Therapy Effects Of Recombinant Adenoassociated Virus Mediated Gene Transfer Of SERCA2a In Chronic Heart Failure Beagles

Although the drug therapies developed over the last decades have improved the prognosis of chronic heart failure(CHF), the condition remains a principal cause of mortality and hospital admission in the world. The hopes for gene therapy in cardiac failure are based on the possibility of acting on the underlying pathophysiological mechanisms by the transfer of genetic material to the failing myocardium. Recent studies in isolated myocardial cells and small animal models have suggested that overexpressing the calcium ATPase of the sarcoplasmic reticulum(SERCA2a) in the failing heart can potentially reverse severe heart failure. Hence we design this study to verify the efficacy and safety of overexpression SERCA 2a with a recombinant adeno-associated virus vector (rAAV2/1) in a large animal CHF model.Firstly, Progressive CHF was induced in beagle dogs over a 4-wk period by rapid right ventricular pacing(230 bpm), then pacing with 180 bpm for maintaining CHF. Clinical manifestation, echocardiographic and hemodynamic parameters significantly exacerbated after 230bpm×4wk pacing compared with those at baseline.Secondly, Twenty animals were randomized into 5 groups (n=4): control group, HF group, HF+EGFP group(infected with rAAV2/l-EGFP, 1×10¹²vg), HF+SERCA2a 1(infected 30d)and 2(infected 60d) group(infected with rAAV2/1-SERCA2a, 1×10¹²vg). Beagles received vectors by directly injecting into myocardium with thoracotomy. Beagles' clinical manifestation, echocardiographic parameters significantly improved after SERCA2a gene transfered 30d(P<0.01). LVSP, +dP/dt_max and -dP/dt_max of HF+SERCA2a 1 group increased respectively 4.12%, 146.81% and 71.52%, while LVEDP lowered 63.43% compared with those of HF+EGFP group; there were not significantly different from those of control group. However, clinical manifestation, echocardiographic and hemodynamic parameters significantly exacerbated after SERCA2a genetransfered 60d.Then, rAAV2/1-SERCA2a sequence was detected by PCR analysis of genomes DNA from major organs, skeletal muscles and sex glands of infected beagles, no rAAV2/1 genomes were detected besides injected myocardium area. SERCA2a mRNA and protein were detected by RT-PCR and Western blot analysis. The results showed the same expression profile in infected and control beagles. We also detected humoral immune responses to rAAV2/1, and found no anti-rAAV2/1 antibody in infected beagles' serum with ELISA. MVO₂ and arrhythmogenesis were not signicantly increased in infected beagles, and CK-MB, cTnT were the same. There was not obvious systemic inflammation because CRP and IL-6 did not increased in transferd group. And no influence showed in liver and kidney function.Finally, the proteome difference from beagles hearts between HF+SERCA2a 1 group and HF group was detected by 2-DE. Ten protein spots that were significantly affected by the experimental protocol were identified by PMF/Tandem MS. They are myosin light chain 1 (embryonic muscle/ atrial iso), slow skeletal muscle troponin T, troponin T; NADH dehydrogenase (ubiquinone) 1 alpha subcomplex; haptoglobin heavy chain, heat shock protein (alpha-crystallin-related, B6), galectin-1 and CG30493-PB isoform 1. The alteration of protein profile indicates that SERCA2a improve cardiac function through restoring contractile proteins to normal phenotype or quantity, improving energy production and regulating expression of stress-related proteins.From all above, gene therapy with adeno-associated viral mediated myocardial transfer of SERCA2a improves cardiac performance in a pacing model of CHF. At the same time, these results indicate that the risk of immune reaction and germline transmission after intramyocardium injection of rAAV2/1-SERCA2a in big animals are relatively low within the range of vector doses studied. We wish this study can form the foundation at big animal level for SERCA 2a gene therapy from bench to bedside.