine AAV, equine AAV, and ovine AAV and any other AAV now known or later discovered or a combination thereof. 
     The heterologous gene may be functionally linked to a heterologous promoter (constitutive, cell-specific, or inducible) capable of driving gene expression in the patient&#39;s target cells under appropriate conditions. Termination signals, such as polyadenylation sites, can also be included. 
     Therefore, in an embodiment, the composition of the present invention comprises rAAV with genome encoding HMGCS2, variants thereof disclosed herein or functional homologues such that a patient&#39;s cells infected with rAAV express HMGCS2, variants thereof disclosed herein or functional homologues as disclosed or shown in the Examples. In another embodiment, the composition of the present invention comprises AAV9 with genome encoding HMGCS2, variants thereof disclosed herein or functional homologues disclosed herein such that a patient&#39;s cells infected with rAAV express HMGCS2, variants thereof disclosed herein or functional homologues in the heart tissue as shown in the Examples. In an embodiment, the genome encoding HMGCS2, variants thereof disclosed herein or functional homologues comprises primers. Such primer may comprise. 
     
       
         
           
               
               
            
               
                   
                 Primer-F→ 
               
               
                   
                 (SEQ ID NO. 2) 
               
               
                   
                 ATACATGGCCAAAAGATGTGGGC 
               
               
                   
               
               
                   
               
               
                   
                 Primer-R→ 
               
               
                   
                 (SEQ ID NO. 3) 
               
               
                   
                 GCACGACGGGACACCGGGCATAC 
               
            
           
         
       
     
     In an embodiment, the rAAV genome comprises nucleotide sequences described above flanked by ITRs. In another embodiment, the nucleotide sequence encoding HMGCS2, variants thereof disclosed herein or functional homologues is functionally linked to a heterologous promoter capable of driving gene expression in the patient&#39;s target cells such as cardiomyocytes. Such promoters can include constitutive, cell-specific or inducible promoters. In an embodiment, the composition of the present invention further comprises αMHC promoter to induce HMGCS2 expression to target cardiomyocyte. In an embodiment the αMHC promoter comprises entire intergenic region between the β-MHC gene (upstream) and the αMHC gene with sequence as detailed in Subramaniam et al. J Biol Chem. 1991 Dec. 25; 266(36):24613-20 which is hereby incorporated in its entirety. 
     In an embodiment, the genome of the rAAV composition of the present invention is lacking one or more rep and cap genes, rendering the rAAV of the present invention unable to reproduce in a patient. The rAAV composition of the present invention may comprise the capsid of any known AAV serotypes such as AAV type 1, AAV type 2, AAV type 3 (including types 3A and  3 B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, avian AAV, bovine AAV, canine AAV, equine AAV, and ovine AAV and any other AAV now known or later discovered or a combination thereof. In another embodiment, since AAV-9 is known to specifically target the heart, in an embodiment, the composition of the present invention comprises rAAV-9 capsid comprising nucleotide sequence encoding HMGCS2, variants thereof disclosed herein or functional homologues. 
     In an embodiment, the composition of the present invention comprises genome fully or partially encapsulated in lipid formulation wherein the genome encodes HMGCS2 or any variants thereof as disclosed and lipid formulation comprises liposomes or polymeric nanoparticles. In another embodiment, the composition of the present invention comprises mRNA housed or encapsulated in lipid formulation wherein the mRNA encodes HMGCS2 or any variants thereof as disclosed and lipid formulation comprises liposomes or polymeric nanoparticles. Methods of preparation of these compositions are disclosed in U.S. Pat. No. 10,086,143 which is hereby incorporated in its entirety. 
     The present invention also provides a method of treatment for cardiac ischemia or heart diseases involving metabolic changes or loss of or injury to cardiomyocytes comprising the step of administering a therapeutically effective amount of any of the disclosed compositions of the present invention to a patient in need. In an embodiment, the present invention comprises a method of treatment for cardiac ischemia or heart diseases involving metabolic changes or loss of or injury to cardiomyocytes comprising the step of parenteral administration of a therapeutically effective amount of rAAV comprising nucleic acid encoding HMGCS2. In an embodiment, the dose range comprises between about 10 7 -10 18 , about 10 11 -10 17  or about 10 12 -10 13  of the rAAV particles comprising nucleic acid encoding HMGCS2. In another embodiment, the present invention comprises a method of treatment for cardiac ischemia or heart diseases involving metabolic changes or loss of cardiomyocytes comprising the step of administration of a therapeutically effective amount of rAAV comprising nucleic acid encoding HMGCS2, variants thereof disclosed herein or functional homologues parenterally at and near the border region of the ischemia. In an embodiment, a method of treatment for cardiac ischemia or heart diseases involving metabolic changes or loss of cardiomyocytes comprising the step of administration of rAAV comprising nucleic acid encoding HMGCS2, variants thereof disclosed herein or functional homologues by perfusion of the heart. 
     In an embodiment, the method of the present invention comprises administration of HMGCS2 enzyme to the patient. In an embodiment, the method of the present invention comprises administration of HMGCS2 enzyme to the heart of the patient. In an embodiment, the method of the present invention comprises administration of HMGCS2 enzyme to the CM injured area of the patient. In an embodiment, the method of the present invention comprises administration of HMGCS2 enzyme to the border region of the CM injured area of the patient. 
     In all of the embodiments of the method of the present invention disclosed herein, the administration time may be prior to the cardiac ischemia. Alternatively, in all of the embodiments of the method of the present invention disclosed herein, the administration time may be after cardiac ischemia such as about 1 hour to about one month after the injury such as about 1 hour, about 3 hours, about 10 hours about 24 hours, about 2 days, about 4 days, about 10 days about 15 days about 20 days, about 25 days or about 30 days including any numbers and number ranges falling within these values. In all of the embodiments of the method of the present invention disclosed herein, the administration method may comprise parenteral administration to the patient and, in some embodiment, to the heart of the patient. 
     It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed. 
     These and other changes can be made to the technology in light of the detailed description. In general, the terms used in the following disclosure should not be construed to limit the technology to the specific embodiments disclosed in the specification, unless the above detailed description explicitly defines such terms. Accordingly, the actual scope of the technology encompasses the disclosed embodiments and all equivalent ways of practicing or implementing the technology. 
     It can be appreciated by those skilled in the art that changes could be made to the examples described without departing from the broad inventive concept thereof. It is understood, therefore, that this invention is not limited to the particular examples disclosed, but it is intended to cover modifications within the spirit and scope of the present invention as defined by the appended claims. 
     Examples 
     Experimental Methods and Materials 
     Material and Methods 
     Animals 
     All animal experiments were conducted in accordance with the Guides for the Use and Care of Laboratory Animals (ARRIVE guidelines), and all of the animal protocols have been approved by the Experimental Animal Committee, Academia Sinica, Taiwan. Myh6-rtTA mice (Stock No: Jam8585) was purchased from MMRRC. Collal-tetO-OSKM mice (Stock No: 011001) and Myh6-CRE (Stock No: 011038) were both purchased from Jackson lab. Conditional HMGCS2 knockout mice were generated by inserting 2 1oxP fragments into the regions before and after exon 2 ( FIG.  4 A ) through CRISPR/Cas9 technique. All mice were housed in individually ventilated cages (IVCs) system in animal core facility at Academia Sinica. Doxycycline treatment (Sigma-Aldrich, D9891) was administrated by intraperitoneal injection at 2 mg per 25 g mouse (Stadtfeld et al., 2010). 
     Adult CM Isolation 
     Adult ventricular CMs were isolated from mice on a Langendorff apparatus. After heparinization for 10 mins, the heart was removed from the anaesthetized mice and then was cannulated for retrograde perfusion with Ca2+-free Tyrode solution (NaCl 120.4 mmol/l, KCl 14.7 mmol/l, KH2PO4 0.6 mmol/l, Na2HPO4 0.6 mmol/l, MgSO4 1.2 mmol/l, HEPES 1.2 mmol/l, NaHCO3 4.6 mmol/l, taurine 30 mmol/l, BDM 10 mmol/l, glucose 5.5 mmol/1). After perfusion, the enzyme solution containing Ca2+-free Tyrode solution supplemented with collagenase B (0.4 mg/g body weight, Roche), collagenase D (0.3 mg/g body weight, Roche), and protease type XIV (0.05 mg/g body weight, Sigma-Aldrich) was perfused to digest the hearts for 10 mins. The ventricle was then cut from the cannula and teased into small pieces in the enzyme solution and then neutralized by the Ca2+-free Tyrode solution containing 10% FBS. Adult CMs were dissociated from the digested tissues by gentle pipetting and collected after removing the debris by filtering through a nylon mesh with 100 μm pores. 
     RNA Isolation and Real-Time PCR 
     Total RNA was isolated from frozen LV tissue or from isolated CMs using Trizol buffer (Invitrogen), and cDNA was synthesized using SuperScript IV reverse transcriptase and random hexamers according to manufacturers&#39; guidelines. Real-time PCR was performed using SYBR green (Bio-Rad), and the primers are described in the Table Si. The mRNA levels in each sample were normalized to GAPDH RNA levels. 
     Flow Cytometry 
     Cells were fixed with 4% paraformaldehyde and permeabilized with 90% methanol on ice. The single cell suspension was further stained with anti-BrdU antibody (ab8152) for 30 mins then washed with PBS. After incubating with secondary antibody conjugated with Alexa fluor-488 or Alexa fluor-568 (Life Technologies) for another 30 mins, samples suspended in PBS were measured by LSRII SORP (Becton Dickinson) and analyzed by FlowJo Software (Treestar, Ashland, Oreg.). 
     Intravital Imaging 
     The multiphoton intravital imaging system was performed following the procedure published in previous study (Vinegoni., 2015). In brief, mice were anesthetized by 1.5% isoflurane (Minrad) and membrane potential dye (Di-2-ANEPEQ) was injected intravenously to examine live imaging of heart tissue was performed using a multi-photon scanning microscope. 
     Immunofluorescence 
     The tissue sections were deparaffinized, rehydrated, and antigens retrieved by boiling twice in sodium citrate solution. The sections were incubated with blocking buffer (5% goat serum and FBS) for 1 hour, and then stained with primary antibody including histone H3 phosphorylated at serine 10 (Millipore), and anti-cardiac troponin T (DSHB) at 4° C. overnight. Samples were incubated in secondary antibodies conjugated with Alexa fluor-488 or Alexa fluor-568 (Life Technology) for 1 h at room temperature. After PBS washing, the nuclei were stained with DAPI (Life Technologies) for 5 min. 
     Transcriptomic Analysis 
     Samples from control or reprogramming CMs were hybridized to a Mouse Oligo Microarray (Agilent) following the manufacturer&#39;s procedure, and arrays were scanned with Microarray Scanner System (Agilent). All CEL files were analyzed by GeneSpring GX software (Agilent) with quantile normalization and median polish probe summarization using the control group as a baseline. The expression levels in the first quantile were filtered out to remove noise. Genes were defined as differentially expressed if they had fold changes of at least ±2 combined with the Student&#39;s t-test (P&lt;0.05) with the Benjamini-Hochberg adjustment for false discovery rate (FDR). Gene Ontology analysis was conducted using DAVID software (Huang et al, 2009). The biological replicates were two for control or reprogramming CM isolated from doxycycline treated CM-OSKM mice. 
     LC-MS Untargeted Profiling 
     Hearts were isolated from control or reprogramming mice at reprogramming day 2. After removing the atria and aorta, samples were frozen in liquid nitrogen and then prepared for LC-MS metabolic profiling. The whole profiling experiments including sample preparation followed a previously published procedure (Wang et al., 2015). 
     13C NMR Spectroscopy and Analysis 
     Mouse hearts were isolated and perfused with unlabeled mixed-substrate buffer (in mM; NaCl 118 mM, NaHCO3 25 mM, KCl 4.1 mM, CaCl2) 2 mM, MgSO4 1.2 mM, KH2PO4 1.2 mM, EDTA 0.5 mM, glucose 5.5 mM, mixed long-chain fatty acids bound to 1% albumin 1 mM, lactate 1 mM, and insulin 50 μU/mL) for 20 minutes and 13C-labeled mixed-substrate buffer for another 40 minutes. 13C-labeled mixed-substrate buffer was divided into 2 groups; one contained [U-13C]glucose and [1,4-13C] OHB and unlabeled mixed FA and Lactate, the other group contained [U-13C] mixed FA and [1,4-13C] Lactate and unlabeled glucose and OHB. After perfusion, the hearts were frozen in liquid nitrogen, homogenized and extracted in perchloric acid, and then neutralized by KOH. The hearts were then lyophilized and dissolved in deuterium oxide (D20) supplemented with internal standard Sodium trimethylsilyl propionate. A Bruker Avance III 600 MHz NMR Spectrometer was used to present proton-decoupled 13C NMR spectra of each heart sample, and spectra were generated by Fourier transformation following multiplication of the free-induction decays (FIDs) by an exponential function. The peak areas of each 13C-metabolites were analyzed using Bruker TopSpin 4.0.2. 
     High Performance Liquid Chromatography 
     An HPLC system Dionex Ultimate 3000 (ThermoFisher Scientific, Waltham, Mass., USA), with a Varian 380-LC (Varian, Palo Alto, Calif., USA) evaporative light-scattering detector was employed. The conditions used followed a published procedure (Heijden et al., 1994). In brief, the condition was used as follows: Column: Hypersil ODS (AMT, Wilmington, Del., USA), 250×4.6 mm, particle diameter 5 μm without precolumn. Solvent system: 0.2 M sodium phosphate buffer, pH 5.0, containing 4.5% (v/v) acetonitrile; flow rate: 1.5 ml/min. The compounds were detected by UV at 254 nm. 
     Transmission Electron Microscopy 
     To monitor mitochondria ultrastructure, transmission electron microscopy was used as described previously (Karamanlidis et al., 2013). Briefly, freshly collected samples from the apex of the mouse hearts were dissected in 1 mm 3  sections and immediately fixed with 2% glutaraldehyde in 0.1 M phosphate buffered saline, and then fixed with 1% osmium tetroxide. After the samples were dehydrated in ethanol and embedded in epon resin, ultrathin sections were prepared and counterstained with uranyl acetate and lead citrate. The stained sections were examined under a Transmission Electron Microscope (JEOL1230). Mitochondrial number was counted in total of 10 images per heart (45 m2 at ×12000 magnification, n=3 hearts per group). Data were expressed as fold changes relative to WT. 
     Mitochondria Isolation 
     Mitochondria were collected from isolated hearts by sequential centrifugation (Boehm et al., 2001). In brief, hearts were isolated and rinsed with mitochondrial isolation buffer (250 mM Sucrose, 10 mM Tris-HCL, and 3 mM EDTA, pH 7.4). Heart tissue was minced in mitochondrial isolation buffer and was homogenized by a homogenizer with Teflon pestle. The homogenate was centrifuged at 800 g for 10 min at 4° C. to remove tissue debris. The supernatant was further centrifuged at 8000 g for 15 min at 4° C. to collect mitochondria. 
     Myocardial Ischemia and Reperfusion 
     C57BL/6 mice (10 weeks old) were randomized and anesthetized by isofluorane inhalation, endotracheally intubated, and placed onto a rodent ventilator. The left anterior descending (LAD) coronary artery was visualized and occluded with a prolene suture for 45 mins after first removing the pericardium. After confirming the whitening region of the left ventricle, the occluded LAD was released. EF % between 55-60% one day after occlusion was considered a successful cI/R model. 
     Determination of Infarct Size 
     Infarct and remote area performed by Myocardial I/R was determined by Evans blue/TTC double staining as described previously (Bohl et al., 2009). In brief, the ligature around the LAD was re-tied after 24 hours of reperfusion. Injection of 1 ml 1% Evans blue dye through heart apex and the heart was excised and then frozen in −20° C. refrigerator for 15 minutes and sliced into four 1 mm-thick slices. The slides were stained with 1% triphenyltetrazolium chloride (TTC, Sigma) in PBS at 37° C. for 10 minutes and photographed. The area at risk (AAR) was identified as red (TTC-stained) and white (infarct) areas. AAR, IR, and total LV area were measured by Image J software (NIH). 
     Western Blot Analysis and Immunoprecipitation 
     Myocardial tissues were frozen and lysed in RIPA buffer with a protease inhibitor cocktail. Protein samples (20 μg) were separated by SDS-PAGE and transferred to a PVDF membrane. The membrane was blocked in 5% skimmed milk and probed with primary antibodies overnight at 4° C.: HMGCS2 (sc-393256) and GAPDH (MAB374), followed by corresponding secondary antibodies. The membrane then was developed with ECL and the signal intensities were visualized by a Supersignal chemiluminescence detection kit (Pierce) and analyzed with Image J software (NIH). 
     Adeno-Associated Virus Production 
     AAV9 was produced by triple-transfection procedures using CMV-HMGCS2/CMV-EGFP plasmid, with a plasmid encoding Rep2Cap9 sequence and an adenoviral helper plasmid pHelper in 293 cells. Virus was purified by two cesium chloride density gradient purification steps through ultracentrifugation followed by dialysis against 5 rounds of PBS buffer change. Viral titers were determined by qPCR. 
     The primers to amplify full gene sequence of HMGCS2 were listed below. 
     
       
         
           
               
               
            
               
                   
                 Primer-F→ 
               
               
                   
                 (SEQ ID NO. 2) 
               
               
                   
                 ATACATGGCCAAAAGATGTGGGC 
               
               
                   
               
               
                   
               
               
                   
                 Primer-R→ 
               
               
                   
                 (SEQ ID NO. 3) 
               
               
                   
                 GCACGACGGGACACCGGGCATAC 
               
            
           
         
       
     
     Lentivirus Production 
     293 cells were seeded in 10-cm-diameter dishes 24 h prior to transfection using PolyJet (SL10068). The PLKO3.1-EGFP or PLKO3.1-HMGCS2 vector plasmids was each cotransfected together with psPAX2 and pMD2.G in a ratio of 5:4:1 (total 9 ag). After 12-18 hours of transfection, the culture medium (DMEM-HG) was changed and the viral supernatant was collected after 48 and 72 hours of transfection. 
     Primers Used in various RNA isolation and Real-Time PCR are listed in Table 1 below 
     
       
         
           
               
               
               
             
               
                 TABLE 1 
               
               
                   
               
               
                   
                 Name 
                 Sequence (5′ to 3′) 
               
               
                   
               
             
            
               
                   
                 GAPDH-F 
                 (SEQ ID NO. 4) 
               
               
                   
                   
                  CAT CAC TGC CAC CCA GAA GAC TG 
               
               
                   
               
               
                   
                 GAPDH-R 
                 (SEQ ID NO. 5) 
               
               
                   
                   
                 ATG CCA GTG AGC TTC CCG TTC AG 
               
               
                   
               
               
                   
                 mOct4-F 
                 (SEQ ID NO. 6) 
               
               
                   
                   
                 CCT GCA GAA GGA GCT AGA ACA GT 
               
               
                   
               
               
                   
                 mOct4-R 
                 (SEQ ID NO. 7) 
               
               
                   
                   
                 TGT TCT TAA GGC TGA GCT GCA A 
               
               
                   
               
               
                   
                 mSox2-F 
                 (SEQ ID NO. 8) 
               
               
                   
                   
                 GCA CAT GAA CGG CTG GAG CAA CG 
               
               
                   
               
               
                   
                 mSox2-R 
                 (SEQ ID NO. 9) 
               
               
                   
                   
                 TGC TGC GAG TAG GAC ATG CTG TAG G 
               
               
                   
               
               
                   
                 mKlf4-F 
                 (SEQ ID NO. 10) 
               
               
                   
                   
                 GAA ATT CGC CCG CTC CGA TGA 
               
               
                   
               
               
                   
                 mKlf4-R 
                 (SEQ ID NO. 11) 
               
               
                   
                   
                 CTG TGT GTT TGC GGT AGT GCC 
               
               
                   
               
               
                   
                 cMyc-F 
                 (SEQ ID NO. 12) 
               
               
                   
                   
                 GCC CCC AAG GTA GTG ATC CT 
               
               
                   
               
               
                   
                 cMyc-R 
                 (SEQ ID NO. 13) 
               
               
                   
                   
                 GTC CTC GTC TGC TTG AAT GG 
               
               
                   
               
               
                   
                 mtDNA-F 
                 (SEQ ID NO. 14) 
               
               
                   
                   
                 CGA AAG GAC AAG AGA AAT AAG G 
               
               
                   
               
               
                   
                 mtDNA-R 
                 (SEQ ID NO. 15) 
               
               
                   
                   
                 CTG TAA AGT TTT AAG TTT TAT GCG 
               
               
                   
               
               
                   
                 mtCox1-F 
                 (SEQ ID NO. 16) 
               
               
                   
                   
                 AGT CTA CCC ACC TCT AGC CG 
               
               
                   
               
               
                   
                 mtCox1-R 
                 (SEQ ID NO. 17) 
               
               
                   
                   
                 TGT GTT ATG GCT GGG GGT TT 
               
               
                   
               
               
                   
                 mtAtp6-F 
                 (SEQ ID NO. 18) 
               
               
                   
                   
                 TCC ACA CAC CAA AAG GAC GAA 
               
               
                   
               
               
                   
                 mtAtp6-R 
                 (SEQ ID NO. 19) 
               
               
                   
                   
                 CCA GCT CAT AGT GGA ATG GCT 
               
               
                   
               
               
                   
                 mtAtp8-F 
                 (SEQ ID NO. 20) 
               
               
                   
                   
                 CAT CAC AAA CAT TCC CAC TGG C 
               
               
                   
               
               
                   
                 mtAtp8-R 
                 (SEQ ID NO. 21) 
               
               
                   
                   
                 TGA GGC AAA TAG ATT TTC GTT CAT T 
               
               
                   
               
               
                   
                 mtCox2-F 
                 (SEQ ID NO. 22) 
               
               
                   
                   
                 GAC GAA ATC AAC AAC CCC GT 
               
               
                   
               
               
                   
                 mtCox2-R 
                 (SEQ ID NO. 23) 
               
               
                   
                   
                 TAG CAG TCG TAG TTC ACC AGG 
               
               
                   
               
               
                   
                 mtNd2-F 
                 (SEQ ID NO. 24) 
               
               
                   
                   
                 CAA GGGATC CCA CTG CAC AT 
               
               
                   
               
               
                   
                 mtNd2-R 
                 (SEQ ID NO. 25) 
               
               
                   
                   
                 AAG TCC TCC TCA TGC CCC TA 
               
               
                   
               
               
                   
                 Hmgcs2-F 
                 (SEQ ID NO. 26) 
               
               
                   
                   
                 GGT GTC CCG TCT AAT GGA GA 
               
               
                   
               
               
                   
                 Hmgcs2-R 
                 (SEQ ID NO. 27) 
               
               
                   
                   
                 ACA CCC AGG ATT CAC AGA GG 
               
               
                   
               
               
                   
                 βMhc-F 
                 (SEQ ID NO. 28) 
               
               
                   
                   
                 GTG CCA AGG GCC TGA ATG AG 
               
               
                   
               
               
                   
                 βMhc-R 
                 (SEQ ID NO. 29) 
               
               
                   
                   
                 GCA AAG GCT CCA GGT CTG A 
               
               
                   
               
               
                   
                 αMhc-F 
                 (SEQ ID NO. 30) 
               
               
                   
                   
                 CCA ACA CCA ACC TGT CCA AGT 
               
               
                   
               
               
                   
                 αMhc-R 
                 (SEQ ID NO. 31) 
               
               
                   
                   
                 AGA GGT TAT TCC TCG TCG TGC AT 
               
               
                   
               
               
                   
                 Pgc1α-F 
                 (SEQ ID NO. 32) 
               
               
                   
                   
                 AGC CGT GAC CAC TGA CAA CGA G 
               
               
                   
               
               
                   
                 Pgc1α-R 
                 (SEQ ID NO. 33) 
               
               
                   
                   
                 GCT GCA TGG TTC TGA GTG CTA AG 
               
               
                   
               
            
           
         
       
     
     Example 1—In Vivo CM-Reprogramming Induces Metabolic Switch, CM Dedifferentiation and In-Creased CM Proliferation 
     In order to examine the process of adult CM reprogramming in vivo, transgenic mice were generated to overexpress mouse OCT4, SOX2, KLF4, and c-MYC (OSKM) specifically in adult CMs after doxycycline induction as shown in  FIG.  1 A .  FIG.  1 B  shows induction of OSKM mRNA expression in isolated transgenic, adult CMs after doxycycline treatment for 2 days. Importantly, this high level of induction was detected only in CMs but not other non-CMs in the heart or other tissues isolated from doxycycline-treated mice ( FIG.  1 R ). Tracking the degree of CM proliferation by BrdU labeling, a three-fold in-crease in BrdU+ CMs was found 2 days following doxycycline administration ( FIGS.  1 C and  1 D ). The proliferative response of adult CMs was highest at reprogramming day 2 compared to day 1 and 4, and six days of doxycycline treatment was lethal. Therefore, reprogramming day 2 was selected as the key time point for further analysis. Using intravital microscopy to investigate the isolated whole hearts with membrane potential dye (Di-2-ANEPEQ) staining, we found that the alignment of CMs was changed after inducing re-programming for 2 days ( FIG.  1 E ). Well-aligned CMs were observed throughout control (Ctrl) hearts, but regions of poorly-aligned CMs were observed in the doxycycline-treated mice ( FIG.  1 F ). In addition, the in vivo morphology of reprogramming CMs was different from Ctrl CMs, maintaining their width but becoming shorter, leading to a different aspect ratio than control CMs ( FIGS.  1 G- 1 I ). By recording each contraction of the Ctrl or reprogramming hearts in vivo using intravital microscopy, areas of disorganized or nonaligned contraction were observed consistent with the disruption of the normal aligned CM structure of the heart ( FIG.  1 S ). Furthermore, heart tissue sectioning was performed to examine the relationship between CM alignment (WGA staining) and CM proliferation (H3P staining). We confirmed that the more proliferative CM population were found in doxycycline-induced hearts and these cells displayed a shortened morphology with poorer cell alignment (around 50-60 μm in length and an aspect ratio of approximately 3) ( FIGS.  1 J- 1 L ). In addition, 2 times more Aurora b kinase (AURKB) positive CMs were shown in reprogramming hearts than in control hearts, showing that reprogramming CMs not only enter mitosis but completing cytokinesis ( FIGS.  1 M and  1 N ). Finally, in order to probe the mechanisms by which adult CMs dedifferentiate to regain their proliferative ability, CMs were isolated from the hearts of mice treated for 2 days with PBS or doxycycline, and RNA was extracted and subjected to microarray analysis ( FIG.  1 O ). Gene Ontology data showed that metabolism-related gene expression was significantly changed in the reprogramming CMs compared to the Ctrl CMs at reprogramming day 2 ( FIG.  1 P ). The gene expression changes included the up-regulation of glucose and amino acid metabolism and down-regulation of nucleotide metabolism. Similar trends were shown in heat map analysis; ketone metabolism-related gene expression was up-regulated and aerobic respiration-related genes were down-regulated in the adult reprogramming CMs compared to the Ctrl CMs ( FIG.  1 Q ). Examining all of the data shown in  FIGS.  1 A- 1 S , temporary CM reprogramming induced dedifferentiation in the form of changes in cell morphology, proliferation, and changes in the expression of genes associated with metabolism. 
     Example 2 Cardiac-Specific Ketogenesis Creates a Systemic and Specific Metabolic Switch Along with Mitochondrial Changes, Inducing CM Dedifferentiation at CM-Reprogramming Day 2 
     Since a metabolic switch appears to be intrinsically linked to adult CM dedifferentiation, it is necessary to clarify the detailed rearrangement of metabolic pathways in adult CMs which are undergoing reprogramming. First, the metabolic profiles of Ctrl and CM-reprogramming hearts were analyzed by liquid chromatography-mass spectrometry (LC-MS) metabolic profiling, and 101 metabolites were detected in both groups ( FIGS.  2 A and  2 B ). Grouping these hits revealed that glucose and ketone body metabolism-related metabolites were up-regulated in CM-reprogramming hearts ( FIG.  2 C ). On the contrary, tricarboxylic acid (TCA) cycle and nucleotide metabolism-related metabolites were down-regulated in CM-reprogramming hearts which is consistent with the microarray data ( FIGS.  2 C and  1 Q ). In order to avoid influence by intermediate products derived from other tissues, a working heart system was set up and carbon NMR was used to detect the 13C-metabolites produced only from the exogenous addition of labeled substrates (Li et al., 2017;  FIG.  2 D ). In NMR analysis, mixed fatty acids (FAs), which are the primary fuel for aerobic respiration, were decreased in the reprogramming hearts compared to the Ctrl hearts ( FIG.  2 E ). Although glucose and ketones slightly increased for oxidation, the aerobic respiration derived from exogenous 13C-metabolites were decreased in the reprogramming hearts ( FIGS.  2 E and  2 T ). In addition, the amounts of Lactate (Lac) and Ala-nine (Ala) were 1.5-2 times higher in reprogramming hearts than in the Ctrl hearts, indicating that glycolysis (anaerobic respiration) was increased in the hearts two days following OKSM induction ( FIG.  2 F ). Interestingly, both β-hydroxybutyrate (OHB, ketone) and Aspartate (Asp) were 2 times higher in the reprogramming hearts than in the Ctrl hearts, indicating that ketogenesis is increased ( FIG.  2 F ). Since ketogenesis and the TCA cycle share the same metabolic substrate, Acetyl-CoA, ketogenesis induction should competitively reduce aerobic respiration in mitochondria. In order to confirm this concept, several techniques were utilized ( FIG.  2 G ). The main intermediate product of ketogenesis is HMG-CoA. Therefore, we isolated mitochondria from Ctrl and reprogramming hearts and quantified HMG-CoA by high-pressure liquid chromatography (HPLC) ( FIG.  2 G ). The amount of HMG-CoA was 2 times higher in the mitochondria isolated from reprogramming hearts than in the Ctrl hearts ( FIG.  2 H ). The end product of ketogenesis, OHB, was measured by an OHB colorimetric assay kit. We found that OHB is more than 1.5 times higher in the reprogramming CMs than Ctrl CMs ( FIG.  2 I ). Using the Sea-horse assay we found that the oxygen consumption rate (OCR) is lower in the adult reprogramming CMs than in the Ctrl CMs ( FIGS.  2 J and  2 K ). HMGCS2, the rate-limiting enzyme of ketogenesis, was up-regulated in adult reprogramming CMs compared to the Ctrl, as determined by microarray analysis. Moreover, the expression of HMGCS2 was significantly increased at both the RNA and protein levels ( FIGS.  2 L and  2 M ). A summary of these changes is shown in  FIG.  2 N . Interestingly, the changes associated with CM reprogramming did not affect overall heart function, as reprogramming hearts showed similar ejection fraction % (EF %) to Ctrl hearts ( FIG.  2 U ). Several metabolic pathways such as ketogenesis and aerobic respiration are carried out in mitochondria, and changes of OCR are always accompanied by mitochondrial differences. Thus, CM mitochondria were assessed by measuring mitochondrial DNA content and mitochondrial RNA expression in the Ctrl and reprogramming CMs. The mitochondrial copy numbers were lower and RNA expression was significantly lower in the reprogramming CMs compared to the Ctrl CMs ( FIGS.  2 O and  2 P ), indicating immature mitochondria were shown in the reprogramming hearts. Transmission electron microscopy (TEM) revealed that mitochondrial area and aspect ratio were both significantly decreased in the reprogramming hearts ( FIGS.  2 Q- 2 S ). Mitochondrial fission is reported to be related to proliferative induction through post-translational phosphorylation of DRP-1 on serine 616 (Marsboom et al., 2012). Indeed, DRP-1 serine 616 phosphorylation was higher in reprogramming CMs compared to the Ctrl CMs ( FIG.  2 V ). These data indicate that during CM reprogramming by OSKM induction, a metabolic switch occurs, including increased ketogenesis and glycolysis and deceased aerobic respiration with immature mitochondrial structure and function. This switch occurs in synchrony with the induction of CM proliferation. 
     Example 3—Forced HMGCS2 Overexpression Effected Before Myocadial Infarction Increases Adult CM Dedifferentiation and Proliferation for Heart Function Improvement after Myocardial Infarction or Under Hypoxia 
     In this section, we aimed to investigate the possible therapeutic role of HMGCS2 on a permanent coronary artery ligation myocardial infarction (MI) model ( FIG.  3 A ). After exogenous HMGCS2 induction by AAV9 induction for 5 weeks, HMGCS2-overexpressing mice showed a higher EF % at D21 following MI surgery than Ctrl AAV9-EGFP mice measured by echocardiography ( FIG.  3 B ). Catheter measurements indicated better heart function in HMGCS2-overexpressing mice 21 days after MI injury compared to Ctrl mice ( FIG.  3 C ). The fibrotic area was also smaller in HMGCS2-overexpressing mice compared to the Ctrl mice ( FIG.  3 D , E). More H3P+ and AURKB+ CMs were found in HMGCS2-overexpressing hearts 3 days after MI injury compared to the Ctrl ( FIGS.  3 F- 3 I ). Taken together, these findings show that exogenous HMGCS2 expression can support cardiac regeneration and improve heart function after MI. Next, we examined whether these findings could be replicated in an in vitro model using hypoxic human induced pluripotent stem cell-derived CMs (hiPSC-CMs) ( FIG.  3 J ). HMGCS2 expression was highly up-regulated in hiPSC-CMs after lentiviral infection (Lenti-HMGCS2) compared to the Ctrl (Lenti-EGFP) ( FIGS.  3 K,  3 R and  3 S ). HMGCS2 overexpression also induces increased ketone production in hiPSC-CMs ( FIG.  3 L ). Furthermore, HMGCS2 overexpressing hiPSC-CMs showed a shorter morphology with a lower length-to-width ratio compared to the Ctrl cells under hypoxia ( FIGS.  3 M- 3 P ). This shows that HMGCS2 overexpression supports human CM dedifferentiation, as we found in adult mouse CMs shown in  FIG.  1   . Finally, HMGCS2 overexpressing hiPSC-CMs showed a two-fold greater proliferative ability compared to Ctrl cells under hypoxic conditions ( FIG.  3 Q ). These data indicate that forced HMGCS2 overexpression supports CM dedifferentiation and facilitates proliferation under hypoxic conditions. 
     Example 4—Forced HMGCS2 Overexpression Effected after Myocardial Infarction Increases Adult CM Dedifferentiation and Proliferation for Heart Function Improvement after Myocardial Infarction 
     In order to test the possible therapeutic role of HMGCS2 on heart regeneration, exogenous HMGCS2 was induced immediately after performing a permanent coronary artery ligation myocardial infarction (MI) model ( FIG.  4 A ). After exogenous HMGCS2 induction by intramyocardial AAV9 injection immediately after MI, HMGCS2-overexpressing mice showed a higher EF % at post-MI D21 than Ctrl AAV9-EGFP mice ( FIG.  4 B ). Catheter measurements indicated better heart function in HMGCS2-overexpressing mice 21 days after MI injury compared to Ctrl mice ( FIG.  4 C ). The infarct area showed no differences in Ctrl or HMGCS2-overexpressing mice 1 day after MI ( FIGS.  4 D and  4 E ), indicating that HMGCS2 overexpression may stimulate regeneration rather than protecting the myocardium. The fibrotic area was also smaller in HMGCS2-overexpressing mice compared to the Ctrl mice ( FIGS.  4 F and  4 G ). More H3P+ CMs were found in HMGCS2-overexpressing hearts 3 days after MI injury compared to controls ( FIGS.  4 H and  4 I ). Taken together, these findings show that exogenous HMGCS2 expression can support cardiac regeneration and improve heart function after MI. 
     DISCUSSION 
     Adult CMs undergoing early OSKM-induced reprogramming display metabolic changes which allow for enhanced dedifferentiation and proliferation in vivo ( FIGS.  1 A to  1 S ). Our previous study investigating early-stage neonatal CM reprogramming in vitro found up-regulation of proliferation-related gene expression (Cheng et al., 2017). However, neonatal and adult CMs differ significantly in their structure, function, metabolism and response to injury (Szibor et al., 2014). In addition, the gene cocktail described in our previous study was unable to efficiently induce proliferation in adult CMs. This indicates that adult CMs and neonatal CMs induce reprogramming via different mechanisms. These data suggest that inducing a metabolic switch of adult CMs, rather than directly inducing cell cycle-related activators, may be a more efficient way for giving rise to the cellular phenotype adaptations necessary to regain proliferative ability ( FIGS.  1 A to  1 S  and  FIGS.  2 A to  2 V ). Since adult CMs are notoriously difficult to maintain in culture, and the reprogramming process may be affected by the cellular microenvironment, this study profiled the changes which reprogrammed adult CMs undergo in vivo. Through specific induction of adult CM reprogramming in vivo, we not only can investigate the transformation of CMs during the process, but its effects on whole mouse can be also detected. This system undoubtedly is a powerful tool to study the reprogramming process specifically at the tissue level in vivo and to explore how reprogramming of specific tissues has systemic effects. 
     Ketogenesis is mainly carried out in liver tissues, where ketones, as water-soluble metabolites, can be easily transferred to other tissues for utilization (Grabacka et al., 2016). Ketone utilization is common as an alternative energy source while fasting or exercising (Puchalska et al., 2017), and ketones are also reported as the preferred metabolic substrate for heart improvement after injury (Anbert et al., 2016; Horton et al., 2019; Nielsen et al., 2019). However, there are few studies clearly defining the role of ketone synthesis in the heart tissue itself. Here, we demonstrate that HMGCS2-induced ketogenesis in adult CMs competitively reduces FA metabolism leading to a metabolic switch and mitochondrial changes ( FIGS.  2 A to  2 V ). Metabolic flexibility allows cells to adapt certain conditions, and primarily occurs due to the antagonism between glucose and FA for providing energy production (Bret., 2017). Besides, ketogenesis plays as a critical regulator to control FA metabolism, Glc metabolism, and TCA cycle for maintaining hepatic metabolic homeostasis (Cotter et al., 2017). The same scenario is presented in our current study, showing that an increase of HMGCS2-induced ketogenesis in adult CMs decreases FA metabolism, and glucose is then used via anaerobic or aerobic respiration, based on the available oxygen. Therefore, ketogenesis-induced adult CM reprogramming can be specifically induced in the border zone but not the remote area of injury hearts. 
     HMGCS2 is up-regulated in the mouse heart ventricle within one week after birth, and its expression is diminished at postnatal day 12 (Talman et al., 2018). However, the role of HMGCS2 in heart function maintenance during development or after injury had not yet been shown. Under certain condition such as reprogramming or injury, exogenous HMGCS2 expression increases adult CM dedifferentiation and proliferation. All these data suggest that HMGCS2 may not be a driver but is required for starting adult CM dedifferentiation and proliferation, and this requirement successfully supports cardiac protection and regeneration after injury ( FIGS.  3 A to  3 S  and  FIGS.  4 A to  4 I ). In previous studies, genes responsible for proliferation such as OSKM always carry a risk of tumor formation, which limits therapeutic applicability (Ohmishi et al., 2014). However, HMGCS2 controls the metabolic flexibility, allowing adult CM dedifferentiation and proliferation during cell stress, thus providing an ideal therapeutic target for heart diseases. 
     Overall, this is the first study to perform and investigate OSKM reprogramming specifically on adult CMs in vivo. We have demonstrated the importance of HMGCS2-induced keto-genesis as a means to regulate metabolic response to CM injury, thus allowing cell dedifferentiation and proliferation as a regenerative response. Furthermore, overlaps between OSKM-induced CM reprogramming, heart development and maturation, and the response to heart injury become readily apparent. Since myocardial infarction remains the greatest cause of death in developed countries, we hope this study provides a foundation for future research, exploiting metabolism as a mechanism to drive myocardial regeneration following injury. 
     
       
         
           
               
               
            
               
                 Sequence Listing 
                   
               
               
                 SEQ. ID NO.: 1 
                   
               
            
           
           
               
               
               
            
               
                 1 
                 aggactcctc cctcaccaaa ctctgcaggctttgaaatca aagttctaaa tgtctcccca 
                   
               
               
                   
               
               
                 61 
                 ggcaatcaga aaaggcaaga cctggcaaat aagaggttgt actaaccagt aacaaaatca 
               
               
                   
               
               
                 121 
                 caaacaacat ttgctcttcc tcttccacag cagactccac aagtaggtgc aatgaaagag 
               
               
                   
               
               
                 181 
                 ccctagattt ggagccaagg ccgtcaaatg ccctcccagc cattgtcact aatcacatat 
               
               
                   
               
               
                 241 
                 ccacaagcca gatcacttaa tctctcaaag cctcaatgtc catatcttcc aaatggggct 
               
               
                   
               
               
                 301 
                 aataattcag gttaactcca tggaactttc atgagaaaag accgtatgca aaagcaactg 
               
               
                   
               
               
                 361 
                 aaaactgata aagcaccaga tatgctagta atgcattagt atcgtgaaat aaacagggct 
               
               
                   
               
               
                 421 
                 catttccaaa ggtacaaaga ccctgcaagt ataaagactt cttcctaggt ctagactttc 
               
               
                   
               
               
                 481 
                 catagaaata gctttcctac ccactttctg atgccgagaa ttttgaaagt tctttttccc 
               
               
                   
               
               
                 541 
                 ttaggttgag atgtaaaggg caaatctgca tgggaaaaga ttgcttcaat ttatcagtca 
               
               
                   
               
               
                 601 
                 tgggaacctg gggtaaatgc attttcagag catttattga aaggagaata gtgggctact 
               
               
                   
               
               
                 661 
                 gaggtagaag agttgcaatc tttatgtggg ctaaaagagg caaatccagg tgcctgggaa 
               
               
                   
               
               
                 721 
                 ccttgtttat agttttgttc tcctacaccg gctcttttgt cagaattgct taaaaaacaa 
               
               
                   
               
               
                 781 
                 acattgtttt tgcaagacct caccctagat gtctaaactt ctaaaatccc tcataatcaa 
               
               
                   
               
               
                 841 
                 tttttctgac ttttaatgct tatctagcag gtaacatgca ttttaaatta atccttttat 
               
               
                   
               
               
                 901 
                 caacacttca gctgaaaagc tgaagtctag gagttgaagg accctaaagt ctcaaatcaa 
               
               
                   
               
               
                 961 
                 aaataaatac atcttttttc atctaggaag tatcaaaatg tgggtttatt taagtatttg 
               
               
                   
               
               
                 1021 
                 ggaggtagta tcttcttcag acacaaatag tgtgttccat tttcttcaac actttgagca 
               
               
                   
               
               
                 1081 
                 attagtagac aaaccagtta tttgattgta tttgaataca attacttgac taagtcatat 
               
               
                   
               
               
                 1141 
                 aaatttcctt cagtatgaaa aactaccacc tcatggtgtt ttactattat ttccctcaat 
               
               
                   
               
               
                 1201 
                 ttatactttg cataatgcat tcctggtgct tcctcaatct acaagttccc ttatcccaaa 
               
               
                   
               
               
                 1261 
                 ggaacaactt aatattagat tggccatata aaatttccac cttcccaagt caaaaatggt 
               
               
                   
               
               
                 1321 
                 tcatgattga ctcaggttat gtgtagagcc agatacctgg attcaaagcc cattcaggcc 
               
               
                   
               
               
                 1381 
                 atttactaga tctaaaacca caaatggtta tataattttc ctgaacctca gtttcctcat 
               
               
                   
               
               
                 1441 
                 ctgtaaaatg ggcttaacaa tagtgccaac ctcagacagt tgtaaaaatt aaatgagata 
               
               
                   
               
               
                 1501 
                 atgaacggaa agtacttagc acagtaccta gcacgcagta attacttagt acatgtcagc 
               
               
                   
               
               
                 1561 
                 ttaaaagaga gaagggaatg aagttgatcc atctatctgt attcccagtg cttatcacag 
               
               
                   
               
               
                 1621 
                 tgccaatttg ttatatacac taattaaaat ttgcattgga ttggatagtt ttggtcttca 
               
               
                   
               
               
                 1681 
                 attctatcaa actgagccat gatgtagcca taatcccgtg tgatgtttgt gtaagagttt 
               
               
                   
               
               
                 1741 
                 aatgtttcta ttgttaaaag taaaaccttg aacaaattaa atttagttga atttatttga 
               
               
                   
               
               
                 1801 
                 gcaaagaaac cattcatgaa taagtcagca ccctgaatta gtaaagattt agagatctcc 
               
               
                   
               
               
                 1861 
                 aatagaaata ttggactgtc agtatttaga gacaaaaata gcttgattgg ttacagctgg 
               
               
                   
               
               
                 1921 
                 catttgcctt acaggaacat gttttggcaa tttgcagcct gcgattgact gaaagcatgg 
               
               
                   
               
               
                 1981 
                 ctgctatgat tggtcaagac tcagctactt gttacatgaa tacactctca ggttaggttg 
               
               
                   
               
               
                 2041 
                 cggtttgttt atatattagg ttaagtaccc tacctaccta ggcagttttg ggccacatta 
               
               
                   
               
               
                 2101 
                 aatttacttt aacactatcg agttttatcc attttcttag tggaataagg aacatgtgga 
               
               
                   
               
               
                 2161 
                 gactacctga gtactccaaa atttagagat cagaaagagg ggagcacctg tggggagtgg 
               
               
                   
               
               
                 2221 
                 ccagggattt ggaggaaaac catgggattg tcaggtctaa gggcaaagtg aaaaaggtgc 
               
               
                   
               
               
                 2281 
                 tttgagaaga agggagagca gccttgccat ttgctgctaa gaggtctagt aagctgaagg 
               
               
                   
               
               
                 2341 
                 ttcaagagca aacactgcat ttggcaataa ggaagccact ggtgaccttg atgagaggga 
               
               
                   
               
               
                 2401 
                 attccttgga gcactggggg caaaagcctt agtggtcaat taaagacaga atgagaggta 
               
               
                   
               
               
                 2461 
                 agcttgtaaa acactgaaag cagacatttt aaataaattt ccctatagat gacagcatag 
               
               
                   
               
               
                 2521 
                 atttggtggc actcagtaag acatatagag tcaagaggag gtatttaaag atgggattgt 
               
               
                   
               
               
                 2581 
                 ataagaacaa taacacaaga agaatgtagt agaaagggaa aatagatcat gagaaagaga 
               
               
                   
               
               
                 2641 
                 ggggaaaact gcaggagcac agccttatgt gagaaacaag cagtacaacc agtgcacaca 
               
               
                   
               
               
                 2701 
                 tggtggtgct ggcccgaggc cagagcaggg actcttcctc tgcatagtga gaaggcagtg 
               
               
                   
               
               
                 2761 
                 agaaggcaat gtgtggggta taaaggcagg caatttgcca gatttgctca tggaaaacgg 
               
               
                   
               
               
                 2821 
                 agttattctt ttctgattgt ttctattttc tcagtgaatt cagagtcaaa gtgatcagct 
               
               
                   
               
               
                 2881 
                 gagaatgagt agaaaggggc tatggcagaa gagaagttgt gactagccct cttgggatgg 
               
               
                   
               
               
                 2941 
                 gagagcaaat ggactgggaa aaggtagtag agttaccagg ccatggtgag ggtccacttg 
               
               
                   
               
               
                 3001 
                 agatgtatgt ttgtaaattt aaagctaaca agttagtaca aagttgtgtt tttcttcatc 
               
               
                   
               
               
                 3061 
                 tatgtttagc tgctcagatg caggcgcaga gtagattaag agttgggttt aaccaaaatt 
               
               
                   
               
               
                 3121 
                 gaaggtttgc taggccagtc cgacagagag cacaaattgc aaagtgtgtg caagggattg 
               
               
                   
               
               
                 3181 
                 cttatggtga ggcaccatgg ttaatctgat ctggataagg agagaaaaga ggtgatgagg 
               
               
                   
               
               
                 3241 
                 tgtaacaaat gctaaaaaca tagaggagtc agtggtggtc tcagtgggag aaaaaggtgt 
               
               
                   
               
               
                 3301 
                 gagggtactt taaacaggag cagggaatat agaggtggtt ggaaattgga atgcatgaaa 
               
               
                   
               
               
                 3361 
                 ctaaaatgtt ggaggtggca tagacactgt aataacaaag tccacattat gactgtggag 
               
               
                   
               
               
                 3421 
                 tgggaggcta aagtcatgtc catgaacacg gacatggctg tgggagcttc agtgagaggc 
               
               
                   
               
               
                 3481 
                 tagggcaggt gaattatctt atgtggagat tgacatctca catccattga gatgactgat 
               
               
                   
               
               
                 3541 
                 ggtggagagg aaagtagtga tctatgtgct taaattttat caatgaggga cagtggaaca 
               
               
                   
               
               
                 3601 
                 tgacagttag ttgactgcaa gaataagggc actgggtggc acagactgta gcatgtgctt 
               
               
                   
               
               
                 3661 
                 taagacagca ggggttttga gaggaggaag aggagaaata ccctggaaga gatagtatga 
               
               
                   
               
               
                 3721 
                 agcaaagagg acacaggcct tgttgtaagc atttaaagtg cattggattg acaagaagca 
               
               
                   
               
               
                 3781 
                 acaggtattt cagagaagag attggaaatg aagaatttca ctgacgacag aactttgcaa 
               
               
                   
               
               
                 3841 
                 aaggctgagt gtaagagcag gaggtgacac agcaaggtag gagattgagt tagaacacca 
               
               
                   
               
               
                 3901 
                 acacaccaag atatatggag ataagaattt aaagataaga tggaagacct agatatcctg 
               
               
                   
               
               
                 3961 
                 gactgctggg ggcacctaga cattctctgt ctgtaggaac atgaagtcaa ctatatcctc 
               
               
                   
               
               
                 4021 
                 ctaaagcagg aaccactcca gctcttttct gtgttcccta cacactcata ttagacaggg 
               
               
                   
               
               
                 4081 
                 ggttggtgat ggggatgact gaatgaacta aggagtgaat gcatgactca caaaaggtag 
               
               
                   
               
               
                 4141 
                 aggaagattg gtgctgtggg aggagtggag ggagacattc atttggaaaa tcagatggca 
               
               
                   
               
               
                 4201 
                 ggagcctttt tattgagtag tagaagagca aagcagaagg accagaatct ggagtcaaaa 
               
               
                   
               
               
                 4261 
                 gacatggttg agttctcatt ctgcaacttc ctagctgcag gtctttggga aaatgactcc 
               
               
                   
               
               
                 4321 
                 tttatacata actctgacct catctataaa gtaaaccttt cctccttagg agattgagct 
               
               
                   
               
               
                 4381 
                 tcaaactgtc acctctttga ggctcctgtc cctttactac aacactaatt tcatcccact 
               
               
                   
               
               
                 4441 
                 tggaaattgt gtagagctgt acaagtaaaa gggtggacaa taaacaggag aaatatagta 
               
               
                   
               
               
                 4501 
                 ggttccacat actctaacgc ccagcccttg gcctatgtgc caacactcac tcccaactcc 
               
               
                   
               
               
                 4561 
                 ttgaaaagct actattaaaa gagtttccct ttggtttaga aagatgtttc ttataatgca 
               
               
                   
               
               
                 4621 
                 tagcacatta aaataataac aactaacacc acagagagga gtgtggaaca cccagtgaga 
               
               
                   
               
               
                 4681 
                 gtaatacaga taaggagcca gggtctaaaa caagacacat agggttacct tgggatgtga 
               
               
                   
               
               
                 4741 
                 tacaacaagg aacatcataa cctcctgctt aggtagctgg gcagaatcaa ggctgccaca 
               
               
                   
               
               
                 4801 
                 gagcctgatg gagtaggagg aacaatgccc agccattccc acacatgctc aggagcaggg 
               
               
                   
               
               
                 4861 
                 cagctatgta catgttggag agatgctgtt tgtctttgac tcgcccgtgt tctgagtgag 
               
               
                   
               
               
                 4921 
                 ccctttgacc cagttttaga agcagactga gccacggtga gcagaggcgg ggcttaggga 
               
               
                   
               
               
                 4981 
                 ggcaggagtc ttggggcttt ataaagtcct gccgggcacc actgggcatc tctttcaagg 
               
               
                   
               
               
                 5041 
                 tttctgctgg gtttctgaac tgctgggttt ctgcttgctc ctctggagat gcagcgtctg 
               
               
                   
               
               
                 5101 
                 ttgactccag tgaagcgcat tctgcaactg acaagagcgg tgcaggaaac ctccctcaca 
               
               
                   
               
               
                 5161 
                 cctgctcgcc tgctcccagt agcccaccaa aggtgagtca ctttctgaga agcaccttgt 
               
               
                   
               
               
                 5221 
                 aactagtaaa agatagtttt tccctgctat tggggaaaac tcactagaat cccactcaaa 
               
               
                   
               
               
                 5281 
                 atttggcaag gcttgtgcac agcagcctta gacaagcaag ttaactttaa agggtctcag 
               
               
                   
               
               
                 5341 
                 ttacctcatc tctaaacaga caatcccttt cagctgtaga gtgagaagag cccaaacctc 
               
               
                   
               
               
                 5401 
                 tgacacatgc tgtgtttgtg agcaatggca acttttactc tgccagctgc atgaagcagt 
               
               
                   
               
               
                 5461 
                 agaaatatca gtaccaggcc acagctttcc tctctacacc accattccca ccttcacccc 
               
               
                   
               
               
                 5521 
                 tagcctctgc ctagaaccac aggaccttgt gccaactgca gtgttagtaa aaccagtgac 
               
               
                   
               
               
                 5581 
                 tttatatcac tgcagcagaa tcagaaatgg actgaggatg agaagctgtg tttgccttgt 
               
               
                   
               
               
                 5641 
                 gttccaattt tatgaaaagg ggaaatgtgt gtttatgtgt gtatgtgtac atgctctttg 
               
               
                   
               
               
                 5701 
                 caagaagaac atgcacactc cttttctttg taaatagtcc ctgaacatgg ctcaagtgct 
               
               
                   
               
               
                 5761 
                 tatgttttcc attgtcagcg atgatggtaa cacagctatc gttagtgcct caggctccca 
               
               
                   
               
               
                 5821 
                 gccacctatg tgtttctgtc taatccccaa accatccact acacattggg actagttctt 
               
               
                   
               
               
                 5881 
                 tatttcctta catttttact ctatattcta tgactactaa atatttagaa aaatgatttt 
               
               
                   
               
               
                 5941 
                 gacctagtgt ctttccttgc caaataccca aggaacctgg gtgtatagat gtgcatggta 
               
               
                   
               
               
                 6001 
                 gaggcaaatg cacatagctt tcttatattt ttcattatgc taccatcatc tcactctccc 
               
               
                   
               
               
                 6061 
                 catgcactgc caaaccctgc atgtgggtta aatgtcccag ctcaggattt aacctgtttc 
               
               
                   
               
               
                 6121 
                 tatatttgtg aagaagagat tgatgtgggt ttcttgtttt aatagcaata gttggccatc 
               
               
                   
               
               
                 6181 
                 agccaaaaga catacatcaa tcctccccaa cattctgact cccttggttc aaactcttgg 
               
               
                   
               
               
                 6241 
                 aatcattccc atttcccttc tggtatattc acagttaatc ccattatgca tggcttgaac 
               
               
                   
               
               
                 6301 
                 taatattgct tttcatgagt caccttttct ctatatgtct aatcgccttt aatccaaccc 
               
               
                   
               
               
                 6361 
                 acattggctc taactccaac ctaaaagaga ctttcatctc agcatctgct ttgctgtctt 
               
               
                   
               
               
                 6421 
                 caaaattcgg taagacttgt gccctccact tactgtattt ctcacatatt gtctccctgc 
               
               
                   
               
               
                 6481 
                 tcccctatac acctgcatct ccagggttcc ttacttgttc agtcaccccc tgccgtggcc 
               
               
                   
               
               
                 6541 
                 actgcccctt cattcccctc cagttcttca ctggcagaag tctgtcatcc atcaaggttt 
               
               
                   
               
               
                 6601 
                 gcctcaaatg cggtctcttc cacgaagctt ctctgatcct ccaacccact gaaatctctg 
               
               
                   
               
               
                 6661 
                 cttcctttga actcctgtag attttgctca cattcctttt tgtgggcctg accacattct 
               
               
                   
               
               
                 6721 
                 gccttgaagt tgggttatat gtgtgcttat cattcctcac actggtcaag gaggtctcaa 
               
               
                   
               
               
                 6781 
                 gaacctcacc ctcttctttt ctttgtccaa cccctttgtt caaccctcac aaacccttcc 
               
               
                   
               
               
                 6841 
                 cagcacagtg cctgaagtgt agtaattaat tttgaaacac aagggaagga ggcaagatgg 
               
               
                   
               
               
                 6901 
                 aatacagaag taaaggtgtg gtgcatgttc ttgaagtggg caacaccagg agaaaaatga 
               
               
                   
               
               
                 6961 
                 tttaaaatta cacaaagtga tcattcttta gagaaagcac aagatgagaa ggatactctt 
               
               
                   
               
               
                 7021 
                 aacttcggtg ggctgaagct tctggaagcc tctccgtgtt aattttcttc aaggctttat 
               
               
                   
               
               
                 7081 
                 aatccatttc tagaaatagc tccccaccaa gacagctaca aaagttacca actgacccat 
               
               
                   
               
               
                 7141 
                 tctaagcttc ttcttgcaag ctttgatttc taactgggaa gaaagggagg gagccagccc 
               
               
                   
               
               
                 7201 
                 agagaagtca gagcgagaat gaggctgaga gaaaggcagc caagctggca ggacaagcgc 
               
               
                   
               
               
                 7261 
                 tggcttaacc attagctccc gggtactggg gaagctcctc cgtaaatatt tgagagtaca 
               
               
                   
               
               
                 7321 
                 aactccagtt atttggaggg agtcaaataa atagggaaga taaataaact ccaaacctct 
               
               
                   
               
               
                 7381 
                 cctgtcagat ataatgtgta tttatcattc tgcctcacta tcttgtgatc atatgatcca 
               
               
                   
               
               
                 7441 
                 cttttgcctc acagctgtcc ttagaagtga ccttgctgct gggagaggct ctagaattct 
               
               
                   
               
               
                 7501 
                 accagaggct cagaatccca aagatgattg atagacacat tcaatctgag ttccagctcc 
               
               
                   
               
               
                 7561 
                 cgtagaatgg agctaaattt ataagcctgg cacccagggc agtgaaggga cagagtattt 
               
               
                   
               
               
                 7621 
                 ctaacacgtg agaaactatg aagttaccct gagtgcatca ctttaccagt gtgtgccttg 
               
               
                   
               
               
                 7681 
                 gtttcactaa ctataaaatg aagaatgttg ctaaagtgaa cagaaggtat aaagtacttt 
               
               
                   
               
               
                 7741 
                 tgtatgggag cagtacagag atcaccaagt tcacctccag tatgctccca tacaaaaggg 
               
               
                   
               
               
                 7801 
                 aacacagatt ttcgccaggg atattaagaa tctgggttaa agagaagtga attggtccag 
               
               
                   
               
               
                 7861 
                 aaaagaaata gatcatctct cccttcttct gctgactcct tccccttcct tttttcctct 
               
               
                   
               
               
                 7921 
                 gctctcgttt agaattgctc tttctgctgt ctgtgttccc tgcatattta gctgtaaaat 
               
               
                   
               
               
                 7981 
                 gtctgcttct ttcactgggc tgtgctctct ttatgggcac aatgcatgtc ttattcactg 
               
               
                   
               
               
                 8041 
                 ctgtgtattt ggactagaac tgtgttgggt gtgctcaata aacattggaa ggccctatca 
               
               
                   
               
               
                 8101 
                 gaaaaatcag ctagcagaaa acttacttaa aagtaggaaa acagtgggta tgttcttgtg 
               
               
                   
               
               
                 8161 
                 tagaaaaaag aaaggagaaa gacatgtaat tagaggtaca cttttaaaat gagtaaagat 
               
               
                   
               
               
                 8221 
                 tgtataatta tgccctataa gggcttataa catgtagaag taaagtatat gacaataatg 
               
               
                   
               
               
                 8281 
                 gttcaaaagg atgcagagag taaataaagt caacctaaag tttttgcagt gttccaaaag 
               
               
                   
               
               
                 8341 
                 taagataagt attaatttaa gtaagattac aacaagccaa ttatgcatgt tataatcttt 
               
               
                   
               
               
                 8401 
                 aaggtcacca gtaaaaggaa aagagggtat aaaatgaata ataaatattt gcttactcta 
               
               
                   
               
               
                 8461 
                 aaaggatatg ggaaaggagg aataaaagaa caaagaacaa atgagacaaa tagaaacaaa 
               
               
                   
               
               
                 8521 
                 taaaaaaata gacttagttc cggctgggcg tggtggctca cgcctgtaat cccagcactt 
               
               
                   
               
               
                 8581 
                 tggaagaccg agatcaggag atcgagacca tcctggctaa cacggtgaaa ccctgtctct 
               
               
                   
               
               
                 8641 
                 actaaaaata caaaaaatta gctgggcgtg gtggcaggtg cctgtagtcc cagctactca 
               
               
                   
               
               
                 8701 
                 ggaggctgag gcaggagaat ggtgtgaacc caggaggcgg agcttgcagt gagcagagat 
               
               
                   
               
               
                 8761 
                 cacgccactg cactccagct tgggtgacag agtgagactc cgggtgacag agtgagactc 
               
               
                   
               
               
                 8821 
                 cgtctcaaaa aagagaaaaa aaaagattta gttccaacta tattagtaat tacaacaaat 
               
               
                   
               
               
                 8881 
                 ataaatggat gaaatactca aattaaaaca ccactattgt tagacttatt aaaatttttt 
               
               
                   
               
               
                 8941 
                 taaaggacta aaatatatat accgatcaca agagatgtat gttaaagata aagacgttaa 
               
               
                   
               
               
                 9001 
                 gaagttgaaa gtaaaaaggg acagaaaaag atgtaccatg gaaacagtaa gcaaaaagct 
               
               
                   
               
               
                 9061 
                 agtgtagcta tatcgacgtc aggaaaggaa actttatgcc aagaatatca caaagatgaa 
               
               
                   
               
               
                 9121 
                 aagggatatt taataagtat agaagggtca attcaatgaa aagataataa caatactaaa 
               
               
                   
               
               
                 9181 
                 tttgtagtca tctgataaca tagcttcaaa atatagaaaa ttaattaaat gattgctatg 
               
               
                   
               
               
                 9241 
                 ttactgtctt ttgaggaaat tgtctacaga ccattagtgg gagtttgact gttatctcca 
               
               
                   
               
               
                 9301 
                 tcacaggttt tctacagcct ctgctgtccc cctggccaaa acagatactt ggccaaagga 
               
               
                   
               
               
                 9361 
                 cgtgggcatc ctggccctgg aggtctactt cccagcccaa tatgtggacc aaactgacct 
               
               
                   
               
               
                 9421 
                 ggagaagtat aacaatgtgg aagcaggaaa gtatacagtg ggcttgggcc agacccgtat 
               
               
                   
               
               
                 9481 
                 gggcttctgc tcagtccaag aggacatcaa ctccctgtgc ctgacggtgg tgcaacggct 
               
               
                   
               
               
                 9541 
                 gatggagcgc atacagctcc catgggactc tgtgggcagg ctggaagtag gcactgagac 
               
               
                   
               
               
                 9601 
                 catcattgac aagtccaaag ctgtcaaaac agtgctcatg gaactcttcc aggattcagg 
               
               
                   
               
               
                 9661 
                 caatactgat attgagggca tagataccac caatgcctgc tacggtggta ctgcctccct 
               
               
                   
               
               
                 9721 
                 cttcaatgct gccaactgga tggagtccag ttcctgggat ggtatgtacg gccacgaacc 
               
               
                   
               
               
                 9781 
                 ttatgtaaga aaggtgctgg aattggaggc tgaatattac cagttttgct tttcagttcc 
               
               
                   
               
               
                 9841 
                 ccaggtggct tcatctagtg aaggaaggac aatatattca cacagctgct gctatcatcc 
               
               
                   
               
               
                 9901 
                 cacaataacc acttagactt atatagcttt acagttaggt agcatgttca catagccatt 
               
               
                   
               
               
                 9961 
                 catttaattc ttacaacagc ctaggaagtg tgtattatac cagatttata gaagagaaca 
               
               
                   
               
               
                 10021 
                 tggaagatct gatagcttac acatagtgag tggcagaggc aaaaatgcca aaccacatct 
               
               
                   
               
               
                 10081 
                 gacatatttc ctattttacc gtacctgttt ctcttaaaca tgtcctaagt ctctgagaga 
               
               
                   
               
               
                 10141 
                 ttggtgatgt tgaaagatgt atgcaagttt agatgttcgg gaaaaaaaca ccttcataga 
               
               
                   
               
               
                 10201 
                 aacaggccca gaaaaccaca agatagactg tgagtatttc tactctttct cccttaggtg 
               
               
                   
               
               
                 10261 
                 gctccttgca tattgctttt tgcttaacat attaacatta ccttgtatct tacttatatc 
               
               
                   
               
               
                 10321 
                 ttctcccagt gctatatttg aggactaacc cctgttgtta cagcaagaaa tgattcaagg 
               
               
                   
               
               
                 10381 
                 gaaacagtac agtatgagag cttgaagcca tagctctatc aataatcatt gataaattcc 
               
               
                   
               
               
                 10441 
                 tgaacctctt tgagcctcag ggttatttgc ctatctgcct tgcttaactt ataagaggac 
               
               
                   
               
               
                 10501 
                 tgaataaaat aattcataga aatgtgaaat tttcataaag atgtgaaaaa acagtatgtt 
               
               
                   
               
               
                 10561 
                 ggcagtagtt aagacactct atatttacta agtttgaaac taggattaaa aaccttagaa 
               
               
                   
               
               
                 10621 
                 accatgataa gcattaatta taaaattaat caaaaagcct taatattggc agagtcctca 
               
               
                   
               
               
                 10681 
                 gagatcatct aattcaatat cttttgcttt agaaaaaaga ggtcaagagg agtgtaacag 
               
               
                   
               
               
                 10741 
                 tttatctctg tacatgcagc aagaccgtgc aattacaaaa gttcattcca ggcttttcca 
               
               
                   
               
               
                 10801 
                 actgccctac ctggctccat cattaacaat tccactgaca tgggatggtc cagtctacat 
               
               
                   
               
               
                 10861 
                 catcaagtct gttcttaaag tgcctctcct acttgatact tgtattacta cctctctagt 
               
               
                   
               
               
                 10921 
                 aacccctacc accattacca ccactgatat gtccaaccaa ttatttagtt gaggagtaga 
               
               
                   
               
               
                 10981 
                 aatgaaaaat aaggggcatt caccagcctt taaccaaaaa tcaaagagcc tattcttgag 
               
               
                   
               
               
                 11041 
                 agcattgtca gccttaagca tgccatttca aatgcgtaga ttcttctgag gggctgggta 
               
               
                   
               
               
                 11101 
                 ttccacagat ggggttgcaa atgcatcttt taaaaaaatg tggtatctag gtataaaagt 
               
               
                   
               
               
                 11161 
                 aaaaatttaa aaaacaagtt attgaaatgt gaatctttag tttgtattta aaacaaaaac 
               
               
                   
               
               
                 11221 
                 agctaagctt gagcctggac actcggacta cataccctgc aggtgacagt aaccaccagg 
               
               
                   
               
               
                 11281 
                 accagaggat gccagtgtga atgagaactc tgcttctgac ctagccagtc attcatctgg 
               
               
                   
               
               
                 11341 
                 ggaccctcag gtgggaggga gtggctctga gactcaggga gttctgaatc actccagaga 
               
               
                   
               
               
                 11401 
                 aaagtggagg ggatgaggaa agagaagagt atttctggct cagattggct gggagtcccc 
               
               
                   
               
               
                 11461 
                 atgttttctt gtgttttttt ttttaaatga aaataattaa aatttatatt tggaaaaaaa 
               
               
                   
               
               
                 11521 
                 catacacata cacaaaagta tataaagcaa agaaagactc ctcatttgac ctgttaccac 
               
               
                   
               
               
                 11581 
                 ttcccaaaat ttaacactga tggtttatat gtattcttcc aatatttttt ctaagtacct 
               
               
                   
               
               
                 11641 
                 gcaagtatac acatatctat tccattttaa acattgtaca aaatattcct catctcttag 
               
               
                   
               
               
                 11701 
                 gtcttagagg taattctgta tcaacatatg taaggtctat ctgattcttt ttaaaaccac 
               
               
                   
               
               
                 11761 
                 aatattcttg atggatatgc caaattttat ttaattaatc ccatattgat ggatatttag 
               
               
                   
               
               
                 11821 
                 ttttttagca atgataaata aagttttaat gaacattgta caatagcttt gtatactttt 
               
               
                   
               
               
                 11881 
                 ggcattgtat tgtaagaata aattcctaga agtggaatat caggataggt tgatttaaaa 
               
               
                   
               
               
                 11941 
                 gtttgataaa atgtgccaaa ttcttctcca aaatgttgta ctaacttaca ttcctacaat 
               
               
                   
               
               
                 12001 
                 gtatatatta tcaaactttc taatctttgt caatttaaca agtaaaatta taatgttttt 
               
               
                   
               
               
                 12061 
                 gatttgcgtt tcttttacta taagaaatct tgaatatttc tatgttgttt attggccttt 
               
               
                   
               
               
                 12121 
                 ttttattata tagcttgcct ttttttattt tttatttatt tattttttta gacagagtct 
               
               
                   
               
               
                 12181 
                 cgatctgttg ccaggctgga gtgcagtggc ggtgatctca gctcactgca acctctgcct 
               
               
                   
               
               
                 12241 
                 cccaggttca agcgattctt ctgtctcagc ctcccgagta gctgggacta caggacccca 
               
               
                   
               
               
                 12301 
                 ccaccacacc cggctcattt tttgtatttt tagtagagat gggatttcac cgtgttagcc 
               
               
                   
               
               
                 12361 
                 aggatagtct tgatctcctg acctcacaat cctcctgcct cggcctcccc aatcgctggg 
               
               
                   
               
               
                 12421 
                 attacaggcg tgagccaccg tgcccggcct agcttgcctt tttaatgaaa cttttataaa 
               
               
                   
               
               
                 12481 
                 tgaagataaa ttgatttttg ttgattgtaa gtattgtaaa tactccccca atttgtcttt 
               
               
                   
               
               
                 12541 
                 tgactttgtt tctgatagaa ggctttgatt tttagataat caaatttact ggccttttcc 
               
               
                   
               
               
                 12601 
                 taaatggatt ctaaatactt ttctatagtt tctaaagttt tcaaaatgtg tgcgtgtgtg 
               
               
                   
               
               
                 12661 
                 tgcttatata caggtagaaa aaagtattgt tttcccttaa ttttatgtat ataaaaatta 
               
               
                   
               
               
                 12721 
                 tatatactta aatatatatt tatatatatt aaatatacca atttacttat actaatatat 
               
               
                   
               
               
                 12781 
                 ttatatatac taaatatata cttacattta tatatttata taaattattt gtatatttat 
               
               
                   
               
               
                 12841 
                 atatatacac acacacacat gcacatagca ttggggaaga aaacaatact ttttcgttga 
               
               
                   
               
               
                 12901 
                 tgttggagtt gggattgtta taattcttaa gagaaggtcc ctggatttca gtgaatttgg 
               
               
                   
               
               
                 12961 
                 gttggagtcc tgactctgaa tccttaccct accatttatt agctatgtgg tttttgggca 
               
               
                   
               
               
                 13021 
                 agtggcttaa attctttagc cctcagtttc ttcatctgta ggatggggat aactatatct 
               
               
                   
               
               
                 13081 
                 gctacataga tttatcatga ggattaaatt atatagaaat gtggctccca aagcagtgct 
               
               
                   
               
               
                 13141 
                 gtgggtgaat actgggagct tcctcacagg tcagaatact aaaattacta ccatatctca 
               
               
                   
               
               
                 13201 
                 cccacaaact tgagtttttg ggacagtact tcttacagat gaaagtggaa cacataatag 
               
               
                   
               
               
                 13261 
                 tcaagaccac aattatttat tgaatactag tctgattatc ataaagttag tgactacgga 
               
               
                   
               
               
                 13321 
                 tcatttactc aatataaact attttcacaa tgaaagtagt gccacacaat tcaaggcacg 
               
               
                   
               
               
                 13381 
                 tggttcagga tccagtcaga actgggtttg aatatcaaaa tccatattaa ctagctatgt 
               
               
                   
               
               
                 13441 
                 gaccttacac tagttactca gtctctcagg aaggcaatgt cttcacttgt gaatgtggat 
               
               
                   
               
               
                 13501 
                 gttacctacc tcattggatt gtttcaagaa ttgtttaagg ttaactagtg tcctactagt 
               
               
                   
               
               
                 13561 
                 gttttaaatg ttagtttccc tccctgtcct ttaccttcta tgatttagga tataatttca 
               
               
                   
               
               
                 13621 
                 ggatcatggt gtgctataag gagatgggta caaacccaaa cctgaattgt ctccaaaagt 
               
               
                   
               
               
                 13681 
                 gcgaattaac acatttttca ctgaagtcag agacagaatt ctgaataaat gagcgtttta 
               
               
                   
               
               
                 13741 
                 cagagtgtca ggacactaaa ttttgacttt acatttcaaa tgtatcatga attgcactag 
               
               
                   
               
               
                 13801 
                 aacataagct ccacaggact gggatttttt attttgttta tcactctata tccaggacct 
               
               
                   
               
               
                 13861 
                 agaattgtgc ctggtacaca gtaggcactc agtctactct agatttggta atgatggtaa 
               
               
                   
               
               
                 13921 
                 atatttcttg tttctcttta caggtcgtta tgccatggtg gtctgtggag acattgccgt 
               
               
                   
               
               
                 13981 
                 ctatcccagt ggtaatgctc gtcccacagg tggggccgga gctgtggcta tgctgattgg 
               
               
                   
               
               
                 14041 
                 gcccaaggcc cctctggccc tggagcgagg tttgtagtaa tccattacca agaggctgtg 
               
               
                   
               
               
                 14101 
                 catggcatag ccaagaacat agatcctaat cccacattgg cacacctgct actcagggct 
               
               
                   
               
               
                 14161 
                 gaggtatgcg tttgaggatg gtattgcttg cctctaaaaa gggctggtct atggagcaga 
               
               
                   
               
               
                 14221 
                 gggaggagag gagaaatggg agaggggaat ccgcgaggct tcctctcttg catcatcagg 
               
               
                   
               
               
                 14281 
                 cattgggata acgatgcatg gaatgagtgg tgcagatgat ggtgaggaat cttagggaac 
               
               
                   
               
               
                 14341 
                 tcttctggca attgaagatt aaaatatata actggatata aagtgaaagt ctttcctttg 
               
               
                   
               
               
                 14401 
                 agactgttgg cttctattct aggttttgtt aagcccatgt aggtgaggaa agggaaatat 
               
               
                   
               
               
                 14461 
                 acatctcatt tttgtaatac caacaacctg tccaactcct tttgaatatg caagggatgt 
               
               
                   
               
               
                 14521 
                 tgaatgggct tgaacttggg catgggacac agataatgac cagaaacctc ctttatatgg 
               
               
                   
               
               
                 14581 
                 ttctctcatc ttttgtgctc aaggtaggct gcattgtgta gtctctgaaa cactttgtgt 
               
               
                   
               
               
                 14641 
                 gcctttccag ggctgagggg aacccatatg gagaatgtgt atgacttcta caaaccaaat 
               
               
                   
               
               
                 14701 
                 ttggcctcgg agtacccaat agtggatggg aagctttcca tccagtgcta cttgcgggcc 
               
               
                   
               
               
                 14761 
                 ttggatcgat gttacacatc ataccgtaaa aaaatccaga atcagtggaa gcaaggtatg 
               
               
                   
               
               
                 14821 
                 agattcagag ggcagaaagt gggggctcta tttacatagg ccaagggttt gtacccaaag 
               
               
                   
               
               
                 14881 
                 gccatgagat ggtcttttct ctcctgcctt gaaaataatg tcaagagaat tgtttcctgt 
               
               
                   
               
               
                 14941 
                 cctctttctt acactcttcc ctgggtctat gctaaaatcc atttggaagt cattcaactt 
               
               
                   
               
               
                 15001 
                 caggtgtaaa attgcttcta acttgagcta aataaaagaa agtaaataat ccagggcaag 
               
               
                   
               
               
                 15061 
                 gcccccagtg tgaaaccaag ggatgtcagc cacctgagaa gatggtgtta agaggctggg 
               
               
                   
               
               
                 15121 
                 cagtcacatt cgacagtggt tggcatttgt ttctggttaa gtcaggcatg gtttggctct 
               
               
                   
               
               
                 15181 
                 tggtttgtgg tttaccatct tttaaagtct cacgttgaga aatcatacct atattttcta 
               
               
                   
               
               
                 15241 
                 tatgctgaag tgttatcagt gatttttctc ttcgtgatgc tactgcaggt tgattttatt 
               
               
                   
               
               
                 15301 
                 ttcaccttta gttttggaat ttccctcctg agaaatatgt actgctttca taagcagaaa 
               
               
                   
               
               
                 15361 
                 ataagcaaat aaatcttcct tttaaaatac agaaaagcag ggagtggtgg ctcacgcctg 
               
               
                   
               
               
                 15421 
                 taatcccagc accttgggaa gctgaggcag gaggattgct tgaacccagg aatttgagac 
               
               
                   
               
               
                 15481 
                 caatgtgggc aacaaagcaa gaccctgtct ctaaaaaaaa aaagtacaaa agttagccag 
               
               
                   
               
               
                 15541 
                 gcatggtggc ataagcctgt agtcccagct actcagaagg ctgagatggg ggaaattgct 
               
               
                   
               
               
                 15601 
                 tgagaccagg agcccatgca gtaagctatg atcaagcaac tgcctccagc ctggactaca 
               
               
                   
               
               
                 15661 
                 gagtgaaaca aaccctgtct ctaaaaacat ataaataaat aaaaataaaa tacagttaaa 
               
               
                   
               
               
                 15721 
                 cctactttaa agacataaat agtattcttg cctgctcagg catgcccaga tgggcatccg 
               
               
                   
               
               
                 15781 
                 caaaagacag attgcagtgt gggagaaggc atggatgcct tgggggtgtc ataaagagct 
               
               
                   
               
               
                 15841 
                 acctcttgtc cctttctact gcagtgggtg ggacaccacc tgccagaggt gaacctcatg 
               
               
                   
               
               
                 15901 
                 ggcaagaagt tgctttgggc ctctctgcct cagtctgtct tctgtaattg gttatttgct 
               
               
                   
               
               
                 15961 
                 cctaactcct ctgaattctt gtggcattta aattttactc cttatttgca tatgtaaggt 
               
               
                   
               
               
                 16021 
                 gacagatgct gctttggatc ccagcactaa aatgtaatat ttcctaaggg cagagattgc 
               
               
                   
               
               
                 16081 
                 attgccctct tcttcagagt gagagagaca gtctgtagag tagagtcaga gacatctgaa 
               
               
                   
               
               
                 16141 
                 cctgaatcca aagccagcct tttcaaagtt ggacagatga caatgttttg tagaccggtt 
               
               
                   
               
               
                 16201 
                 cctcctctgg caaatgaaga aaattatata acacaaggtt gatttgagcc aagtatcata 
               
               
                   
               
               
                 16261 
                 gaggctggta atagtagata caaaggcttt gtttctttcc cttctttcct tattcgtaga 
               
               
                   
               
               
                 16321 
                 gattgcttag taagtgcatg taaaatgaat aaataaagct catatgtgtt tgcaggaggt 
               
               
                   
               
               
                 16381 
                 gggaagtagt tccctgggag gcctggagaa actcggcaca gttaaatctc agggaggata 
               
               
                   
               
               
                 16441 
                 tctaaatggc tcgcccctca tgccccatcc ttgccttcac gcttcctctt ccagctggca 
               
               
                   
               
               
                 16501 
                 gcgatcgacc cttcaccctt gacgatttac agtacatgat ctttcataca cccttttgca 
               
               
                   
               
               
                 16561 
                 agatggtcca gaagtctctg gctcgcctga tgttcaatga cttcctgtca gccagcagtg 
               
               
                   
               
               
                 16621 
                 acacacaaac cagcttatat aaggggctgg aggctttcgg gtgagttctc ttcttgggga 
               
               
                   
               
               
                 16681 
                 gcctagaggc tggtgaggtg tgagcaagaa ggaggcttct tcatgcctta agtctagacc 
               
               
                   
               
               
                 16741 
                 accagcaccc ctgtggggga caaatggcaa tcctccagca gaacaggaac aatcccaggt 
               
               
                   
               
               
                 16801 
                 ccttccacgg ggtagtgggt tattgtctgg gtagggccct ccatgagtta ttgcagggaa 
               
               
                   
               
               
                 16861 
                 acatggggga tttggcagca ctgcaggatc aaggggcagt aagaaactac agaggataaa 
               
               
                   
               
               
                 16921 
                 gaaagaaaga gagaaaggga gaaagagagg aagggagaaa gagagtagct aaatcattca 
               
               
                   
               
               
                 16981 
                 gtcaataaac attttctgaa catgttatgt gctagacatc gtattaacct ctcaggatac 
               
               
                   
               
               
                 17041 
                 taaaatgaat gtgactccat ggtccctgcc ctagagcatc tcacagccta tacagacaca 
               
               
                   
               
               
                 17101 
                 aacacacaga agcaaatgat cacactacag ggtagcaatt tgagaagtgt caggtcccat 
               
               
                   
               
               
                 17161 
                 tctcatttgc cattgtctta attcatgtcc tgcttttgct tttctcccat ctataaaatg 
               
               
                   
               
               
                 17221 
                 gggatgttcc agctcatccc cttagatgtg aaaaagcaga aagaatgctg tttattgatt 
               
               
                   
               
               
                 17281 
                 cactacacta atacactaat atttacaaag aaatgtcttc aatacagttt ccactgggaa 
               
               
                   
               
               
                 17341 
                 aggaatcttt ccctttcttc ttggtacctg tttatttcaa attttggtca attttatcaa 
               
               
                   
               
               
                 17401 
                 cagtagaata ggctaccaag tgtagcccct gttactaact agtactccta accctgccac 
               
               
                   
               
               
                 17461 
                 taactaaaac atcaaaatta gcacaaacac tgcttgtaag accagcccta tcgaaacaaa 
               
               
                   
               
               
                 17521 
                 aagtataaca tataccaaag atactagctt aatatcttta atatataaag atattatcaa 
               
               
                   
               
               
                 17581 
                 taataaaata aataccctaa tagaaaaatg agcaaaggat atgaacagaa aattttctca 
               
               
                   
               
               
                 17641 
                 aagaagacat gtatatgatc acattttaaa tatgcatatt cattaataaa aagtttactc 
               
               
                   
               
               
                 17701 
                 aagttaccat tttctctaga tagtcttttt aaaatgtgaa tacccagaat tgtcaagcct 
               
               
                   
               
               
                 17761 
                 gtgggaaaat gggcagtagt ggatgcgtaa atggatcagg tgttttgagg agtaacagga 
               
               
                   
               
               
                 17821 
                 gagcatgaag ccaaagcctt aaagatgtgc aaactttggg ctcagtaatc ccatgtgttt 
               
               
                   
               
               
                 17881 
                 aaaagaacac ctacctattc gctgtagtgt tttaactagt gaggaacagg agcaggaaga 
               
               
                   
               
               
                 17941 
                 tgggttaaag tgcggtacat cctgtcatgg accattcttc agcctttaca aataatgtta 
               
               
                   
               
               
                 18001 
                 tagaatgtca tggaaaaaaa atatatatat atatacacac acacactaag ttaagaaagt 
               
               
                   
               
               
                 18061 
                 atgctaacca caacacatag catgattttc tttctaattt tctagtaagc tctataaaat 
               
               
                   
               
               
                 18121 
                 tagggatgga ttccaccaga aaaataagcc ctaagtactc tctctgaatg gtaaggccat 
               
               
                   
               
               
                 18181 
                 tagtggtatg ttctcctctg tactgttctg tatttccaaa tattgtagga aaaacatgcg 
               
               
                   
               
               
                 18241 
                 ccccaaagtc ctctccagaa gctgttactt ttcccccttg ctccctgcct cccgtcccct 
               
               
                   
               
               
                 18301 
                 ggcctctcac atggctacct ctggctacct cacagggggc taaagctgga agacacctac 
               
               
                   
               
               
                 18361 
                 accaacaagg acctggataa agcacttcta aaggcctctc aggacatgtt cgacaagaaa 
               
               
                   
               
               
                 18421 
                 accaaggctt ccctttacct ctccactcac aatgggaaca tgtacacctc atccctgtac 
               
               
                   
               
               
                 18481 
                 gggtgcctgg cctcgcttct gtcccagtga gtactgcatc tggctccatg tcctccatgc 
               
               
                   
               
               
                 18541 
                 acaccctcag cctccgcccc cgtgggctgc agggtcaaca aagttgggtt tctcttttgg 
               
               
                   
               
               
                 18601 
                 ctcagaaatt taaaagaaag gaaggggcct ggtgtagtgg ctcatgcctg taatctcagc 
               
               
                   
               
               
                 18661 
                 atttggggag gtttaggcgg gcagatcgcc tgaacctagg agttcgagac ccgcctgggc 
               
               
                   
               
               
                 18721 
                 aacgtggtga aacctcatct ctacaaaaat tagctgagca tggttgtgtg cacgtgtggt 
               
               
                   
               
               
                 18781 
                 cccagctgct cgggaggctg aagtgggagg atggtgtgag cccaggagtg gaaggttgca 
               
               
                   
               
               
                 18841 
                 gtgagccatg attgtgtcat tggactccaa cctggatgac agaatgagat cctgtcataa 
               
               
                   
               
               
                 18901 
                 ataaataaat aaatataaaa gaaaggaaag gagggagaag gcaggaaaag gaaggaagat 
               
               
                   
               
               
                 18961 
                 gaaagaaact cgtaccaaag gtgtatgtat aggcagattt acagtctgta tcagacagtg 
               
               
                   
               
               
                 19021 
                 gtctccaaag tgaagtacat gatgtcaagg gatgggcaag atctgtttgg gcacatcaag 
               
               
                   
               
               
                 19081 
                 aaaacagtag ctttggtatg catatttttg tctcatttat ttaaaatctc tatacttagt 
               
               
                   
               
               
                 19141 
                 agagcatggt ggttaaatgg gtctgacttt agagcccaca acctgggttc aaattttttt 
               
               
                   
               
               
                 19201 
                 aaccaattat tagggttgac tttggataat acttaacctc aatgcacctc accttcccca 
               
               
                   
               
               
                 19261 
                 actgtagcat gtgtgcaatc acaatacctg tgttctactg tttttatgag cattaagtat 
               
               
                   
               
               
                 19321 
                 ctaaaacaat taaaatagca gtgcttagca ggtgctcaaa tgttggatgt tatttctatt 
               
               
                   
               
               
                 19381 
                 cattttctgt tttgtgggtt ttataaggaa gtactgcatc taacataaga aagggctcat 
               
               
                   
               
               
                 19441 
                 gaagtggctc atgcctataa tcctagcact ttggaaggct caggcaggag gatctcttga 
               
               
                   
               
               
                 19501 
                 gctcaggagt ttgagaccag ccttgggaac agagggaggc cccatctcta caaaattttt 
               
               
                   
               
               
                 19561 
                 ttaaacaatt agccatggat gttcacggtg gctcatgcct gtaataccaa cactttggga 
               
               
                   
               
               
                 19621 
                 ggccaaggtg ggaagatcac ctgaggtcag gagtttgaga acagcctggc caacatggca 
               
               
                   
               
               
                 19681 
                 aaaccccttc tctactaaaa atacaaacat cagctgggca tggtggtacg tgcctgtagt 
               
               
                   
               
               
                 19741 
                 cccagcaact cgagaggctg aggcatgaga attgcttgaa cccgggaggc agaagttgca 
               
               
                   
               
               
                 19801 
                 gtgagctgag atcgagctac tgcactatag cttgggtgac agagtgagac tctgtctcaa 
               
               
                   
               
               
                 19861 
                 aaaaaaaaaa aaaattagct gggtgtggca gcttgcacct gtagtcccag ctactcagga 
               
               
                   
               
               
                 19921 
                 tcctgaatcc tgaggtggga ggatcacttg agcccaggag gtaaaggctg cagtgagcca 
               
               
                   
               
               
                 19981 
                 tgatcacgcc actgcattcc gggcactcca ggctgggcaa cagagcaaga ctctgccaaa 
               
               
                   
               
               
                 20041 
                 aaaagaaaaa aaaaacgggc aggaaaaagt gcttatgggt gaacttgatc aaattattac 
               
               
                   
               
               
                 20101 
                 tcacagggga tgatcaaaaa gttatgactg ctgaaccatt accaatcaac atgggagcct 
               
               
                   
               
               
                 20161 
                 gaagggtgag tccagtggtc tgatctccat ctggagacac cttcagaatg cactgaattt 
               
               
                   
               
               
                 20221 
                 accctgtcct catgagaggg gagaagctct atgtacacca aaaattatct tgtgttttct 
               
               
                   
               
               
                 20281 
                 ctgccttata tatcttggat attagctgct ttccttttgg caaggtttcc tacacaaagg 
               
               
                   
               
               
                 20341 
                 cctgtccctg gggtctacca gaagtccctc tttatgtagg gtgcctggaa cccatttcta 
               
               
                   
               
               
                 20401 
                 gttgcatgag gtagacaggg agaagatcgg gatgataggc tgttgttcta tttgaagtgc 
               
               
                   
               
               
                 20461 
                 agaatataat atatatatac atatatgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt 
               
               
                   
               
               
                 20521 
                 gttttatttg atttctttcc ccacagccac tctgcccaag aactggctgg ctccaggatt 
               
               
                   
               
               
                 20581 
                 ggtgccttct cttatggctc tggtttagca gcaagtttct tttcatttcg agtatcccag 
               
               
                   
               
               
                 20641 
                 gatgctgctc caggtgagtg tcatctttct agtaggcctt cctgacaaga ttcatctggt 
               
               
                   
               
               
                 20701 
                 agaataacca tcttcttccc caccattact gaggctgcca tcttgacaga gttacgttat 
               
               
                   
               
               
                 20761 
                 tattaatagc aaagtaaatc actgaaggga tttaagcatg gagtaagttt gtttaattta 
               
               
                   
               
               
                 20821 
                 tgtgtttaaa gcacttattt ggctactact tagagactag attgaaaagg aacaaagctg 
               
               
                   
               
               
                 20881 
                 gatatgggga aaccacttag attgttccag taactagttc aggcaagagg taatggtggt 
               
               
                   
               
               
                 20941 
                 ttgattgcaa ctgattaaag agaagttgat ggatttgaga tacctaataa gaatttattg 
               
               
                   
               
               
                 21001 
                 attattttgt gattgatgtg attaaggaca tgcatttaag tactatgtgg catacacctt 
               
               
                   
               
               
                 21061 
                 gaccaaatca gtgtgtctgc ctgcatgttt tgctaacaag tatgcttgct tatcatttct 
               
               
                   
               
               
                 21121 
                 tggtattcta agccacacac accacacgtt cctccagggt gtaacctccc acagaacctg 
               
               
                   
               
               
                 21181 
                 gctctctgtt gaactcgtga ttggcaatag tgataatgac aatgaaaaag gtgtaacaat 
               
               
                   
               
               
                 21241 
                 cttgcttttg cttcccaggc tctcccctgg acaagttggt gtccagcaca tcagacctgc 
               
               
                   
               
               
                 21301 
                 caaaacgcct agcctcccga aagtgtgtgt ctcctgagga gttcacagaa ataatgaacc 
               
               
                   
               
               
                 21361 
                 aaagagagca attctaccat aagggtaaga aaaaagtcag gaagagagga agagagaccc 
               
               
                   
               
               
                 21421 
                 cattccagta gctgggagcc agggatttct ttggaaatct agaatttagt agtccagggt 
               
               
                   
               
               
                 21481 
                 caagactttt acgagatatg gttgggagaa gatttgctag aagatctgtt gtccaaaggg 
               
               
                   
               
               
                 21541 
                 gcaagaagtg ggtggggaaa cagaagatag agttgggaag agggaggcag gatgcagctt 
               
               
                   
               
               
                 21601 
                 cccagtatag aatatagcta aacacccaga atgtgtagtc ccatggaagc cagaagtata 
               
               
                   
               
               
                 21661 
                 gtctttgaaa ataccatctg caacagttga aagagtacag actttagagc tagatatcca 
               
               
                   
               
               
                 21721 
                 aatctaaccc tgagctgtgc cactcactag ctgtttatct ttggaaaaat ggttgaactt 
               
               
                   
               
               
                 21781 
                 ttctcagttg tcttatttct aaaatcatac cgattttgca ggatttccaa acaaattaaa 
               
               
                   
               
               
                 21841 
                 tgaattactc tatataaata tgttatcgac aaatattact gtcccctcca aattgccctc 
               
               
                   
               
               
                 21901 
                 tttctccacc aaacataaaa acaaaaaaca aaatattgct ccaaaagcaa caaatgaaag 
               
               
                   
               
               
                 21961 
                 gaaaatgaaa cccaaaggta atactagagt gattagttgg tggttttaaa accatagtaa 
               
               
                   
               
               
                 22021 
                 tacacagttt taccatgatt tctacaggtt ttatatatat tctcaagcaa aacttgggat 
               
               
                   
               
               
                 22081 
                 gcatgttgtt ttgcagcatg gtctcaaaag gagacagaat atacggaatt ggaaatgttc 
               
               
                   
               
               
                 22141 
                 cagaaaacct agacctagtg gtcattgatc tcttctggac cagtggatat gttatagcaa 
               
               
                   
               
               
                 22201 
                 agaaagacaa tgaaaataaa aatggagcag ggcacagtgg ctcacgcctg taatgctagt 
               
               
                   
               
               
                 22261 
                 cctttgggag gcagaggcag gtggatcact tgaggccagg agtttgagac cagcctggcc 
               
               
                   
               
               
                 22321 
                 aacatggtga aaacccatct ctactaaaaa tataaaaatt ataaaaatat gaatataata 
               
               
                   
               
               
                 22381 
                 aaaaaaataa aattatgtaa aaattagccg agtgtggtgg cacacacctc taatctcagc 
               
               
                   
               
               
                 22441 
                 tactcaggag gctgaggaga attacttgaa cccaggaggc agaggatgca gtgaactgag 
               
               
                   
               
               
                 22501 
                 atcacaccac cacactctag cctgggtgac acagaaagac tctgtctcaa aacaaaaaaa 
               
               
                   
               
               
                 22561 
                 aaaaaaagaa gaaaagaaaa ataggacctc tgagacaaac gttaacggac aaagcactga 
               
               
                   
               
               
                 22621 
                 aatactgcaa tgaatcagaa ccagaaaatt tagagtttag aaggacgtgt ctgttaggaa 
               
               
                   
               
               
                 22681 
                 acaggaagct gggaattacg tctcaaagta ggaactattg gcaaaaggat gggatgaaga 
               
               
                   
               
               
                 22741 
                 tttcaatgga ggaaggctat gtttactgta ggaaaatgtt gtactcttat aataaaagtc 
               
               
                   
               
               
                 22801 
                 ttaatagact tttattaagg ccttaagtgc tagattcaag atggctgccc ctcttgttct 
               
               
                   
               
               
                 22861 
                 gtgggtccag tgttctattt ggtggactaa gggtgacctt gcagcccctt acagcccagc 
               
               
                   
               
               
                 22921 
                 caagagagct tcactgtgaa ggggcagaca tcttcattac tattttctct tccaaaaact 
               
               
                   
               
               
                 22981 
                 catataactc tttgtgagta ctgcctcttc tcctcattcc acagtgaatt tctccccacc 
               
               
                   
               
               
                 23041 
                 tggtgacaca aacagccttt tcccaggtac ttggtacctg gagcgagtgg acgagcagca 
               
               
                   
               
               
                 23101 
                 tcgccgaaag tatgcccggc gtcccgtcta aaggtggtga gtgagagttt gcagagttgg 
               
               
                   
               
               
                 23161 
                 tggcataaaa ccctaatgtc ttcctctgag taacaacaca gagagagaag gtggggacag 
               
               
                   
               
               
                 23221 
                 gtgcagggag aagaaagttt aatggaagag gattggggtg acaggagaaa tgggagaatt 
               
               
                   
               
               
                 23281 
                 atctgtggaa tttttaaaag gaaaagcaag tattcagaat aggaatcttg tagtttggga 
               
               
                   
               
               
                 23341 
                 acattaacca ggccagggag ggttcacagc tttcaaacta atcagaagtg gggatttgta 
               
               
                   
               
               
                 23401 
                 ccataaagac caattaaaac tcttggggct ctttgccttg gaaaggcaaa agctggggga 
               
               
                   
               
               
                 23461 
                 gaaacatgtt ctgaaatctt gaatgtgaaa aataggagct ggatttgttt acctgatctg 
               
               
                   
               
               
                 23521 
                 ctgaagatag gaagctctcc tagaagcttg acagattagc attcagagca tccgttgagt 
               
               
                   
               
               
                 23581 
                 gaacaggctg tgaacctgaa cctatagaaa tcattactcc agggggatga gatcaacaga 
               
               
                   
               
               
                 23641 
                 tctgatgagc aacagaacaa ccaagatgaa cagccccaaa acctcagaaa tggtacacac 
               
               
                   
               
               
                 23701 
                 caatgtgtgg gagacagatt cataaggaat ggggcggttg aagattctgt taaagccaga 
               
               
                   
               
               
                 23761 
                 tacttctgct ggagggagtt ttaggctaag ggtcatgtaa caattcttat atcatgggat 
               
               
                   
               
               
                 23821 
                 tccttctggg gagaagcaat gaggttcagg aaattcgtgg acacaaggat agggagaaga 
               
               
                   
               
               
                 23881 
                 gagcaaggtg aaagaggatt gcggtgacag gagaaatggg agatattctt tatgatcgtt 
               
               
                   
               
               
                 23941 
                 tttaaaggaa agcaaacatt caaaaataag aatcttatat gaacccaggt agctgccttc 
               
               
                   
               
               
                 24001 
                 agttgaccaa ataggtagga taagcagaat gatagagtga gaagagattt attttacaac 
               
               
                   
               
               
                 24061 
                 ccataaattt taattagtgc agtctccatg ctcaagtttt taagattttc ccctcctttt 
               
               
                   
               
               
                 24121 
                 ggtagatgga gagggaagaa gaaaaaggtg tgccgaggca gggaaggagc agaggaaggg 
               
               
                   
               
               
                 24181 
                 aaggaagaag tcagtgggtg gcagagatgc acagatacag ccacctgaga ggaagcagag 
               
               
                   
               
               
                 24241 
                 gtgcgggtgg aggggccctg ggttcattcc ttaccgctgg gatattggca ggtgctaggc 
               
               
                   
               
               
                 24301 
                 tgttgcagcc cagatgttgt tagggctagg agaggtggac aagtgggctg agggccgcag 
               
               
                   
               
               
                 24361 
                 gatgcctttg agaggacgag ctcagttagc agccctgaag actgtggtac tgcccgggag 
               
               
                   
               
               
                 24421 
                 cctgtgtgca tgttggaaat acggttctta agggcaggtc agtagcaaag aggggctgtt 
               
               
                   
               
               
                 24481 
                 aaatgtgtca acttagttca ttcatcagaa gaagagtggg agaaataggg agggaggggg 
               
               
                   
               
               
                 24541 
                 gaaagggaga gagagaggtt ggggagagag tcagcgggag ggggagagag aaagagaaat 
               
               
                   
               
               
                 24601 
                 ttggaatttt taaaggagaa tttccacgtc agcctccctc cctctcatgg tagacaagct 
               
               
                   
               
               
                 24661 
                 tcttgcaagt gcttaggcag aattatacct gaaaaaaaaa gctggaactc ttgacctttt 
               
               
                   
               
               
                 24721 
                 ctcatgttga ttattaatat gagcagtgaa cttccaacaa tgagatttta gcagaaatga 
               
               
                   
               
               
                 24781 
                 agggctgctg tcagtgcagt gctcatggtg gagctctaca ggtctctgca gcgccctagc 
               
               
                   
               
               
                 24841 
                 ctgcctctcc tgctctccta tcacaggcag atgtgcgacg gggaccctgc ctacccccag 
               
               
                   
               
               
                 24901 
                 ccttggctcc agtagcattg ggcacagatc cctcaggtgt ccaggcttgg cacagggtgc 
               
               
                   
               
               
                 24961 
                 atagtgggag caccctcagg atgcagttag gggagcccct ctgcacagcc acacctcggg 
               
               
                   
               
               
                 25021 
                 caagaagcag gtactggggg cagggtgccc aagaggagac ccatgattga atgacttttt 
               
               
                   
               
               
                 25081 
                 gtttatttaa gttctgcaga tccatggaaa gcttcctggg aaacgtatgc tagcagagct 
               
               
                   
               
               
                 25141 
                 tctccccgtg aatcatattt ttaagatccc actcttagct ggtaaatgaa tttgaatcga 
               
               
                   
               
               
                 25201 
                 catagtagcc ccataagcat cagccctgta gagtgaggag ccatctctag cgggcccttc 
               
               
                   
               
               
                 25261 
                 attcctctcc atgctgcaat cactgtcctg ggcttatggt gctatggact aggggtcctt 
               
               
                   
               
               
                 25321 
                 tgtgaaagag caagatggag caatggagag aagacctctt cctgaatcac tggactccag 
               
               
                   
               
               
                 25381 
                 aaatgtgcat gcagatcagc tgttgccttc aagatccaga taaactttcc tgtcatgtgt 
               
               
                   
               
               
                 25441 
                 tagaacttta ttattattaa tattgttaaa cttctgtgct gttcctgtga atctccaaat 
               
               
                   
               
               
                 25501 
                 tttgtacctt gttctaagct aatatatagc aattaaaaag agagaaagag gaaatgattc 
               
               
                   
               
               
                 25561 
                 ctgcgtttct tggaacccag aatacaaacc cagcctaaca tgcagcaagc ctgctagacc 
               
               
                   
               
               
                 25621 
                 ttgtgggtca gagggctggg tccttgcctc acaggctgcc tctgtcccct tgcaattcca 
               
               
                   
               
               
                 25681 
                 ttctatttct gccacatgcc aagtgctatg acaggtacaa ggcaaataag aacggtagaa 
               
               
                   
               
               
                 25741 
                 cacagcttcc cccagcccac ttccctgttc taaagacacc acatagacag agagcagcag 
               
               
                   
               
               
                 25801 
                 acaggggcca gcaggagctg tagttcagat cttcttggtc attccttgcc gctgttattt 
               
               
                   
               
               
                 25861 
                 gaacaaataa acacagcgca aaggttaaca agtttttgcc ttctatagcc aaaaataaaa 
               
               
                   
               
               
                 25921 
                 aaataaataa attttgatgc ctggcaggaa attattccat tacaggatct ttcccccttg 
               
               
                   
               
               
                 25981 
                 ggggagggca ctgcttcttc tagggtcctc ttataaaata gcaatggttc aggcagatgg 
               
               
                   
               
               
                 26041 
                 ggattgagct gaggacggga gtgggaggag agggaaagta tcagggtgtt gtcatcactt 
               
               
                   
               
               
                 26101 
                 ccttttagaa agtttcctca gtcaccccca tgaggaaagg gcaccttgga aaagagagag 
               
               
                   
               
               
                 26161 
                 gatgctttcc attggcgggg agcagagctg gtgggggcag gggaggagga ggggaggagg 
               
               
                   
               
               
                 26221 
                 aggaggagga gaagcagggg aggcttaagg ctcccttaag cctcagggag cgcttaagaa 
               
               
                   
               
               
                 26281 
                 tggccccaca ggaatgagaa gctgggtctg ttcccttcac tgttttgctc aaggctgttc 
               
               
                   
               
               
                 26341 
                 atgtcacaac aaatcccaga taagccccaa tttgctcaga gaatccagca ttagctgact 
               
               
                   
               
               
                 26401 
                 gccttcccag gcctctctca aggtgcctgc aaaactctac tcatcacacc agctgcagcc 
               
               
                   
               
               
                 26461 
                 gctgcttagc agcccctctt tgctaccctc ttgctgcctg cacctcctca gcaagatgtt 
               
               
                   
               
               
                 26521 
                 taggggccct caacctggtt ggcatcccta gcagaacaac atgtgccttt cggtatctgt 
               
               
                   
               
               
                 26581 
                 gtgcagggga gaaaacccag cactaacctt agctctggag acaagaggcc tcgggcctgg 
               
               
                   
               
               
                 26641 
                 ccttctatcc acacagaagc tcactgtgca gtgttggtgc tgaaactctc tccatcagcc 
               
               
                   
               
               
                 26701 
                 tcagtcagcc tcagcaacca gaacttccca tacttcctgc atcagaggcc aggcctgtct 
               
               
                   
               
               
                 26761 
                 ccactaggga ggcatttgag cacaaatgga atgatattaa acattcgaca accaggttgt 
               
               
                   
               
               
                 26821 
                 caagggctga ccaattgaat ggacactgcc cacagcccac acaccagctg ggcatcagca 
               
               
                   
               
               
                 26881 
                 ctggctccct ccaacttcct tattcaccaa cttttatact gagcccgagg ccttcctctg 
               
               
                   
               
               
                 26941 
                 gcagctctgg gacactgatg cctgcctgct ctgaacaaag ccctctcccc catgtaaggt 
               
               
                   
               
               
                 27001 
                 cagcacacga gggaatgagt tgccaatggc tcagtcaaca ttttcaccct aaagtctaca 
               
               
                   
               
               
                 27061 
                 gataccatac aaataaagac tttccctgtg ggcaaaaatt cacacagggt gacctagggc 
               
               
                   
               
               
                 27121 
                 aggagagagg acggcagatt gggcaagtgt tgggctatga tacactcatt caaacgggaa 
               
               
                   
               
               
                 27181 
                 tactcaacat gtgatgttaa aactgatgca aaagatggcc ccgccactga ccatgagaca 
               
               
                   
               
               
                 27241 
                 agcccaagct ctagggggac acactgatca caacttcagg agtcagcaca ttgaggcaga 
               
               
                   
               
               
                 27301 
                 ttctgtgcgt ggcccagctt ttgccctgcc tccaccctga gctcacagcc agccttctgc 
               
               
                   
               
               
                 27361 
                 tgtgtgtgca caagaatgaa cttctactct aaaggggcag tgaagagatg ccacatgcca 
               
               
                   
               
               
                 27421 
                 caaagaacat gagggagtcc atggcaccct ccctgtagcc ctagctggat ttttcaaaaa 
               
               
                   
               
               
                 27481 
                 tttcattgta tatatttgag ggatagaaca tgacgttgta agatatatat atgtagtaaa 
               
               
                   
               
               
                 27541 
                 atggttactg taacggaaca aattaacata ttcattattt tacaaagtta cccatccccc 
               
               
                   
               
               
                 27601 
                 gccaccatgg caagagcagc tgcaatctac taatttagga aaaatcctca gtacaataca 
               
               
                   
               
               
                 27661 
                 ctgttattaa ctatagtcct caggttgtac atcagatctt ttgacttact caccctatgt 
               
               
                   
               
               
                 27721 
                 attttctact ttacattctt tgacctgtat ctccctagac acccccctca actacttttc 
               
               
                   
               
               
                 27781 
                 tagttcctat gtcaatatat ttgacctctt ttttgggggg ggattccaca tataaatgag 
               
               
                   
               
               
                 27841 
                 taagtgcaat aattttcttt ttgtgtctgg cctatttact tagtcatcag ggaaatgcaa 
               
               
                   
               
               
                 27901 
                 atcaaaacca cggtgagata ccacctcaca cctgtta 
               
               
                 //