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R. R. Klimczak, D. Dalkara, J. Koerber, K. Kolstad, D. V. Schaffer, J. G. Flannery; Molecular Engineering of Adeno-Associated Virus Yields a Novel Variant With Efficient Intravitreal Transduction of Müller Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(13):3012.
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© ARVO (1962-2015); The Authors (2016-present)
Gene therapy has great potential for treating and possibly curing a variety of retinal diseases. Neuroprotective strategies that, through gene delivery, provide sustained production of a therapeutic protein or an RNA product present a promising and broad approach towards the treatment of these disorders. For retinal gene therapy, the transduction of Müller glial cells from the vitreous would be advantageous in secreting neuroprotective factors to mediate independent protection of the entire retina through a simple and minimally invasive approach. Unfortunately, naturally occurring AAV serotypes are inefficient in intravitreally (IV) transducing Müller cells as these vectors selectively infect neurons in the retina. Our goal is to create, using a directed evolutionary approach, AAV variants to effectively transduce Müller cells in vivo through IV injection and further explore their therapeutic potential in animal disease models.
The AAV cap gene from serotypes 1-9 were subjected to error-prone PCR and DNA shuffling and subsequently repackaged to create a diverse library of variants. Variants were then selected against glial cells in vitro through multiple iterations and subsequently subjected to a further round of error-prone PCR to create a successive generation of permissive variants for further selection. After multiple generations, variants from the resulting pool were cloned into recombinant form and packaged with a GFP transgene to assay Muller-infectivity. AAV tropism of novel variants was assessed by fluorescence microscopy.
An AAV variant derived from AAV6 created through directed evolution is dramatically more specific and efficient at infecting Müller cells in the rat through IV injection throughout the retina relative to AAV 2 as assayed through GFP expression while AAV6 shows no visible expression.
Directed evolution is a powerful approach to manipulate the tropism of AAV vectors in the retina. This isolated variant provides a new therapeutic tool to treat retinal degenerative diseases as well as providing new approaches to studying Müller cell physiology.
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