Gene Transfer During LVAD Support. University of Pittsburgh Medical Center Pittsburgh, PA
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1 Gene Transfer During LVAD Support University of Pittsburgh Medical Center Pittsburgh, PA
2 Heart Failure Major cause of morbidity and mortality In the United States each year, more than 1,000,000 hospitalizations and 500,000 deaths Ischemic versus non-ischemic etiology Mortality remains high despite recent advances, including β-blockers, ACE inhibitors, ICDs, BiV Pacers, Ventricular Assist Devices (VADs), Transplants
3 Calcium Handling in Heart Failure Smaller, delayed calcium transients Decreased amount and activity of the Sarcoplasmic Reticulum Calcium ATPase (SERCA2a) Altered ratio of SERCA2a and phospholamban
4
5 SERCA2a Expression & Activity in Non-Failing SERCA2a / Phospholamban Non-Failing Human Failing Hearts Failing p<0.005 Failing SERCA2a Phospholamban Diastolic [Ca 2+ ] ATPase Activity (nmol/mg.min) NonFailing Failing ATPase SR Activity ATPase (mmol/mg.min) Activity SR ATPase Activity [Ca 2+ ] (µmol/l) NonFailing Failing
6 Ventricular Assist Devices LVADs, RVADs, BiVADs Morbidity from infection, bleeding, clotting, device failure Most patients go to transplant, but some recover Ideal to study novel heart failure treatments More than 50 heart transplants & 50 LVADs/year at the University of Pittsburgh & Massachusetts General Hospital
7 Methods: Devices Novacor LVAS Heartmate LVAS VE Thoratec VAD Electrically powered Electrically powered Pneumatically powered 10 L/min 10 L/min 7.2 L/min
8 VAD Implantations (n=125) Ischemics 58 (46%) Non-Ischemics 67 (54%) Transplanted 92 (74%) Died on Device 25 (20%) Weaned 8 (6%) Addition of RVAD 13 (10%) Pump Replacements 7 (6%) Mean Time to Transplant 118 days (2 548)
9 Hypothesis In patients on Ventricular Assist Devices (LVADs) with end-stage heart failure, gene delivery of SERCA2a by direct intracardiac injection of AAV6-CMV-SERCA2a can safely: restore SERCA2a protein levels and activity to normal improve local molecular and cellular indices of cardiac function improve global indices cardiac function and ability to wean off VAD support
10 AAV6-CMV-SERCA2a ITR CMV SD/SA Human Serca2a polya ITR paav-serca2a 4791 bp
11 AAV6-CMV-SERCA2a Long-term expression of gene product, driven by CMV promoter Produced by an adenovirus-free, triple plasmid transfection method Purified by a heparin affinity FPLC
12 Assaying AAV6-CMV-SERCA2a Titers: Dot blot (v.g./ml); Infectious center assay; immunostaining; SDS PAGE Sterility: inoculation in tissue culture medium Human genomic DNA Wild-type or wild-type-like AAV Other proteins by Western blot
13 Gene Transfer to Myocardium using Different Viral Vectors 80 %X-GAL Positive Myocytes E1- E4 deleted Adenovirus Adeno- associated Virus Lentivirus E1- deleted Adenovirus DAYS POST INFECTION
14 Gene Transfer into Rat Myocardium: Superior Efficiency with AAV X-Gal Activity week 2 weeks 4 weeks 12 weeks 36 weeks
15 SERCA2a Gene Transfer: Efficacy in Heart Failure Ad-CMV-SERCA2a improves contractility in human myocytes isolated from failing hearts Ad-CMV-SERCA2a improves function in a rat aortic-banded model of heart failure Ad-CMV-SERCA2a and AAV6-CMV-SERCA2a improve function in a porcine mitral regurgitation model of heart failure
16 Adenoviral Gene Transfer of SERCA2a in a Rat Pressure-Overload Model Ad.βgal Aortic Banding POH Heart Failure 0.5 Ad.SERCA2a Transition from Compensated Hypertrophy to Failure Fractional Shortening (%) Weeks Post Banding
17 LVP (mmhg) SERCA2a Overexpression Improves the Pressure-Dimension Relationship Sham + Ad.βgal Failing + Ad.SERCA2a Failing + Ad.βgal LV Dimension (mm) LV Dimension (mm)
18 SERCA2a Gene Transfer Improves Survival in Pressure-Overload Rats 120 %Survival Sham Failing + Ad.SERCA2a Dobutamine Failing Failing + Ad.GFP Gene Transfer DAYS
19 AAV6-CMV-SERCA2a Gene Transfer: Preclinical Data Intracoronary delivery of AAV6-CMV-SERCA2a in a swine model of mitral regurgitation with heart failure Mitral regurgitation created using percutaneous technique 30 days: Gene transfer using AAV6-CMV-SERCA2a or AAV6-CMV-βgal 60 to 120 days: Invasive hemodynamics, functional studies, molecular studies
20 Mitral valve cordes rupture to create MR using a percutaneous biotome Mitral valve (MV) P A Papillary muscles
21 Intracoronary Delivery of 5 x /ml AAV6-CMV-SERCA2a LAD LCX Great cardiac vein Catheter in Coronary sinus
22 SERCA2a Protein Expression in LV Sections 6 Weeks Post-Delivery 0.5 Arbitrary Units Saline AAV.βgal AAV.SERCA2a
23 SR ATPase Activity in LV Sections 12 weeks Post-Delivery pmol/mg SR protein.min AAV6.CMV.βgal AAV6.CMV.SERCA2a pca
24 Porcine Model of Heart Failure Chronic mitral regurgitation QuickTime and a Cinepak decompressor are needed to see this picture. QuickTime and a Cinepak decompressor are needed to see this picture. QuickTime and a Cinepak decompressor are needed to see this picture. Mitral Regurgitation 3-month post Mitral Regurgitation +AAV-SERCA
25 Hemodynamic Parameters: AAV6-CMV-SERCA2a Gene Transfer Control MR MR + AAVβgal MR + AAVSERCA2a Heart Rate (bpm) 102 ± ± ± ± 6 Fractional Shortening (%) 42.3 ± ± ± ± 4 Stroke volume (ml) 34.2 ± ± ± ± 2 +dp/dt (mmhg/sec) 1865 ± ± ± ± 344 -dp/dt (mmhg/sec) ± ± ± ± 101 LV Systolic Pressure (mmhg) 92 ± 5 89 ± 5 82 ± 4 98 ± 4 LV End-diastolic Pressure (mmhg) 7 ± 2 14 ± 1 19 ± 4 10 ± 3 # of animals
26 AAV6-CMV-SERCA2a Gene Transfer: Canine Pacing Study Direct injection of AAV6-CMV-SERCA2a during cardiopulmonary bypass in a canine pacing model of heart failure Transvenous Pacemaker, Pacing at 230 bpm 30 days: Gene transfer using AAV6-CMV-SERCA2a (n=8) or AAV6-CMV-βgal (n=4) by direct injection Pacing at 190 bpm, serial blood testing, EKG, echocardiographic and hemodynamic studies 75 days: Cellular, molecular, histological, immunological, tissue distribution studies
27 AAV6-CMV-SERCA2a Gene Transfer: Enrollment Subjects with nonischemic cardiomyopathy, age 18-65, will be enrolled at the time of LVAD placement at the University of Pittsburgh or Massachusetts General Hospital Decompensated heart failure as a bridge to transplant Randomize 8 patients to AAV6-CMV-SERCA2a and 8 subjects to saline injection
28 AAV6-CMV-SERCA2a Gene Transfer: Exclusion Criteria Acute myocarditis (signs, symptoms, biopsy) Coronary artery disease (>50% lesion) Postpartum (6 months) Fever, leukocytosis, positive blood cultures Unable to provide consent
29 AAV6-CMV-SERCA2a Gene Transfer: Gene Delivery Two 3x3 grids: LV anterior and lateral walls 5x10 11 /ml in low dose grid 5x10 12 /ml in high dose grid 1 cm between each injection site 5 mm deep 0.1 mls per injection
30 AAV6-CMV-SERCA2a Gene Transfer: Follow-up Testing on LVAD Serum (weekly x 4, then biweekly) Myocardial toxicity (CPK, Troponin) Kidney, liver function tests Inflammation (WBC, CRP, ESR) Antibodies to SERCA2a, AAV6 Viral DNA
31 AAV6-CMV-SERCA2a Gene Transfer: Weaning Protocol 4-6 weeks after implantation, good clinical status Metabolic stress testing MVO 2 > 17 ml O 2 /kg/min on VAD MVO 2 > 15 ml O 2 /kg/min off VAD Tissue-Doppler Echocardiography (automated border detection, regional wall motion) On 90% support, EF > 40%; Off support Right Heart Catheterization PCW < 20 mmhg, CI > 2.2 l/min, HR stable
32 AAV6-CMV-SERCA2a Gene Transfer: Analysis at Time of Transplant Tissue (high dose, low dose, uninjected areas) SERCA2a expression (RNA, protein, activity) Inflammation, fibrosis (Histology, immunohistochem) Myocytes size, apoptosis Expression of muscle genes (Microarray: NCX, ANF, phospholamban, myosin heavy chain, collagen) Isolated Myocytes Shortening velocity, systolic and diastolic calcium
33 AAV6-CMV-SERCA2a Gene Transfer: Endpoints Safety Inflammation, kidney, liver damage Worsened heart function (increased RVADs) Arrhythmias Local Efficacy Tissue, myocytes (vs. controls, function of dose) Regional wall motion by echocardiography Global Efficacy Ability to wean off LVAD
34 Acknowledgements University of Pittsburgh Joseph Glorioso PhD Xiao Xiao PhD Dennis M. McNamara MD Charles F. McTiernan PhD Kenneth McCurry MD Robert Kormos MD Michael Mathier MD Arthur M. Feldman MD PhD Mass General Hospital Roger Hajjar, MD G. William Dec, MD Thomas Disalvo, MD Marc Semigran, MD Joren Madsen, MD PhD Thomas McGillvray, MD
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