Tang Qiushi, Keeler Allison M, Zhang Songbo, Su Qin, Lyu Zhuoyao, Cheng Yangfan, Gao Guangping, Flotte Terence R
Department of Pediatrics, University of Massachusetts Medical School, Worcester, Massachusetts, USA.
Horae Gene Therapy Center, University of Massachusetts Medical School, Worcester, Massachusetts, USA.
Biores Open Access. 2020 Oct 16;9(1):219-228. doi: 10.1089/biores.2020.0031. eCollection 2020.
A number of packaging systems are available for production of recombinant adeno-associated virus vectors (rAAVs). Among these, the use of a two-plasmid cotransfection system, in which Rep and Cap genes and Ad helper genes are on the same plasmid, has not been frequently employed for good manufacturing practices (GMP) production, even though it presents some practical advantages over the common three-plasmid (triple) transfection method. To confirm and expand the utility of the two-plasmid system, we generated GMP-compatible versions of this system and used those package reporter genes in multiple capsid variants in direct comparison with triple transfection. Vector yields, purity, and empty-to-full ratios were comparable between double and triple transfection methods for all capsid variants tested. We performed an side-by-side comparison of double and triple transfection vectors following both intravenous injection and intramuscular injection in mice. Expression and transduction were evaluated in muscle and liver 4 weeks after injection. Additional studies of bioactivity were conducted using packaged vectors carrying a variety of cargos, including the therapeutic transgene, microRNA, and single- or double-stranded vector. Results showed that cargos packaged using double transfection were equivalently bioactive to those packaged using a triple transfection system. In conclusion, these data suggest the utility of midrange (1E12-1E16) GMP-compatible packaging of adeno-associated virus (AAV) vectors for several AAV capsids.
有多种包装系统可用于生产重组腺相关病毒载体（rAAV）。其中，使用双质粒共转染系统（Rep和Cap基因以及腺病毒辅助基因位于同一质粒上）在药品生产质量管理规范（GMP）生产中并不常用，尽管它与常见的三质粒（三重）转染方法相比具有一些实际优势。为了证实并扩大双质粒系统的实用性，我们构建了该系统的GMP兼容版本，并在多个衣壳变体中使用这些包装报告基因，与三重转染进行直接比较。对于所有测试的衣壳变体，双转染和三重转染方法之间的载体产量、纯度以及空壳与满壳比率相当。我们在小鼠静脉注射和肌肉注射后对双转染和三重转染载体进行了并排比较。在注射后4周评估肌肉和肝脏中的表达和转导情况。使用携带多种货物（包括治疗性转基因、微小RNA以及单链或双链载体）的包装载体进行了额外的生物活性研究。结果表明，双转染包装的货物与三重转染系统包装的货物具有同等的生物活性。总之，这些数据表明了适用于几种腺相关病毒（AAV）衣壳的中等范围（1E12 - 1E16）GMP兼容的腺相关病毒载体包装的实用性。
