Biological bypass through induced angiogenesis by vascular endothelial growth factor D (VEGF-D) gene therapy (GT) is a new concept for the treatment of cardiac ischemia. Serotype 5 adenoviruses are used in the clinical trials for transferring the VEGF-D cDNA into the ischemic myocardium. However, the presence of replication-competent vectors in the adenovirus products is a widely recognized problem that may pose a potential safety risk to the treated patients. We compared three different VEGF-D GT production lots containing different levels of replication-competent adenoviruses (RCA) tested in 3 × 10¹⁰ viral particles (vp): <10 RCA (VEGF-D L-RCA1), 10–100 RCA (VEGF-D H-RCA2), and 100–200 RCA (VEGF-D H-RCA3), as measured by a novel droplet digital polymerase chain reaction (PCR) RCA assay in a preclinical rabbit model (n = 21). β-galactosidase encoding nonclinical-grade preparation was used as a nonangiogenic control. Each preparation was injected into the right semimembranosus muscle using dose of 1 × 10¹¹ vp. Efficacy of the products was tested by the combination of contrast pulse sequencing ultrasound and modified Miles assay as well as quantifying the total cross-sectional area of capillaries. Safety, immunogenicity, toxicity, biodistribution, and shedding were assessed by general histology, serial measurements of C-reactive protein, white blood cell count and body temperature as well as using quantitative real-time PCR with primers targeted to the VEGF-D and replication-permitting E1 sequences. We found no significant differences in the efficacy or safety between the study groups. Most importantly, no detectable presence of RCA-specific E1 sequence was found in any samples tested, indicating that no detectable vector replication took place in vivo. We conclude that relatively low levels of RCA in adenoviral GT products may not be as important major safety issue as previously anticipated.

中文翻译：

---

含有残留复制能力的腺病毒的临床级人血管内皮生长因子-DΔNΔC基因治疗的疗效和安全性

通过血管内皮生长因子D（VEGF-D）基因治疗（GT）诱导血管生成的生物旁路是治疗心脏缺血的新概念。血清型 5 腺病毒用于将 VEGF-D cDNA 转移到缺血心肌的临床试验。然而，腺病毒产品中存在复制能力强的载体是一个广泛认可的问题，可能对接受治疗的患者构成潜在的安全风险。我们比较了三个不同的 VEGF-D GT 生产批次，它们含有不同水平的可复制腺病毒 (RCA)，在 3 × 10 ^10中测试病毒颗粒 (vp)：<10 RCA (VEGF-D L-RCA1)、10-100 RCA (VEGF-D H-RCA2) 和 100-200 RCA (VEGF-D H-RCA3)，由一种新型临床前兔模型中的液滴数字聚合酶链反应 (PCR) RCA 测定 ( n  = 21)。编码非临床级制剂的β-半乳糖苷酶用作非血管生成对照。^{使用 1 × 10 11}的剂量将每种制剂注射到右侧半膜肌副总裁。产品的功效通过对比脉冲测序超声和改良的 Miles 测定相结合以及量化毛细血管的总横截面积来测试。安全性、免疫原性、毒性、生物分布和脱落通过一般组织学、C 反应蛋白的系列测量、白细胞计数和体温以及使用针对 VEGF-D 和复制的引物的定量实时 PCR 进行评估- 允许 E1 序列。我们发现研究组之间的疗效或安全性没有显着差异。最重要的是，在任何测试的样本中都没有发现可检测到的 RCA 特异性 E1 序列，这表明在体内没有可检测到的载体复制发生. 我们得出结论，腺病毒 GT 产品中相对低水平的 RCA 可能不像先前预期的那样重要的主要安全问题。