Zhong Li, Li Weiming, Yang Zuocheng, Qing Keyun, Tan Mengqun, Hansen Jonathan, Li Yanjun, Chen Linyuan, Chan Rebecca J, Bischof Daniela, Maina Njeri, Weigel-Kelley Kirsten A, Zhao Weihong, Larsen Steven H, Yoder Mervin C, Shou Weinian, Srivastava Arun
Department of Microbiology and Immunology, Walther Oncology Center, Walther Cancer Institute, Indiana University School of Medicine, Indianapolis, IN 46202, USA.
Hum Gene Ther. 2004 Dec;15(12):1207-18. doi: 10.1089/hum.2004.15.1207.
Controversies abound concerning hematopoietic stem cell transduction by recombinant adeno-associated virus 2 (AAV) vectors. For human hematopoietic cells, we have shown that this problem is related to the extent of expression of the cellular receptor for AAV. At least a small subset of murine hematopoietic cells, on the other hand, does express both the AAV receptor and the coreceptor, yet is transduced poorly. In the present study, we have found that approximately 85% of AAV genomes were present in the cytoplasmic fraction of primary murine c-Kit(+)Lin- hematopoietic cells. However, when mice were injected intraperitoneally with hydroxyurea before isolation of these cells, the extent to which AAV genomes were detected in the cytoplasmic fraction was reduced to approximately 40%, with a corresponding increase to approximately 60% in the nuclear fraction, indicating that hydroxyurea facilitated nuclear transport of AAV. It was apparent, nonetheless, that a significant fraction of the AAV genomes present in the nuclear fraction from cells obtained from hydroxyurea-treated mice was single stranded. We next tested whether the single-stranded AAV genomes were derived from virions that failed to undergo uncoating in the nucleus. A substantial fraction of the signal in the nuclear fraction of hematopoietic cells obtained from hydroxyurea-treated mice was also resistant to DNase I. That AAV particles were intact and biologically active was determined by successful transduction of 293 cells by virions recovered from murine hematopoietic cells 48 hr postinfection. Although hydroxyurea facilitated nuclear transport of AAV, most of the virions failed to undergo uncoating, thereby leading to only a partial improvement in viral second- strand DNA synthesis and transgene expression. A better understanding of the underlying mechanism of viral uncoating has implications in the optimal use of recombinant AAV vectors in hematopoietic stem cell gene therapy.
关于重组腺相关病毒2（AAV）载体对造血干细胞的转导存在诸多争议。对于人类造血细胞，我们已经表明这个问题与AAV细胞受体的表达程度有关。另一方面，至少一小部分小鼠造血细胞确实同时表达AAV受体和共受体，但转导效率却很低。在本研究中，我们发现大约85%的AAV基因组存在于原代小鼠c-Kit(+)Lin-造血细胞的细胞质部分。然而，在分离这些细胞之前给小鼠腹腔注射羟基脲后，在细胞质部分检测到的AAV基因组的程度降低到了大约40%，而在细胞核部分相应增加到了大约60%，这表明羟基脲促进了AAV的核转运。尽管如此，很明显，从羟基脲处理的小鼠获得的细胞的细胞核部分中存在的相当一部分AAV基因组是单链的。接下来我们测试了单链AAV基因组是否来自未能在细胞核中进行脱壳的病毒粒子。从羟基脲处理的小鼠获得的造血细胞核部分中的大部分信号也对DNase I有抗性。通过感染后48小时从鼠造血细胞中回收的病毒粒子成功转导293细胞，确定了AAV颗粒是完整且具有生物活性的。虽然羟基脲促进了AAV的核转运，但大多数病毒粒子未能进行脱壳，从而仅导致病毒第二链DNA合成和转基因表达有部分改善。更好地理解病毒脱壳的潜在机制对于重组AAV载体在造血干细胞基因治疗中的最佳应用具有重要意义。
