Novel Properties of Tyrosine-mutant AAV2 Vectors in the Mouse Retina

Department of Ophthalmology, University of Florida, Gainesville, FL, USA.
Molecular Therapy (Impact Factor: 6.23). 11/2010; 19(2):293-301. DOI: 10.1038/mt.2010.234
Source: PubMed


Vectors based on adeno-associated virus serotype 2 (AAV2) have been used extensively in many gene-delivery applications, including several successful clinical trials for one type of Leber congenital amaurosis in the retina. Many studies have focused on improving AAV2 transduction efficiency and cellular specificity by genetically engineering its capsid. We have previously shown that vectors-containing single-point mutations of capsid surface tyrosines in serotypes AAV2, AAV8, and AAV9 displayed significantly increased transduction efficiency in the retina compared with their wild-type counterparts. In the present study, we evaluated the transduction characteristics of AAV2 vectors containing combinations of multiple tyrosine to phenylalanine mutations in seven highly conserved surface-exposed capsid tyrosine residues following subretinal or intravitreal delivery in adult mice. The multiply mutated vectors exhibited different in vivo transduction properties, with some having a unique ability of transgene expression in all retinal layers. Such novel vectors may be useful in developing valuable new therapeutic strategies for the treatment of many genetic diseases.

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    • "Recent efforts to develop viruses that can penetrate to the outer retina when injected into the vitreous have shown promising results (Petrs-Silva et al. 2009; Dalkara et al. 2013). AAV viruses with tyrosine mutations exposed at the surface allow the virus to escape degradation by cellular pathways, and in rodents these viruses penetrated and transduced photoreceptors when injected into the vitreous (Petrs-Silva et al. 2009; Petrs-Silva et al. 2011). However, in our preliminary experiments there was limited improvement in the extent or pattern of cells in the primate retina (macaque and baboon) transduced by tyrosine-mutated AAV2 as opposed to wild-type AAV2, or for tyrosine-mutated AAV5 compared with wild-type AAV5 (ARVO abstract) (Di et al. 1987). "
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    ABSTRACT: It has been possible to use viral-mediated gene therapy to transform dichromatic (red-green color-blind) primates to trichromatic. Even though the third cone type was added after the end of developmental critical periods, treated animals acquired red-green color vision. What happened in the treated animals may represent a recapitulation of the evolution of trichromacy, which seems to have evolved with the acquisition of a third cone type without the need for subsequent modification to the circuitry. Some transgenic mice in which a third cone type was added also acquired trichromacy. However, compared with treated primates, red-green color vision in mice is poor, indicating large differences between mice and monkeys in their ability to take advantage of the new input. These results have implications for understanding the limits and opportunities for using gene therapy to treat vision disorders caused by defects in cone function.
    Cold Spring Harbor Perspectives in Medicine 08/2014; 4(11). DOI:10.1101/cshperspect.a017418 · 9.47 Impact Factor
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    • "AAV vectors with multiple tyrosine-to-phenylalanine modifications infect many cell types following intravitreal or subretinal injection (Petrs-Silva et al. 2011). Photoreceptor specific expression of transgenes can be achieved using several different promoters, including that of rhodopsin kinase and interphotoreceptor retinoid binding protein. "
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    ABSTRACT: Mutations in the gene for rhodopsin, RHO, cause autosomal dominant retinitis pigmentosa, a disease characterized by death of rod photoreceptor cells. At the end stage, when most rods are gone, cones die too, taking central vision with them. One goal of gene therapy, therefore, is to preserve central vision by promoting rod survival in the vicinity of the macula. Dominance in RHO mutations is associated with two phenomena: interference with the function of normal rhodopsin and intrinsic toxicity of the mutant protein. In the case of interference, increased production of the wild-type protein may be therapeutic, but in the case of toxicity, suppression of the mutant protein may also be needed. RHO augmentation has made use of advances in gene delivery to the retina using adeno-associated virus (AAV). Several strategies have been developed for suppression of rhodopsin expression, but because of the heterogeneity of RHO mutations they are not specific for the mutant allele: They suppress both mutant and wild-type RHO. Experiments in autosomal dominant retinitis pigmentosa (adRP) mouse models suggest that both RHO augmentation and supplementation plus suppression preserve the survival of rod cells.
    Cold Spring Harbor Perspectives in Medicine 07/2014; 4(9). DOI:10.1101/cshperspect.a017400 · 9.47 Impact Factor
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    • "All vectors intended for in vitro analyses were separately packaged in AAV2 or AAV2(tripleY-F) capsid containing three phenylalanine-to-tyrosine substitutions (Y444F, Y500F, Y730F) (Petrs-Silva et al., 2011). AAV2-based vectors were chosen for in vitro experiments based on their increased transduction efficiency relative to other serotypes (Ryals et al., 2011). "
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