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H. Petrs-Silva, S. H. Min, L. Zhong, S. Zolotukhin, A. Srivastava, A. Lewin, W. W. Hauswirth; Improving Retinal Ganglion Cell Transduction by Using scAAV Type 8 or Type 9 Vectors Containing Capsid Mutations. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5350. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
The transduction efficiency of AAV vectors varies greatly in different cells and tissues in vitro and in vivo. Administration of an AAV serotype 2 vector (AAV2) results in the transduction of diverse cell types in the murine retina whereas other serotypes have more restricted retinal cell tropisms. However, no other serotype tested so far is as effective at transducing retinal ganglion cells (RGCs) as AAV2 injected in the vitreous. Surface exposed capsid amino acid residues are key elements in AAV-mediated transduction since they define not only cell tropism, perhaps also the onset and intensity of passenger gene expression.One reason is that modification of the AAV capsid surface upon viral entry appears to mediate cytoplasmic degradation of AAV vectors. In an attempt to modulate AAV tropism, tyrosine (Y) residues mapping to the capsid surface were modified to phenylalanine (F) to alter vector transduction characteristics. Our goal was to evaluate RGC transduction properties of wild type (WT) self complementary adeno-associated virus vector (scAAV) serotypes 8 and 9 in parallel with single Y-F capsid mutants of each.
Intravitreal injections of scAAV type 2, 8 and 9 WT and capsid mutant vectors with identical small CBA promoters expressing enhanced green fluorescent protein (EGFP) were performed in adult mice. EGFP expression was evaluated in retinal whole mounts and in retinal sections by GFP immunohistochemistry 2 weeks after vector treatment.
scAAV8 and scAAV9 vectors transduce retinal ganglion cells poorly compared with scAAV2 vectors. In contrast, scAAV8-Y733F, scAAV9-Y436F and Y731F mediated greatly enhanced RGC transduction compared with their parental wild type vectors. The level of improvement for each different mutant vector relative to its wild type parent was similar. Comparison of these mutants to wild type and Y-F mutant scAAV2 vectors is currently being evaluated.
scAAV8 and scAAV9 vectors do not transduce retinal ganglion cells efficiently compared with scAAV2 vectors following intravitreal delivery. However Y-F capsid mutants at position 733 for type 8, and 436 or 731 for type 9 promoted a significant increase of EGFP expression in the ganglion cell layer. These results suggest that RGC transduction can be enhanced by specific capsid surface modifications, and this concept opens new possibilities for AAV variants that may improve ganglion cell transduction.
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