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Native molecular state of AAV vectors revealed by single molecule sequencing.
Hum Gene Ther. 2011 Aug 29;
Authors: Xiao W
Abstract
The single-stranded genome of adeno-associated virus (AAV) vectors is one of the key factors leading to a slow-rising but long-term transgene expression kinetics. Previous molecular studies have established what is now considered as a textbook molecular model of AAV genomes with two copies of inverted-tandem repeats at either end. In this study, We profiled hundreds of thousands of individual molecules of AAV vector DNA directly isolated from the capsids using single-molecule sequencing (SMS) that avoids any intermediary steps such as plasmid cloning. The sequence profile at 3' ends of both the regular and oversized vector did show the presence of an ITR, which provided a direct confirmation that AAV vector packaging initiates from its 3' end. Furthermore, the vector 5' termini profile showed inconsistent termination for oversized vectors. Such incomplete vectors would not be expected to undergo the canonical synthesis of the second strand of their genomic DNA and thus could function only via annealing of complementary strands of DNA. Furthermore, low levels of contaminating plasmid DNA were also detected. SMS may become a valuable tool during the development phase of vectors that are candidates for clinical use and for facilitating/accelerating studies on vector biology.
PMID: 21875357 [PubMed - as supplied by publisher]
More...
Native molecular state of AAV vectors revealed by single molecule sequencing.
Hum Gene Ther. 2011 Aug 29;
Authors: Xiao W
Abstract
The single-stranded genome of adeno-associated virus (AAV) vectors is one of the key factors leading to a slow-rising but long-term transgene expression kinetics. Previous molecular studies have established what is now considered as a textbook molecular model of AAV genomes with two copies of inverted-tandem repeats at either end. In this study, We profiled hundreds of thousands of individual molecules of AAV vector DNA directly isolated from the capsids using single-molecule sequencing (SMS) that avoids any intermediary steps such as plasmid cloning. The sequence profile at 3' ends of both the regular and oversized vector did show the presence of an ITR, which provided a direct confirmation that AAV vector packaging initiates from its 3' end. Furthermore, the vector 5' termini profile showed inconsistent termination for oversized vectors. Such incomplete vectors would not be expected to undergo the canonical synthesis of the second strand of their genomic DNA and thus could function only via annealing of complementary strands of DNA. Furthermore, low levels of contaminating plasmid DNA were also detected. SMS may become a valuable tool during the development phase of vectors that are candidates for clinical use and for facilitating/accelerating studies on vector biology.
PMID: 21875357 [PubMed - as supplied by publisher]
More...