Our current study demonstrated that both AAVrh8 and AAVrh10 vectors can strongly transduce cells in the INL. Other more widely used AAV serotypes, such as AAV2 vectors have shown the most promise for transducing cells in the GCL and ONL(Allocca et al., 2006
; Martin et al., 2002
; Surace and Auricchio, 2008
), while serotypes such as AAV8 (AAV vectors with an AAV2 genome and AAV8 capsid) have shown some ability to transduce cells in the inner retina (Lebherz et al., 2008
). AAV2 vectors have previously shown the most promise for transducing cells in the GCL and ONL.AAVrh8 appeared to transduce amacrine cells particularly well, based on the location and morphology of the labeled cells. However, AAV2 has also been shown to efficiently transduce amacrine cells (Lebherz et al., 2008
). Both AAVrh8 and AAVrh10 were able to transduce presumptive bipolar cells, also based on their location and morphology. However, we did not find any co-labeling with PKCa-IR, a rod bipolar cell marker, and therefore they were probably cone bipolar cells.
Perhaps the most interesting finding was that both AAVrh8 and AAVrh10 robustly transduced horizontal cells, and particularly with AAVrh10 the transduction appeared to span the entire OPL. Recent work by Dalkara et al, (2009)
suggests that the inability of many AAV vectors to effectively transduce the inner retina from an intravitreal injection is due to the barrier created by the inner limiting membrane (ILM). However, our results suggest that at least the AAVrh8 and AAVrh10 serotypes can efficiently transduce beyond the GCL without any obvious barriers. Quantitative analysis of the intensity of EGFP suggested that AAVrh10 transduced horizontal cells nearly 3.5 fold better than AAVrh8. This suggests an interesting opportunity to selectively target horizontal cells. Since both serotypes did not exclusively transduce horizontal cells, additional specificity may be obtained by using horizontal cell-specific promoters, such as connexin 57 (Janssen-Bienhold et al., 2009
). However it should be noted that EGFP expression beyond horizontal cells was inconsistent depending on the retinal region observed. Therefore, use of these viral vectors for other neuronal cell types may be useful to study local connections and environments, but may not be useful to transduce the retina as a whole beyond the horizontal cells.
The receptors for AAVrh8 and AAVrh10 are not yet known, but since both have similar cellular tropism, they may share some cell-surface receptor preference. Indeed, there are many other factors that affect AAV transduction, such as intracellular trafficking to the nucleus, decapsidation, and conversion of the single stranded DNA genome to double stranded DNA (Ding et al., 2005
). Since both vectors used the same promoter and an AAV2 backbone, the slight differences in cellular tropism and the large difference in horizontal cell transduction may reflect properties conferred by their respective capsids. For instance, the differences in the capsid proteins might allow multiple viruses to tranduce individual cells because there could be a cellular difference in the density of cell surface receptors for different AAV capsids. It is also possible that differences in the viral ssDNA sequences could allow for faster conversion to dsDNA and elevated transgene expression.
Our study focused only on the transduction of the mouse retina over the course of 4 weeks, which was determined as a reasonable incubation time based on our initial pilot study. Previous studies have shown recombinant AAV vectors, such as AAV5 reaches its peak transduction at 5 weeks, however AAV2 may take up to 15 weeks (Auricchio et al., 2001
; Yang et al., 2002
). A more recent study in retina shows that AAV1, 4, 5, 7, 8 and 9 all reach their peak transduction efficiency by 2 weeks and AAV2 by 3 weeks (Lebherz et al., 2008
). Considering that AAV mediated transgene expression remains stable for years in rodent and primate retina (Lebherz et al., 2005
; Riviere et al., 2006
), we decided to conduct our experiments at 4 weeks post-injection, after most AAV serotypes would have reached their peak transduction in retina (Lebherz et al., 2008
). This was also consistent with our pilot study, which showed a nearly identical transduction pattern from 3–9 weeks post-injection. We also felt that 4 weeks reflected a reasonable time point for short-term neurochemical studies involving such techniques as gene knockdown by RNAi. However, we cannot discount the possibility that transduction efficiency may have increased over a longer incubation and future experiments should assess AAVrh8 and AAVrh10 over prolonged periods of time.
The increase in GFAP-IR levels in transduced areas of AAVrh8 and AAVrh10 was disconcerting but not totally disappointing. Some areas of the same transduced retinas did not show any evidence of GFAP elevation or stress, even if there were some EGFP positive cells, indicating that it was a localized phenomenon. This could have been due to mechanical damage from the injection, however we saw no evidence of retinal damage and the increased GFAP expression was apparent throughout large transduced areas of the retina that were not associated with the injection site. Further neurochemical tests could be done to determine if the retinal areas with elevated GFAP expression were functioning normally. It would also be useful in future studies to look for microglial activation in response to the viral transduction by using immunocytochemistry to look for evidence of microglial activation using markers such as CD11b or CD45(Santos et al., 2010
). Additional tests should also be done to determine if the high viral titer or the EGFP itself caused the stress response. However, EGFP has been used extensively in retina with AAV and it was not previously found to be toxic (Lebherz et al., 2008
; Petrs-Silva et al., 2009
; Rex et al., 2005
We used relatively high viral titers (1 × 1013
gc/ml) and this could have contributed to the elevated GFAP expression. Indeed, our initial pilot study used ~1 × 10 11
gc/μl and found a similar transduction pattern. We could also potentially use lower titers and obtain the same transduction pattern by developing self-complementary genomes. Wild-type AAV contains a single-stranded DNA genome which must be converted to double-stranded DNA in the host nucleus before its genes can be expressed. This is a rate limiting step for AAV transduction and can be overcome by using self complementary AAV vectors (McCarty, 2008
). Infusion of these scAAV vectors in mouse retina yields a higher number of transduced cells and faster transgene expression kinetics at considerably lower vector doses (Natkunarajah et al., 2008
; Yang et al., 2002
). Also in a recent study, Petrs-Silva et al. (2009)
demonstrated that scAAV vectors produced with AAV capsids carrying point mutations of different surface tyrosines (Y-F mutants), which were previously shown to be involved in the ubiquitination of AAV2 and subsequent targeting to proteosome degradation (Zhong et al., 2008
), were exceptionally efficient in mouse retina at considerably lower doses than we used in the present study. The combination of scAAV vectors with Y-F mutants of AAVrh8 and AAVrh10 capsids (both carry highly homologous tyrosine residues) may yield new vectors with exceptional potency and broad applicability to probe the molecular biology of retina.
The results of this study showed that AAVrh8 and AAVrh10 are promising new candidates to better study the amacrine, horizontal and bipolar cells, all of which have been largely overlooked in previous studies relying on viral gene delivery to retina. While future work is needed to further optimize the use of these AAV vectors, their ability to transduce inner retina will undoubtedly make AAVrh8 and AAVrh10 promising candidates for gene delivery to the retina.