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Tissue Tropisms of AAV Vectors Deficient in Receptor Binding

Please use this identifier to cite or link to this item: http://hdl.handle.net/1811/440

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Title: Tissue Tropisms of AAV Vectors Deficient in Receptor Binding
Creators: Crumrine, Jennette K.
Advisor: Bartlett, Jeffery S.; Waller, Kathy
Issue Date: 2005-06
Abstract: Adeno-associated virus (AAV) is a single stranded DNA virus with an icosahedral 20-25 nm capsid comprised of three structural proteins, VP1, VP2, and VP3. AAV is of great interest as a gene therapy vector for the treatment of cancer and genetic diseases due to its ability to transduce both mitotic and postmitotic tissues, be produced at high titers, and mediate long-term gene expression without pathogenicity. However, one concern is that the AAV2 receptor, heparan sulfate proteoglycan (HSPG), is expressed on a wide variety of human tissues. This can cause unwanted gene transfer and expression when only a specific cell type needs to be transduced. Changing arginine amino acids (a.a.) at positions 585 and 588, to alanines (R585A,R588A) has been shown to effectively eliminate AAV binding to HSPG, while the insertion of small peptide epitopes (<20 aa) that bind alternative cellular receptors has been shown to effectively retarget AAV vectors to these new receptors. The amino acid sequence of the major capsid protein VP3 is contained within the two larger and less abundant capsid proteins VP1 and VP2, thus mutations within VP3 are also present in VP1 and VP2. PCR-based site directed mutagenesis of pACG2, which expresses all of the AAV capsid proteins, was used to change the arginines at positions 585 and 588 within the VP1, VP2, and VP3 proteins to alanines. Similarly, these arginine residues were also changed to alanines in a second plasmid that expresses only VP1 and VP3 proteins. Different peptides that target the vasculature endothelial growth factor receptor 2(VEGFR2) were then incorporated into the R585A, R588A AAV2 capsid constructs. The ability of wild-type AAV2 vectors, R585A, R588A AAV2 vectors, VEGFR2-targeted AAV2 vectors, and an AAV2 vector containing the R585A, R588A mutation on only the VP1 and VP3 proteins to bind to polystyrene beads coated with heparin and to HSPG-expressing Hela C12 cells were assessed. Our preliminary data suggest that AAV2 vectors comprised of capsids with R585A, R588A mutations were unable to bind to HSPG. The wild-type AAV2 was the only virion that successfully bound to the beads and Hela cells. An affinity assay was performed showing that with a competitor in 100-fold excess, AAV2 did not bind as well to the Hela C12 cells similar to the heparan mutants. These preliminary findings suggest that AAV2 does naturally bind to heparan, and that the rAAV2 mutants were deficient in receptor binding. This can lead to successful elimination of unwanted gene transfer and expression and the retargeting of the rAAV2 vector to tissues that were once thought untargetable.
Series/Report no.: The Ohio State University. Medical Technology Division Honors Theses; 2005
Keywords: AAV
HSPG
VEGF
specific binding
non-specific binding
URI: http://hdl.handle.net/1811/440
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