Impaired nuclear transport and uncoating limit recombinant adeno-associated virus 2 vector-mediated transduction of primary murine hematopoietic cells

Li Zhong, Weiming Li, Zuocheng Yang, Keyun Qing, Mengqun Tan, Jonathan Hansen, Yanjun Li, Linyuan Chen, Rebecca J. Chan, Daniela Bischof, Njeri Maina, Kirsten A. Weigel-Kelley, Weihong Zhao, Steven H. Larsen, Mervin C. Yoder, Weinian Shou, Arun Srivastava

Research output: Contribution to journalArticle

49 Scopus citations

Abstract

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 ∼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 ∼40%, with a corresponding increase to ∼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.

Original languageEnglish (US)
Pages (from-to)1207-1218
Number of pages12
JournalHuman gene therapy
Volume15
Issue number12
DOIs
StatePublished - Dec 2004

ASJC Scopus subject areas

  • Molecular Medicine
  • Molecular Biology
  • Genetics

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    Zhong, L., Li, W., Yang, Z., Qing, K., Tan, M., Hansen, J., Li, Y., Chen, L., Chan, R. J., Bischof, D., Maina, N., Weigel-Kelley, K. A., Zhao, W., Larsen, S. H., Yoder, M. C., Shou, W., & Srivastava, A. (2004). Impaired nuclear transport and uncoating limit recombinant adeno-associated virus 2 vector-mediated transduction of primary murine hematopoietic cells. Human gene therapy, 15(12), 1207-1218. https://doi.org/10.1089/hum.2004.15.1207