Abstract
Purpose: :
Adeno-associated virus (AAV) vectors are promising vehicles for retinal gene transfer. AAV efficiently transduces dividing and non-dividing cells, exhibits low immunogenicity, and mediates long-term transduction in several retinal cell types. AAV serotypes (AAV 1-9) exhibit varying abilities to infect specific cell types (tropism) in the retina. The interaction of AAV with cellular receptors enables entry of the virus into the cell. Manipulation of the virus-receptor interaction offers potential for engineering targeted delivery to specific classes of retinal neurons and glia. Our initial goal is to understand the tropism profiles of the nine known AAV serotypes by studying their localization and distribution in the retina following intravitreal injection. This localization can then be correlated with the serotype specific expression patterns to understand the mechanisms of, and trafficking barriers to, vector transduction in Muller glia.
Methods: :
AAV serotypes 1, 2, 5 and 9 were produced via the triple transfection method. Purified, high titer AAV (1012-1013 vg/ml) was covalently labeled with the fluorescent dye Cy3 and injected intravitreally (5µl) to wt rats at p30. The localization patterns of the labeled AAV serotypes were elucidated using confocal microscopy after cryosectioning. In parallel, GFP expression was analyzed.
Results: :
AAV serotype 2 particles localized predominantly in the ganglion cell layer, with a corresponding gene expression pattern in RGC cell bodies, axons and dendrites after intravitreal injection. All other serotypes exhibited similar particle distribution, with the majority of the labeled particles observed in the ganglion cell layer and limited distribution into the INL and ONL. The virus particle distribution is mirrored by their pattern of inefficient transduction of neurons and glia in the proximal retinal layers following intravitreal injection. This transduction pattern is likely related not only to receptor mediated viral tropism, but to intracellular diffusion barriers in the retina.
Conclusions: :
Surface glycoproteins on AAV viral capsids are crucial determinants of AAV expression, efficiency and tropism. Florescent labeling of AAV capsids aids in understanding the physical barriers associated with AAV mediated retinal gene delivery and to track the progressive steps of vector distribution, surface recognition and transduction at the cellular and molecular levels. These data on different AAV serotypes thru labeled capsid tracking can aid in the rational design of novel AAV capsids giving rise to variants targeted to specific cell types.
Keywords: gene transfer/gene therapy • Muller cells • proteoglycans/glycosaminoglycans