title: Targeting photoreceptors via intravitreal delivery using novel, capsid-mutated AAV vectors.
abstract: Development of viral vectors capable of transducing photoreceptors by less invasive methods than subretinal injection would provide a major advancement in retinal gene therapy. We sought to develop novel AAV vectors optimized for photoreceptor transduction following intravitreal delivery and to develop methodology for quantifying this transduction in vivo. Surface exposed tyrosine (Y) and threonine (T) residues on the capsids of AAV2, AAV5 and AAV8 were changed to phenylalanine (F) and valine (V), respectively. Transduction efficiencies of self-complimentary, capsid-mutant and unmodified AAV vectors containing the smCBA promoter and mCherry cDNA were initially scored in vitro using a cone photoreceptor cell line. Capsid mutants exhibiting the highest transduction efficiencies relative to unmodified vectors were then injected intravitreally into transgenic mice constitutively expressing a Rhodopsin-GFP fusion protein in rod photoreceptors (Rho-GFP mice). Photoreceptor transduction was quantified by fluorescent activated cell sorting (FACS) by counting cells positive for both GFP and mCherry. To explore the utility of the capsid mutants, standard, (non-self-complementary) AAV vectors containing the human rhodopsin kinase promoter (hGRK1) were made. Vectors were intravitreally injected in wildtype mice to assess whether efficient expression exclusive to photoreceptors was achievable. To restrict off-target expression in cells of the inner and middle retina, subsequent vectors incorporated multiple target sequences for miR181, an miRNA endogenously expressed in the inner and middle retina. Results showed that AAV2 containing four Y to F mutations combined with a single T to V mutation (quadY-F+T-V) transduced photoreceptors most efficiently. Robust photoreceptor expression was mediated by AAV2(quadY-F+T-V) -hGRK1-GFP. Observed off-target expression was reduced by incorporating target sequence for a miRNA highly expressed in inner/middle retina, miR181c. Thus we have identified a novel AAV vector capable of transducing photoreceptors following intravitreal delivery to mouse. Furthermore, we describe a robust methodology for quantifying photoreceptor transduction from intravitreally delivered AAV vectors.
authors: Department of Ophthalmology, University of Florida College of Medicine, Gainesville, Florida, United States of America.
authors: Christine N
authors: CN
authors: Kay
authors: Renee C
authors: RC
authors: Ryals
authors: George V
authors: GV
authors: Aslanidi
authors: Seok Hong
authors: SH
authors: Min
authors: Qing
authors: Q
authors: Ruan
authors: Jingfen
authors: J
authors: Sun
authors: Frank M
authors: FM
authors: Dyka
authors: Daniel
authors: D
authors: Kasuga
authors: Andrea E
authors: AE
authors: Ayala
authors: Kim
authors: K
authors: Van Vliet
authors: Mavis
authors: M
authors: Agbandje-McKenna
authors: William W
authors: WW
authors: Hauswirth
authors: Sanford L
authors: SL
authors: Boye
authors: Shannon E
authors: SE
authors: Boye
mesh_terms: D000818:Animals; D002213:Capsid / Q000378:metabolism*; D002460:Cell Line; D002645:Chickens; D000229:Dependovirus / Q000235:genetics* / Q000502:physiology; D051606:G-Protein-Coupled Receptor Kinase 1 / Q000235:genetics; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000008:administration & dosage*; D006801:Humans; D058449:Intravitreal Injections; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D035683:MicroRNAs / Q000378:metabolism; D009154:Mutation / Q000235:genetics*; D020419:Photoreceptor Cells, Vertebrate / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D012703:Serotyping; D014161:Transduction, Genetic; D019076:Transgenes / Q000235:genetics; D056189:Viral Tropism
chemical_list: D035683:MicroRNAs; D051606:G-Protein-Coupled Receptor Kinase 1; C496764:GRK1 protein, human
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