Ein lentiviraler Vektor für die Transduktion von G0-arretierten Zellen. Analyse genetischer Determinanten

慢病毒载体比小鼠白血病病毒(MLV)来源的伽马逆转录病毒载体优越，因为它们允许非有丝分裂细胞的转导。然而，它们不能有效地介导基因转移到静止的原代人类细胞，如未刺激的淋巴细胞、单核细胞或CD34造血祖细胞，这可能是由于这些细胞的低激活状态和GO细胞周期状态。相反，一种来自猴免疫缺陷病毒(SIV)smmPBj的慢病毒载体已被证明可以介导停滞在细胞周期GO状态的人类细胞系(造血干细胞被认为处于该细胞周期状态)和人类原代细胞，如GQ中新分离的单核细胞。因此，PBJ载体将极大地扩展人类基因治疗的前景。由于这种载体的亲本病毒是一种高致病性的SIV毒株，会导致猴子的急性肠病，与其他慢病毒衍生的载体相比，SIVsmmPBJ载体也提供了加速安全性研究的机会，这些慢病毒不会在这些物种中引发急性疾病。基于最小的PBJ载体的开发，它可以有效地将基因转移到其他慢病毒载体无法有效转导的人类造血细胞中，我们的目标是(I)识别基因转移到GO细胞(如原代单核细胞或干细胞)的遗传病毒和细胞决定因素，(Ii)构建第三代PBJ载体，以及(Iii)测试PBJ载体在动物体内的插入致癌安全性。

Lentiviral vectors (LVs) are superior to gammaretroviral vectors derived from murine leukaemia virus (MLV) since they allow transduction of non-mitotic cells. However, they are inefficient in mediating gene transfer into particular quiescent primary human cells such as non-stimulated lymphocytes, monocytes, or CD34+ haematopoietic progenitor cells, probably due to the low activation status and the GO cell cycle state of these cells. In contrast, a lentiviral vector derived from simian immunodeficiency virus (SIV) smmPBj has been shown to mediate transduction of human cell lines arrested in the Go state of the cell cycle (haematopoietic stem cells are believed to be in that cell cycle state) and of human primary cells such as freshly isolated monocytes in GQ. PBj vectors will therefore significantly extend human gene therapy perspectives. Since the parental virus of this vector is a highly pathogenic SIV strain which causes an acute enteropathy in monkeys, SIVsmmPBj vectors offer also the chance of accelerated safety studies compared to vectors derived from other lentiviruses which do not induce an acute disease in these species. Based on the development of a minimal PBj vector which allows efficient gene transfer into human haematopoietic cells which cannot be efficiently transduced by other lentiviral vectors, we aim at (i) identifying genetic viral and cellular determinants of gene transfer into Go cells such as primary monocytes or stem cells, (II) generation of a third-generation PBj vector and (iii) testing PBj vector safety in animals with respect to insertional oncogenesis.