Herpes simplex virus 1 (HSV-1)-derived amplicon vectors are unique in their ability to accommodate large DNA molecules allowing whole genomic loci to be included with all of their regulatory elements. Additional advantages of these amplicons include their minimal toxicity and ability to persist as episomes, with negligible risk of insertional mutagenesis, being particularly well-suited for gene therapy of neurological disorders due to their outstanding ability to deliver genes into neurons and other neural cells. However, extensive gene therapy application has been hindered by difficulties in vector production. This work improved HSV-1 amplicons production by genetic modification of the packaging cell line and optimization of the culture medium. A stably-transfected Vero 2-2 cell line overexpressing the anti-apoptotic Bcl-2 protein was generated, exhibiting an increased resistance to apoptosis, prolonged culture duration, and a significant improvement in viral vector production. Additionally, supplementation of the growth medium with antioxidants, polyamines, amino acids, and reduced glutathione further increased the yield of packaged amplicon vectors. With these modifications, HSV-1 amplicons could be isolated from culture supernatants instead of cell lysates, leading to vector preparations with higher titer and purity and paving the way for generation of stable cell lines that are capable of continuous herpesviral vector production.

单纯疱疹病毒1（HSV-1）衍生的扩增子载体具有独特的能力，可容纳较大的DNA分子，从而使整个基因组位点都包含在其所有调控元件中。这些扩增子的其他优点包括其最小的毒性和作为附加体持久的能力，而插入诱变的风险可忽略不计，由于其将基因传递至神经元和其他神经细胞的出色能力，特别适合神经系统疾病的基因治疗。然而，由于载体生产中的困难，广泛的基因治疗应用受到了阻碍。这项工作通过包装细胞系的遗传修饰和培养基的优化提高了HSV-1扩增子的产量。产生了稳定表达的过表达抗凋亡Bcl-2蛋白的Vero 2-2细胞系，表现出对凋亡的抵抗力增强，延长的培养持续时间以及病毒载体生产的显着改善。另外，用抗氧化剂，多胺，氨基酸和减少的谷胱甘肽补充生长培养基进一步提高了包装的扩增子载体的产量。通过这些修饰，可以从培养物上清液中分离HSV-1扩增子，而不是从细胞裂解物中分离出来，从而可以制备具有更高滴度和纯度的载体，并为能够连续生产疱疹病毒载体的稳定细胞系铺平了道路。减少的谷胱甘肽进一步提高了包装扩增子载体的产量。通过这些修饰，可以从培养物上清液中分离HSV-1扩增子，而不是从细胞裂解物中分离出来，从而可以制备具有更高滴度和纯度的载体，并为能够连续生产疱疹病毒载体的稳定细胞系铺平了道路。减少的谷胱甘肽进一步提高了包装扩增子载体的产量。通过这些修饰，可以从培养物上清液中分离HSV-1扩增子，而不是从细胞裂解物中分离出来，从而可以制备具有更高滴度和纯度的载体，并为能够连续生产疱疹病毒载体的稳定细胞系铺平了道路。