Follenzi A, Ailles L E, Bakovic S, Geuna M, Naldini L
Laboratorie for Gene Transfer and Therapy, IRCC, Institute for Cancer Research and Treatment, University of Torino Medical School, Candiolo (Torino), Italy.
Nat Genet. 2000 Jun;25(2):217-22. doi: 10.1038/76095.
Gene-transfer vectors based on lentiviruses are distinguished by their ability to transduce non-dividing cells. The HIV-1 proteins Matrix, Vpr and Integrase have been implicated in the nuclear import of the viral genome in non-dividing cells. Here we show that a sequence within pol is also required in cis. It contains structural elements previously associated with the progress of reverse transcription in target cells. We restored these elements in cis within late-generation lentiviral vectors. The new vector transduced to a much higher efficiency several types of human primary cells, when both growing and growth-arrested, including haematopoietic stem cells assayed by long-term repopulation of NOD/SCID mice. On in vivo administration into SCID mice, the vector induced higher plasma levels of human clotting factor IX (F.IX) than non-modified vector. Our results indicate that nuclear translocation of the genome is a rate-limiting step in lentiviral infection of both dividing and non-dividing cells, and that it depends on protein and nucleic acid sequence determinants. Full rescue of this step in lentivirus-based vectors improves performance for gene-therapy applications.
基于慢病毒的基因转移载体以其转导非分裂细胞的能力而著称。HIV-1蛋白基质、Vpr和整合酶与病毒基因组在非分裂细胞中的核输入有关。在此我们表明，pol基因内的一个序列在顺式作用中也是必需的。它包含先前与靶细胞中逆转录进程相关的结构元件。我们在新一代慢病毒载体中顺式恢复了这些元件。当人类原代细胞处于生长和生长停滞状态时，新载体对几种类型的细胞转导效率要高得多，包括通过对NOD/SCID小鼠进行长期再增殖检测的造血干细胞。在对SCID小鼠进行体内给药时，该载体诱导的人凝血因子IX（F.IX）血浆水平高于未修饰的载体。我们的结果表明，基因组的核转位是慢病毒感染分裂细胞和非分裂细胞的限速步骤，并且它取决于蛋白质和核酸序列决定因素。在基于慢病毒的载体中完全挽救这一步骤可提高基因治疗应用的性能。
