title: 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.
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 ssAAV vectors has implications not only for the basic biology of AAV but also for the optimal use of these vectors in human gene therapy.
  
  
  OBJECTIVE
  The results of the studies described here not only have provided novel insights into some of the critical steps in the life cycle of a human virus, the adeno-associated virus (AAV), that causes no known disease but have also led to the development of novel recombinant AAV vectors which are more efficient in allowing increased levels of gene expression. Thus, these studies have significant implications for the potential use of these novel AAV vectors in human gene therapy.
authors.affiliation: Division of Cellular and Molecular Therapy, Department of Pediatrics, University of Florida College of Medicine, Gainesville, Florida, USA Powell Gene Therapy Center, University of Florida College of Medicine, Gainesville, Florida, USA lingchen@peds.ufl.edu aruns@peds.ufl.edu.
authors.forename: Chen
authors.initials: C
authors.lastname: Ling
authors.affiliation: Division of Cellular and Molecular Therapy, Department of Pediatrics, University of Florida College of Medicine, Gainesville, Florida, USA Powell Gene Therapy Center, University of Florida College of Medicine, Gainesville, Florida, USA Department of Traditional Chinese Medicine, Changhai Hospital, Second Military Medical University, Shanghai, China Shanghai University of Traditional Chinese Medicine, Shanghai, China.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Department of Orthopedics & Rehabilitative Medicine, University of Florida College of Medicine, Gainesville, Florida, USA.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Lu
authors.affiliation: Division of Cellular and Molecular Therapy, Department of Pediatrics, University of Florida College of Medicine, Gainesville, Florida, USA Powell Gene Therapy Center, University of Florida College of Medicine, Gainesville, Florida, USA Department of Traditional Chinese Medicine, Changhai Hospital, Second Military Medical University, Shanghai, China.
authors.forename: Lina
authors.initials: L
authors.lastname: Wang
authors.affiliation: Department of Haematology and Centre for Stem Cell Research, Christian Medical College, Vellore, India.
authors.forename: Giridhara R
authors.initials: GR
authors.lastname: Jayandharan
authors.affiliation: Division of Cellular and Molecular Therapy, Department of Pediatrics, University of Florida College of Medicine, Gainesville, Florida, USA Powell Gene Therapy Center, University of Florida College of Medicine, Gainesville, Florida, USA.
authors.forename: George V
authors.initials: GV
authors.lastname: Aslanidi
authors.affiliation: Division of Cellular and Molecular Therapy, Department of Pediatrics, University of Florida College of Medicine, Gainesville, Florida, USA Powell Gene Therapy Center, University of Florida College of Medicine, Gainesville, Florida, USA.
authors.forename: Baozheng
authors.initials: B
authors.lastname: Li
authors.affiliation: Division of Cellular and Molecular Therapy, Department of Pediatrics, University of Florida College of Medicine, Gainesville, Florida, USA Department of Traditional Chinese Medicine, Changhai Hospital, Second Military Medical University, Shanghai, China.
authors.forename: Binbin
authors.initials: B
authors.lastname: Cheng
authors.affiliation: Division of Cellular and Molecular Therapy, Department of Pediatrics, University of Florida College of Medicine, Gainesville, Florida, USA Powell Gene Therapy Center, University of Florida College of Medicine, Gainesville, Florida, USA.
authors.forename: Wenqin
authors.initials: W
authors.lastname: Ma
authors.affiliation: Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
authors.forename: Thomas
authors.initials: T
authors.lastname: Lentz
authors.affiliation: Department of Traditional Chinese Medicine, Changhai Hospital, Second Military Medical University, Shanghai, China Shanghai University of Traditional Chinese Medicine, Shanghai, China.
authors.forename: Changquan
authors.initials: C
authors.lastname: Ling
authors.affiliation: Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA Division of Molecular Pharmaceutics, University of North Carolina School of Pharmacy, Chapel Hill, North Carolina, USA.
authors.forename: Xiao
authors.initials: X
authors.lastname: Xiao
authors.affiliation: Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
authors.forename: R Jude
authors.initials: RJ
authors.lastname: Samulski
authors.affiliation: Powell Gene Therapy Center, University of Florida College of Medicine, Gainesville, Florida, USA Department of Molecular Genetics & Microbiology, University of Florida College of Medicine, Gainesville, Florida, USA Genetics Institute, University of Florida College of Medicine, Gainesville, Florida, USA.
authors.forename: Nicholas
authors.initials: N
authors.lastname: Muzyczka
authors.affiliation: Division of Cellular and Molecular Therapy, Department of Pediatrics, University of Florida College of Medicine, Gainesville, Florida, USA Powell Gene Therapy Center, University of Florida College of Medicine, Gainesville, Florida, USA Department of Molecular Genetics & Microbiology, University of Florida College of Medicine, Gainesville, Florida, USA Genetics Institute, University of Florida College of Medicine, Gainesville, Florida, USA Shands Cancer Center, University of Florida College of Medicine, Gainesville, Florida, USA lingchen@peds.ufl.edu aruns@peds.ufl.edu.
authors.forename: Arun
authors.initials: A
authors.lastname: Srivastava
mesh_terms: D000818:Animals; D002460:Cell Line; D000229:Dependovirus / Q000235:genetics* / Q000502:physiology; D015870:Gene Expression; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors; D022781:Hepatocytes / Q000821:virology*; D006801:Humans; D008297:Male; D008810:Mice, Inbred C57BL; D017384:Sequence Deletion; D019076:Transgenes; D019065:Virus Assembly