Enhanced transgene expression from recombinant single-stranded D-sequence-substituted adeno-associated virus vectors in human cell lines in vitro and in murine hepatocytes in vivo

Chen Ling, Yuan Wang, Yuan Lu, Lina Wang, Giridhara R. Jayandharan, George V. Aslanidi, Baozheng Li, Binbin Cheng, Wenqin Ma, Thomas Lentz, Changquan Ling, Xiao Xiao, R. Jude Samulski, Nicholas Muzyczka, Arun Srivastava

Research output: Contribution to journalArticlepeer-review

21 Scopus citations

Abstract

We have previously reported that the removal of a 20-nucleotide sequence, termed the D sequence, from both ends of the inverted terminal repeats (ITRs) in the adeno-associated virus serotype 2 (AAV2) genome significantly impairs rescue, replication, and encapsidation of the viral genomes (X. S. Wang, S. Ponnazhagan, and A. Srivastava, J Mol Biol 250:573-580, 1995; X. S. Wang, S. Ponnazhagan, and A. Srivastava, J Virol 70:1668-1677, 1996). Here we describe that replacement of only one D sequence in either ITR restores each of these functions, but DNA strands of only single polarity are encapsidated in mature progeny virions. Since most commonly used recombinant AAV vectors contain a single-stranded DNA (ssDNA), which is transcriptionally inactive, efficient transgene expression from AAV vectors is dependent upon viral second-strand DNA synthesis. We have also identified a transcription suppressor sequence in one of the D sequences, which shares homology with the binding site for the cellular NF-κB-repressing factor (NRF). The removal of this D sequence from, and replacement with a sequence containing putative binding sites for transcription factors in, single-stranded AAV (ssAAV) vectors significantly augments transgene expression both in human cell lines in vitro and in murine hepatocytes in vivo. The development of these genome-modified ss AAV vectors has implications not only for the basic biology of AAV but also for the optimal use of these vectors in human gene therapy.

Original languageEnglish (US)
Pages (from-to)952-961
Number of pages10
JournalJournal of virology
Volume89
Issue number2
DOIs
StatePublished - 2015
Externally publishedYes

Bibliographical note

Publisher Copyright:
© 2015, American Society for Microbiology.

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