Self-complementary recombinant adeno-associated viral vectors: packaging capacity and the role of rep proteins in vector purity.
Self-complementary adeno-associated viral (scAAV) vectors bypass the requirement for viral second-strand DNA synthesis, but the packaging capacity of these vectors ( approximately 2.4 kb) is significantly smaller than that of conventional AAV vectors ( approximately 4.8 kb). We constructed human recombinant green fluorescent protein (hrGFP) expression cassettes ranging from 2.3 to 4.1 kb. Each vector was biologically active, but the transduction efficiency of vectors containing <3.3-kb genomes was significantly higher than those containing 3.5-kb genomes or larger. However, scAAV vectors containing up to approximately 3.3-kb genomes also contained single-stranded genomes, and 3.5-kb and larger genomes were packaged only as single-stranded DNA. These data suggest that the maximum packaging capacity of scAAV vectors is approximately 3.3 kb. The production of single-stranded genomes was not due to repair of the terminal resolution site (trs) in the inverted terminal repeats in the AAV genome, but rather was partly due to the use of AAV helper plasmid, known to lead to higher levels of expression of Rep proteins. The use of a helper plasmid known to lead to reduced levels of Rep proteins led to the generation of scAAV vectors that contained approximately 90% of the viral genomes in double-stranded forms. These studies demonstrate the feasibility of achieving encapsidation of larger genomes into scAAV vectors than was suggested originally, but underscore the need to exercise caution in using the appropriate helper plasmid to generate scAAV stocks capable of high-efficiency transduction that are relatively free of single-stranded DNA-containing vectors.
Wu
Jianqing
J
Division of Cellular & Molecular Therapy, Department of Pediatrics, University of Florida College of Medicine, Gainesville, FL 32610, USA.
Zhao
Weihong
W
Zhong
Li
L
Han
Zongchao
Z
Li
Baozheng
B
Ma
Wenqin
W
Weigel-Kelley
Kirsten A
KA
Warrington
Kenneth H
KH
Srivastava
Arun
A
D018076:DNA, Complementary / Q000235:genetics; D004274:DNA, Recombinant / Q000235:genetics*; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D000229:Dependovirus / Q000235:genetics* / Q000502:physiology*; D005822:Genetic Vectors / Q000235:genetics / Q000302:isolation & purification*; D016679:Genome, Viral; D006367:HeLa Cells; D006801:Humans; D017384:Sequence Deletion; D014161:Transduction, Genetic; D014764:Viral Proteins / Q000378:metabolism*; D019065:Virus Assembly / Q000502:physiology*; D014779:Virus Replication
D018076:DNA, Complementary; D004274:DNA, Recombinant; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D014764:Viral Proteins; C068734:rep proteins, Adeno-associated virus 2