AAV2-Mediated Transduction of the Mouse Retina After Optic Nerve Injury

Abstract

PurposeGene therapy of retinal ganglion cells (RGCs) has promise as a powerful therapeutic for the rescue and regeneration of these cells after optic nerve damage. However, early after damage, RGCs undergo atrophic changes, including gene silencing. It is not known if these changes will deleteriously affect transduction and transgene expression, or if the therapeutic protein can influence reactivation of the endogenous genome.MethodsDouble-transgenic mice carrying a Rosa26-(LoxP)-tdTomato reporter, and a mutant allele for the proapoptotic Bax gene were reared. The Bax mutant blocks apoptosis, but RGCs still exhibit nuclear atrophy and gene silencing. At times ranging from 1 hour to 4 weeks after optic nerve crush (ONC), eyes received an intravitreal injection of AAV2 virus carrying the Cre recombinase. Successful transduction was monitored by expression of the tdTomato reporter. Immunostaining was used to localize tdTomato expression in select cell types.ResultsSuccessful transduction of RGCs was achieved at all time points after ONC using AAV2 expressing Cre from the phosphoglycerate kinase (Pgk) promoter, but not the CMV promoter. ONC promoted an increase in the transduction of cell types in the inner nuclear layer, including Müller cells and rod bipolar neurons. There was minimal evidence of transduction of amacrine cells and astrocytes in the inner retina or optic nerve.ConclusionsDamaged RGCs can be transduced and at least some endogenous genes can be subsequently activated. Optic nerve damage may change retinal architecture to allow greater penetration of an AAV2 virus to transduce several additional cell types in the inner nuclear layer.

Mitochondrial Transfer of the Mutant Human ND6T14484C Gene Causes Visual Loss and Optic Neuropathy

“…These findings were consistent with previous reports that AAV2 efficiently infects RGCs after intravitreal injection of adult rodent eyes. 33 – 35 No hND6 was detected in any retinal layer of mCherry - injected mice. PCR using primers targeting the hND6T14484C construct showed bands of the expected size in the whole retina ( Fig.…”

Progress in Gene Therapy to Prevent Retinal Ganglion Cell Loss in Glaucoma and Leber’s Hereditary Optic Neuropathy

“…Numerous studies support this observation, with both CMV and CAG promoters increasing expression primarily in RGCs, although some amacrine, Müller glial, and bipolar cells are also transduced [ 10 , 14 , 25 ] ( Figure 2 ). Interestingly, a different profile of transduction is observed when the AAV2-CMV vector is injected at birth as opposed to adulthood.…”

“…Promoter choice is also important for cell-specific expression and the strength of transgene expression within the targeted cell. While several RGC-specific promoters exist ( Table 1 ), many vectors designed for optic neuropathies employ a ubiquitous CMV [ 10 – 14 ] or a hybrid CMV early enhancer/chicken b-actin promoter (CAG) [ 15 – 17 ] due to their small size and high levels of transgene expression [ 18 , 19 ]. Promoter size is particularly important when looking to incorporate larger genes into an AAV.…”

Stimulation of Retinal Pigment Epithelium With an α7 nAChR Agonist Leads to Müller Glia Dependent Neurogenesis in the Adult Mammalian Retina

“…In some experiments, conditioned media from PNU-282987– or MLA+PNU-282987–treated RPE cells was collected as described above, and 1 μL of media was injected into the vitreal chamber of mice; 1 μL is the standard volume injected into the vitreal chamber of adult mice, as the total vitreous volume is relatively small 26–28. Other eyes received an injection of control RPE media (untreated), an injection of a saline vehicle, or an injection of RPE media obtained immediately after PNU-282987–treated RPE cells were thoroughly washed.…”