腺相关病毒介导心肌肌浆网三磷酸腺苷酶基因治疗心力衰竭犬的安全性分析

Biosafety assessment of recombinant adeno-associated viral vectormediated SERCA2a gene therapy of HF beagles

目的研究心肌注射重组腺相关病毒(rAAV)_(2/1)介导心肌肌浆网Ca^(2+)-ATP酶(SERCA2a)基因治疗慢性心力衰竭犬的安全性。方法选取成年比格犬8只,随机分为对照组和SERCA2a基因转导组(转导组),每组4只。分别采用PCR、RT PCR和Western blot法从DNA、RNA和蛋白水平检测rAAV_(2/1)SERCA2a转导后在犬体内的分布;采用双夹心ELISA法检测转导组犬的血清。结果除注射rAAV_(2/1)的心肌区域外,未注射rAAV_(2/1)的心肌区域、主要脏器、骨骼肌和性腺中均未检测到rAAV_(2/1)-SERCA2a。对照组与转导组犬心肌、腰大肌、咀嚼肌和睾丸表现出相同的SERCA2a蛋白表达谱,未在其他组织中检测到。转导组犬血清内未检测到抗rAAV_(2/1)抗体。结论 rAAV_(2/1)介导的基因转导是相对安全的,未整合到心脏以外的其他组织,也未在犬体内引发体液免疫。

Objective To study the safety of intramyocardium injection of recombinant adeno-associated virus（rAAV）-SERCA2a to rescue chronic heart failure beagles. Methods rAAV-SERCA2a sequence was examined by PCR analysis of genomes DNA from main organs（myocardium,liver, spleen,lung,kldney） ,skeletal muscles（major psoas muscle,masseter muscle, diaphragmatic mus- cle,gastrocnemius muscle） and sex glands（ovaries,testicle） of infected beagles. SERCA2a mRNA and protein were detected by RT-PCR and Western blot analysis. Results No rAAV2/1 genomes were detected in main organs, skeletal muscles and sex glands besides injected myocardium area. The same SERCA2a protein expression profile（myoeardium, major psoas muscle,masseter mus- cle,testicle） was observed in infected and control beagles. Humoral immune response to rAAV2/1 was also detected. No anti-rAAV2n antibody was detected in infected beagles＇ serum with ELISA. Conclusion These results indicate that intramyocardium injection of rAAV-SERCA2a is relatively safe.