经腹超声引导的AAV9-GFP胎猪递送:子宫内胎儿基因治疗的翻译和微创模型。

IF 4.5 3区 医学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY
Alessia Di Donfrancesco, Alessia Adelizzi, Anastasia Giri, Roberto Duchi, Simona Boito, Maria Barandalla, Giulia Massaro, Chiara Santanatoglia, Enrica Cappellozza, Andrea Perota, Ivano Di Meo, Valeria Tiranti, Emanuela Bottani, Cesare Galli, Nicola Persico, Dario Brunetti
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引用次数: 0

摘要

子宫内胎儿基因治疗(IUFGT)有可能在发生不可逆损害之前纠正严重的单基因疾病。尽管在小型和大型动物模型中取得了可喜的结果,但由于技术挑战、安全问题以及缺乏相关疾病模型物种的标准化方案,其向临床实践的转化仍然受到限制。我们建立并验证了一种微创超声引导方法,利用CAG启动子下编码GFP的自互补AAV9载体,对胎猪进行全身基因传递。注射在不同胎龄(GA 80和GA 108)通过心内或脐静脉途径进行。监测产后结局,并通过qPCR、ddPCR、免疫荧光和Western blotting评估转基因生物分布和表达。通过细胞因子分析和组织学分析评估炎症反应、毒性和产妇安全性。该手术耐受性良好,没有明显的产妇发病率或一次早产以外的不良产科结果。生物分布分析显示,载体广泛存在于外周组织中,在肝脏和心脏中表达强劲的GFP。重要的是,在分析的任何器官中没有明显的组织毒性、坏死或纤维化的证据。观察到促炎细胞因子(GM-CSF, GRO-α, IFN-γ)轻度增加,但与组织病理学改变无关。在仔猪或母猪血清中未检测到抗aav9衣壳抗体,表明对该载体的免疫反应很小。这些发现证明了超声引导的IUFGT在猪身上的安全性、可行性和有效性,支持了其作为治疗性基因传递给严重先天性疾病胎儿的翻译平台的潜力。该模型为产前干预的进一步临床前开发提供了一个有价值的框架,特别是对早期发病的疾病,如线粒体疾病。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Transabdominal ultrasound guided AAV9-GFP delivery in fetal pigs: a translational and minimally invasive model for in utero fetal gene therapy.

In utero fetal gene therapy (IUFGT) has the potential to correct severe monogenic disorders before irreversible damage occurs. Despite promising results in small and large animal models, its translation to clinical practice remains limited by technical challenges, safety concerns, and the lack of standardized protocols in relevant disease models species. We established and validated a minimally invasive, ultrasound-guided approach for systemic gene delivery in fetal pigs using a self-complementary AAV9 vector encoding GFP under a CAG promoter. Injections were performed at different gestational ages (GA 80 and GA 108) via intracardiac or umbilical venous routes. Postnatal outcomes were monitored, and transgene biodistribution and expression were assessed by qPCR, ddPCR, immunofluorescence, and Western blotting. Inflammatory response, toxicity, and maternal safety were evaluated through cytokine profiling and histological analyses. The procedure was well tolerated, with no significant maternal morbidity or adverse obstetric outcomes beyond one preterm delivery. Biodistribution analysis revealed widespread vector presence in peripheral tissues, with robust GFP expression in liver and heart. Importantly, there was no evidence of significant tissue toxicity, necrosis, or fibrosis in any of the organs analyzed. Mild increases in pro-inflammatory cytokines (GM-CSF, GRO-α, IFN-γ) were observed but were not associated with histopathological changes. No anti-AAV9 capsid antibodies were detected in sera from piglets or sows, suggesting a minimal immune response to the vector. These findings demonstrate the safety, feasibility, and efficacy of ultrasound-guided IUFGT in pigs, supporting its potential as a translational platform for therapeutic gene delivery in fetuses affected by severe congenital diseases. This model offers a valuable framework for further preclinical development of prenatal interventions, particularly for disorders with early onset, such as mitochondrial diseases.

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来源期刊
Gene Therapy
Gene Therapy 医学-生化与分子生物学
CiteScore
9.70
自引率
2.00%
发文量
67
审稿时长
4-8 weeks
期刊介绍: Gene Therapy covers both the research and clinical applications of novel therapeutic techniques based on a genetic component. Over the last few decades, significant advances in technologies ranging from identifying novel genetic targets that cause disease through to clinical studies, which show therapeutic benefit, have elevated this multidisciplinary field to the forefront of modern medicine.
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