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Kristina Ertel, Chris Reid, Daniel M Lipinski; Intravitreal transduction profile of recombinant adeno-associated virus in murine and human retina. Invest. Ophthalmol. Vis. Sci. 2017;58(8):291.
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© ARVO (1962-2015); The Authors (2016-present)
The ability to target cells of the inner and outer retina via intravitreal injection is vital for treatment of diseases where detachment of the retina may be detrimental. It has previously been demonstrated that the tropism of recombinant adeno-associated virus (rAAV) vectors can be altered through mutation of the virion by site-directed mutagenesis or inclusion of short targeting peptides. Herein, we compare the transduction profiles of three rAAV2-based capsid mutant vectors –rAAV2.7m8, rAAV2.QuadYF-TV and a hybrid vector termed rAAV.MAX– following intravitreal injection in mice. Additionally, we use human retinal tissue to determine the ability of each vector to transduce macular cone photoreceptors – a critical consideration for clinical translation of intravitreal gene delivery.
rAAV vectors packaging a ubiquitously expressing mCherry reporter gene were injected intravitreally into adult NRL-EGFP and WT mice. Retinal fluorescence levels were quantified in vivo using a custom multiline confocal scanning laser ophthalmoscope (m-cSLO) allowing dual color fluorescence imaging. Post-mortem, double positive retinal cells were quantified by flow cytometry to determine the percent of photoreceptors transduced. Human retinal tissue from the Lion’s Eye Bank of Wisconsin was maintained in organotypic culture following administration of capsid mutant vectors packaging a GFP reporter gene. WT mouse eyes and human retinal explants were cryosectioned to histologically evaluate tropism.
rAAV2.MAX exhibited greater retinal penetrance and photoreceptor transduction following intravitreal injection than either unmodified rAAV2 or the original capsid mutant vectors. Dual color m-cSLO imaging facilitated normalization of retinal fluorescence levels in vivo, allowing for quantification of rAAV-mediated transgene expression. rAAV2-MAX vector administration on human macular tissue resulted in substantial transduction of central photoreceptors.
The increased tissue penetrance and photoreceptor transduction properties of the rAAV2.MAX vector demonstrates the effects of capsid mutation and peptide insertion within the AAV capsid are cumulative. rAAV2.MAX was able to mediate gene transfer to both murine and human photoreceptors from the vitreous thus encouraging for the future treatment of retinal diseases where subretinal gene delivery may represent an unacceptable surgical risk.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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