Transfer of contaminants in adeno-associated virus vector stocks can mimic transduction and lead to artifactual results.
The potential of adeno-associated virus (AAV) vectors for gene transfer and gene therapy applications is currently being intensively investigated. Although much progress has been made in defining AAV vector biology, inconsistencies remain in the literature regarding the efficiency of AAV transduction in various cell types. In the course of exploring these differences, we have identified a problem associated with the use of AAV vector stocks that results in overestimation of gene transfer efficiencies. We show here that biologically active vector-encoded proteins can contaminate AAV vector stocks, especially cell lysate preparations that have not been further purified, and can be transferred in a virion-independent manner to target cells, a phenomenon called pseudotransduction. This observation is significant because impure cell lysate stocks have been widely employed in the AAV literature, and we demonstrate here that this phenomenon can occur with commonly used reporter proteins such as beta-galactosidase and alkaline phosphatase. We conclude that although there are many potential explanations for apparently conflicting results in the literature, the possibility of pseudotransduction must be considered, especially when cell lysate stocks of AAV vectors have been employed. This artifact can be avoided by further vector purification.
Alexander
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IE
Gene Therapy Research Unit, New Children's Hospital and Children's Medical Research Institute, Parramatta, NSW, Australia.
Russell
D W
DW
Miller
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AD
D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism; D002455:Cell Division; D002457:Cell Extracts / Q000737:chemistry; D002478:Cells, Cultured; D000229:Dependovirus / Q000235:genetics* / Q000302:isolation & purification; D005347:Fibroblasts; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005819:Genetic Markers / Q000235:genetics; D005822:Genetic Vectors / Q000302:isolation & purification; D006412:Hematopoietic Stem Cells / Q000378:metabolism; D006651:Histocytochemistry; D006801:Humans; D010920:Placenta / Q000201:enzymology; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection / Q000235:genetics; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D002457:Cell Extracts; D005819:Genetic Markers; D011506:Proteins; D000469:Alkaline Phosphatase; D001616:beta-Galactosidase