Rod Outer Segment Development Influences AAV-Mediated Photoreceptor Transduction After Subretinal Injection

Plasmid construction, AAV vector production, and purification

Recombinant AAV2/5-CMV-GFPd, AAV2/5-CMV-H2bGFP, AAV2/5-CMV-Cre, AAV2/5-pQCMV-H2bGFP, and AAV2/5-mCAR-H2bGFP were produced using the pAAV2-CMV-GFPd, pAAV2-CMV-H2bGFP, pAAV2-CMV-Cre, pAAV2-pQCMV-H2bGFP, or pAAV2-mCAR-H2bGFP plasmids, respectively. The pAAV2-CMV-GFPd plasmid expresses the destabilized GFP cDNA under the control of a human CMV enhancer/promoter, a human β-globin intron and an SV40 polyA signal, flanked by two AAV2 inverted terminal repeat sequences. The pAAV2-CMV-H2bGFP plasmid expresses a histone 2B fused GFP (nuclear GFP). It was constructed by replacing the GFPd cDNA with the H2bGFP sequence derived from the parental pQCMV-H2bGFP plasmid. The pAAV2-CMV-Cre plasmid expresses a Cre recombinase. It was constructed by replacing the GFPd cDNA with the Cre sequence derived from the parental pQCMV-H2bGFP-I-Cre plasmid. The pAAV2-mCAR-H2bGFP plasmid was made by replacing the CMV promoter with the mouse cone arrestin (mCAR) promoter from the parental pQCMV-H2bGFP plasmid. ³⁵

scAAV1, -2, -3b, -4, -5, -6, -7, -8, -9, rh8, rh10, rh39, and rh43 expressing enhanced GFP (eGFP) under the control of the CB6 promoter were produced using the pAAVsc-CB6-eGFP plasmid, which bears a CB6 promoter, a rabbit globin polyA, and engineered ITRs for scAAV vector. The CB6 sequence was previously described ³⁶ and includes a CMV enhancer/beta-actin (CB) promoter with a CMV IE enhancer.

AAV2/5-CMV-GFPd, AAV2/5-CMV-H2bGFP, AAV2/5-CMV-Cre, AAV2/5-pQCMV-H2bGFP, and AAV2/5-mCAR-H2bGFP were produced in the Punzo laboratory by triple transfection of 293 cells according to previously reported methods ³⁷ and purified by two rounds of CsCl₂ ultracentrifugation. scAAV vector production was carried out by the Vector Core of the Horae Gene Therapy Center of UMASS Medical School (Worcester, MA). ³⁸ Viral vector titers were determined simultaneously for all batches by quantitative real-time PCR (qPCR) with primers directed toward SV40pA (forward: 5′-CGAGTGCTTTATTTGTGAAATTTG-3′; reverse: 5′-GGGGTTCCTTGTAGTTAATGA-3′) or eGFP (forward: 5′-AGCAAAGACCCCAACGAGAA-3′; reverse: 5′-GGCGGCGGTCACGAA-3′) and expressed as vector genome per milliliter (vg/mL). The final vector titers were between 8.1 × 10¹² and 5.1 × 10¹³ vg/mL (Table 1).

Animals

The CD1, Ai9 Cre reporter mice, and Pde6β^rd1/rd1 mice were purchased from the Jackson Laboratory. The M-opsin-Cre mice ³⁹ (cone-specific Cre line) and the rhodopsin knockout (Rho ^−/−) mice ⁴⁰ were provided by Yun Z. Le (University of Oklahoma Health Sciences Center) and Janis Lem (Tufts University, Boston), respectively. Ai9^+/−_MCre⁺ mice were generated by crossing MCre⁺ mice with the Ai9 Cre reporter mice. Pde6β^rd1/+_Ai9^+/− mice were generated by crossing rd1 mice and Ai9 mice. Heterozygotes were mated to produce Pde6β^rd1/rd1/+_Ai9^+/− mice.

All animals were maintained at UMASS Medical School under a 12 hour/12 hour light/dark cycle with unrestricted access to food and water. Lighting conditions were kept constant in all cages, with illumination ranging between 10 and 15 lux. All experiments involving mice were conducted in compliance with the Association for Research in Vision and Ophthalmology statement for the use of animals in ophthalmic and vision research. All procedures were approved by Institutional Animal Care and Use Committee of UMASS Medical School.

Subretinal administration of rAAV vectors

The same experimenter (L.P.) performed all subretinal injections of AAV vectors. Subretinal injection of AAV vectors was performed using a trans-scleral approach through the posterior part of the sclera, as previously described, ⁴¹ with the following modifications. Injections were performed using thin-wall beveled glass micropipettes (Clunbury Scientific LLC) without sclerotomy. In mice older than PND1, a small hole was made at the transition of the cornea and sclera with a 33-gauge needle before the injection in order to release intraocular pressure and allow for the formation of a vector bleb upon subretinal injection. This procedure allowed the vector bleb to occupy 40–60% of the retinal surface without injection-related damage. Fast green dye was added to the AAV preparations at a final concentration of 0.1% as a tracer to visualize the location of injection and thus ensure that AAV vectors were injected into the subretinal space. Mice received 0.5–0.75 μL of vectors at PND1, 1–1.5 μL of vectors at PND5–10, and 1.5–2.5 μL of vectors at PND21.

Intravitreal administration of rAAV vectors

Intravitreal injections were performed in PND1 mice, as previously described, ⁴¹ using the same glass micropipettes (Clunbury Scientific LLC) introduced through the cornea-scleral margin. Fast green dye was added to AAV preparations at a final concentration of 0.1% as a tracer to visualize the location of injection and thus ensure that AAV vectors were injected into the intravitreal cavity. Mice received 0.75–1 μL of vectors.

Quantification of cone and rod transduction

In all cases, the analysis was performed 3 weeks post AAV injection. The total number of retinas analyzed in each experimental group is indicated in Table 1. The efficiency of retinal transduction was first assessed under an inverted fluorescent microscope, and only retinas that displayed native GFP fluorescence in >30% of the total retinal surface were processed for further analysis. All images were acquired with the Leica DM5500 fluorescent microscope equipped with a motorized stage for tiling and z-stack image acquisition and deconvolution software for confocal-like image quality.

Quantification of cone and rod transduction in selected retinas was performed using three independent methods. First, for each group of injected mice, a minimum of two retinas were entirely sectioned (20 μm/section) and collected serially on five slides, such that each slide contained a representation of the entire eye. A minimum of two slides were processed for immunohistochemistry using Cy3-labeled peanut agglutinin lectin (PNA; 1:500; Vector Laboratories) and rabbit anti-cone arrestin (1:500; EMD Millipore). Images were taken from 5–10 sections at 40× over the entire transduced area at different focal plans (z stacks) using epifluorescence microscopy (Supplementary Fig. S1; Supplementary Data are available online at www.liebertpub.com/hum). The acquired epifluorescent images were then deconvolved in order to obtain confocal-like resolution by eliminating the out-of-focus signal that could be misinterpreted as colocalization (Supplementary Fig. S1a). Cone quantification was performed in one selected z stack for each image, based on the colocalization of the GFP signal with the cone arrestin marker (usually at the level of cell nuclei). Rod quantification was performed within the same z stack for each image by manually counting the number of total GFP⁺ PRs and the number of GFP⁺ cones per image, and by determining through an automated counting algorithm the total number of PR nuclei in the outer nuclear layer (ONL) of the same image using the Imaris software package (Supplementary Fig. S1b). Rod transduction was then calculated as an average per eye and per group by determining the ratio of the number of total GFP⁺ PRs – number of GFP⁺ cones to the total number of PR nuclei – number of cone cells per image.

Second, for groups of mice injected with AAV-dGFP or AAV-eGFP vectors, cone quantification was also performed on retinal flat mounts stained with the following lectins/antibodies: Cy3-labeled PNA (1:500; Vector Laboratories), rabbit anti-cone arrestin (1:500; EMD Millipore; AB15282), rabbit anti-LM opsin (1:500; EMD Millipore; AB5405), and goat anti-S opsin (1:500; Santa Cruz Biotechnology; SC-14363). For each flat mount, four to six representative 100 μm² areas within the transduced area (center and periphery) were analyzed. The percentage of transduced cones was determined as an average per eye and per group by quantifying the number of GFP⁺ cones over the total number of cones in the same areas. This quantification was performed at the level of cone segments, where GFP fluorescence in cones was unambiguously surrounded by PNA staining (Supplementary Fig. S1c).

Third, AAV5-CMV-H2bGFP and AAV5-pQCMV-H2bGFP vectors were also evaluated in Ai9⁺_MCre mice, which express CRE-mediated td-Tomato (red) in the entire cone cell bodies (Supplementary Fig. S1d).

Cell lines

Two different Chinese hamster ovary (CHO) cell lines were used to confirm the specificity of the lectins used in this study. These include the parental cell line Pro5 and sialic-acid deficient cell line Lec2. Both cell lines were kindly provided by Miguel Sena-Esteves (UMASS Medical School). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and 1% streptomycin and maintained at 37°C with 5% CO₂. Cells were seeded at 10⁵ cells/well in 12-well plates prior to treatment for 1 h at 37°C with DMEM alone or DMEM supplemented with 50 mIU/mL of neuraminidase type III from Vibrio cholerae (Sigma–Aldrich; N7785). Cells were then washed three times with DMEM, once with PBS-1× and fixed with 4% PFA for 10 min. Cells were subjected to staining for 1 h in PBS-1 × at room temperature (RT) using the following lectins: Cy2- or Cy3-labeled PNA (1:500), Cy2-conjugated Maackia amurensis lectin (MAL1; 1:500), biotinylated Erythrina cristagalli lectin (ELC; 1:500), and Cy5-conjugated wheat germ agglutinin (WGA; 1:500) all from Vector Laboratories. After three washes with ice-cold PBS-1× to remove unbound lectins, ECL was visualized using Streptavidin-Cy5 (Molecular Probes). Cells were imaged using an inverted fluorescence microscope.

Lectin analysis of the inter-photoreceptor matrix

Analysis of the inter-photoreceptor matrix (IPM) was performed on retinal sections, flat mounts, and explants from Ai9_MCre ⁺ mice euthanized at PND1, PND5, PND10, PND14, and PND21, and on retinal flat mounts from Rho ^−/− mice euthanized at PND21. Three eyes were used for each experimental group. Retinal sections and retinal flat mounts were processed and stained, as previously described, for the cell culture experiments, with the following panel of lectins in PBS1X: PNA (1:500; Vector Laboratories), MAL1 (1:500; Vector Laboratories), ELC (1:500; Vector Laboratories), and WGA (1:500; Vector Laboratories). For retinal explants treated with neuraminidase, retinas were dissected in cold DMEM and incubated in either DMEM alone or DMEM with 50 mIU/mL of neuraminidase type III from V. cholerae (Sigma–Aldrich; N7785) for 1 h at 37°C. Retinal explants were then washed three times with cold DMEM, once with cold PBS-1 × , and fixed in 4% PFA for 30 min. Retinal explants were subjected to lectin staining for 1 h in PBS-1× at RT. After three washes with ice-cold PBS-1× to remove unbound lectins, retinal explants were flat mounted and analyzed by fluorescence microscopy (Leica DM5500).

AAV-binding assay and qPCR

AAV-binding assay was performed on three biological samples and repeated two to four times with retinal explants from PND1 and PND21 CD1 mice. Retinas were dissected in cold DMEM and incubated in either DMEM with or without neuraminidase for 1 h at 37°C. Retinal explants were then washed three times with cold DMEM, and pre-chilled at 4°C for 1 h in 500 μL of DMEM. AAV vectors were added at 1 × 10¹⁰ vg/well, and explants were incubated at 4°C for 1 h. Retinal explants were then washed three times with cold DMEM and once with cold PBS-1× to remove unbound AAV particles. Total DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen). AAV genome copies were quantified by real-time PCR with the aforementioned primers for the SV40pA or eGFP sequence. AAV vector genomes (vg) were normalized to the number of retinal cells, using primers complementary to the mouse beta actin DNA (forward: 5′-ACTGGGACGACATGGAGAAG-3′; reverse: 5′-GGGGTGTTGAAGGTCTCAAA-3′). Samples were run in triplicate. The results were expressed as mean AAV vg per genome DNA.

Localization of AAV binding to retinal photoreceptors

Localization of AAV particles was assessed after subretinal injection of PND1 and PND21 Ai9_MCre ⁺ mice with scAAV8 or scAAV9 vectors. Eyes injected with PBS-1× were used as negative controls. One hour post injection, animals were euthanized and retinas processed for flat mount, as previously described. ⁴¹ Retinas were incubated overnight at 4°C with polyclonal rabbit antibodies raised against intact capsids of AAV8 or AAV9 at 1:500 in blocking solution (kindly provided by G.G.). Sections were then incubated with a Cy2-conjugated secondary anti-rabbit antibody (Jackson ImmunoResearch; 1:500) for 2 h at RT. Results were analyzed by fluorescence microscopy (Leica DM5500).

Statistical analysis

All data are expressed as mean ± standard deviation (SD). All statistical comparisons use Student's t-test, with p < 0.05 considered as statistically significant in all comparisons.

Subretinal injection of AAV5 at PND1 predominantly targets cone photoreceptors

Recent reports showed that subretinal injection of AAV8 into PND0 mice results primarily in cone transduction, whereas the vast majority of transduced cells after subretinal injection in adult mice are rods. ^{32 –34} To demonstrate further that PR postnatal development impacts the pattern of PR transgene expression, neonatal mice (PND1) were subretinally injected with 6 × 10⁸–1.2 × 10⁹ vg of single-stranded (ss) AAV2/5-CMV-dGFP (Table 1 and Supplementary Fig. S2a). AAV5 was chosen because this serotype (i) has been shown to significantly target PR cells in the adult mouse, ^14,15,18 cat, ²⁶ dog, ^23,42,43 pig, ^15,22 and nonhuman primate ²¹ ; (ii) has been used successfully to restore retinal function and preserve retinal structure in various models of PR diseases ¹³ ; and (iii) is currently planned in upcoming Phase I–II clinical trials for the treatment of PR diseases. ¹³ Mice with successful subretinal injections were euthanized 3 weeks post injection (wpi), at PND21, after PR differentiation and maturation is completed (Fig. 1 and Supplementary Fig. S2). As previously described, ^30,33,34 subretinal injections at PND1 yielded GFP expression throughout the entire retinal surface (Fig. 1a and Supplementary Fig. S2a), presumably because the interactions between the RPE and the PR outer segments that could limit the spread of the vector have not yet formed at PND1. Robust transduction of RPE was observed across the retina (Supplementary Fig. S2b). High levels of GFP were also detected in the ONL, which contains rod and cone PR nuclei (Fig. 1c and Supplementary Fig. S2b), and in the corresponding PR segments (Fig. 1a and c and Supplementary Fig. S2b). Immunostaining with cone-specific markers (PNA and cone arrestin) revealed that the majority of the GFP⁺ cells were also positive for the cone-specific markers (Fig. 1a and c and Supplementary Fig. S2b). Hence, as predicted, the transduction of PRs was essentially relegated to cones when considering the percentage of GFP⁺ cells (cones: 69.1 ± 7.4%; rods: 2.7 ± 3.1%). L/M and S cones were equally transduced (Supplementary Fig. S3).

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Figure 1.

Photoreceptor transduction in the neonatal mouse retina by adeno-associated virus serotype 5 (AAV5) vectors. Mice injected on postnatal day 1 (PND1) were subretinally injected with similar doses of AAV2/5-CMV-dGFP, AAV2/5-CMV-H2bGFP, AAV2/5-CMV-CRE, AAV2/5-pQCMV-H2bGFP, AAV2/5-mCAR-H2bGFP, or scAAV2/5-CB6-eGFP vectors. (a) Representative retinal flat-mounts and (c, d) cryosections 3 weeks post injection (wpi) labeled with an antibody raised against anti-cone arrestin (CA) or with peanut agglutinin lectin (PNA; red) and counter stained with DAPI (blue: removed from 60% of panels to visualize red and green staining better) (c,d). AAV-mediated GFP was detected by its native fluorescence (green). AAV-mediated CRE expression was detected in Ai9⁺ mice with the CRE-mediated expression of td-Tomato (white, native fluorescence). In retinal sections, retinal pigment epithelium (RPE) cells were artificially removed using Photoshop. Asterisks indicate GFP⁺ or CRE⁺ (td-Tomato⁺) cones, while arrowheads indicate GFP⁺ or CRE⁺ (td-Tomato⁺) rods, which are mainly found in the inner most rows of the outer nuclear layer (ONL). Quantitative analysis of the percentage of GFP⁺ or CRE⁺ photoreceptors in vector-exposed area is shown in (b). Error bars represent standard deviation (SD). Numbers in bars represent the number of retina analyzed. *p < 0.05; **p < 0.01; ***p < 0.001 by Student's t-test. (d) After injection of higher dose of AAV2/5-pQCMV-H2bGFP, a higher number of GFP⁺ rods is observed throughout the ONL (representative section). Scale bars = 25 μm. INL, inner nuclear layer; IS, inner segments; OS, outer segments; sc, self-complementary; ss, single-strand.

To rule out an influence of factors such as inhibition of CMV promoter activity or dGFP steady state level on the final transgene expression pattern, PR transduction was analyzed in mice injected at PND1 with five additional AAV5 vectors. These vectors carry different transgenes and/or ubiquitous or PR-specific promoters: AAV5-CMV-H2bGFP, AAV5-CMV-Cre, AAV5-mCAR-H2bGFP, AAV5-pQCMV-H2bGFP, as well as a self-complementary (sc) AAV5-CB6-eGFP (Table 1). Though some construction-dependency was noted, tropism was similar for all vectors examined (Fig. 1b and c and Supplementary Fig. S2b). Hence, the findings suggest that the absence of efficient rod transduction following subretinal administration at PND1 is not dependent on factors that alter vector genome expression, as well as second-strand synthesis in the transduced cells, but rather on differences in entry of AAV into cones and rods at the time of delivery. Consistent with that, AAV8 vectors carrying different promoters, such as AAV-hRK1-GFP, AAV-mCAR-GFP, or AAV-CMV-GFP, have also been seen to drive transgene expression predominantly in cones after subretinal injection in PND0 neonate mice. ^33,34 Moreover, efficient transfection of rods has been previously obtained after PND0 electroporation of pCMV-H2bGFP, ⁴¹ a plasmid DNA that carries the same expression cassette as the AAV5-CMV-H2bGFP. Finally, a higher number of GFP⁺ rods was observed after administration of higher doses of AAV5-CMV-H2bGFP (9 × 10⁹ vg; Fig. 1d and Table 1) and AAV5-mCAR-H2bGFP (>1 × 10¹⁰ vg) ⁴¹ at PND1, indicating that efficient transduction of immature rods is possible at this age but dependent on the quantity of the vector injected (Fig. 1d and Table 1).

Rod development coincides with increased rod transduction after subretinal delivery

In mice, cones start to undergo their terminal mitosis to begin their subsequent differentiation before rods. Most rods in mouse are born over an extended developmental time period (E12 to PND5) with the peak of rod production around birth and their differentiation into mature rods continuing well into the postnatal period. ⁴⁴ To examine further how the PR transduction pattern correlates with rod PR development, GFP expression was investigated in mice injected at three additional ages, corresponding to three different stages of PR development: PND5 (end of PR birth and early development of PR processes), PND10 (intermediate development of PR outer segments), and PND21 (PR development completed; Table 1). Injected retinas were all harvested at 3 wpi.

The histological analyses of three different AAV2/5 vectors (Fig. 2 and Supplementary Figs. S4 and S5) revealed a progressive shift in tropism from cones to rods that correlated with the differentiation of the retina. Indeed, after injection of AAV5-CMV-dGFP (Fig. 2a and b and Supplementary Fig. S4), AAV5-CMV-H2bGFP (Fig. 2a), or scAAV5-CB6-eGFP (Supplementary Fig. S5a and Fig. 2b), the percentage of GFP⁺ rods increased with the age of the mouse at the time of injection, whereas the percentage of GFP⁺ cones decreased. As a result, injections of the three vectors at PND1 and PND5 directed GFP expression predominantly in cones, while a preferential transduction of rods was observed for injections at PND10 and PND21 (Table 1).

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Figure 2.

Efficiency of cone and rod photoreceptor transduction with AAV2/5 vectors at different times after birth. AAV2/5-CMV-dGFP, AAV2/5-CMV-H2bGFP, and scAAV2/5-CB6-eGFP were subretinally injected in mice at PND1, PND5, PND10, and PND21. In all cases, analysis was performed 3 wpi. (a) Representative retinal flat mounts (upper row) and cryosections (middle row) from mice injected with AAV2/5-CMV-dGFP and cryosections (bottom row) from mice injected with AAV2/5-CMV-H2bGFP and immunostained for PNA or CA (red) and counterstained with DAPI (blue: removed from 60% of panels to visualize red and green staining better). AAV-mediated GFP was detected by its native fluorescence (green). RPE cells were artificially removed using Photoshop. Dotted horizontal lines divide the ONL in three sublayers showing that early injections (PND1 and PND5) result in the majority of transduced rods (arrowheads) located in the most inner part of the ONL. Arrowheads indicate GFP⁺ rods (b). Quantification of cone, rod, and total photoreceptor transduction within the vector-exposed area after injection of AAV2/5-CMV-dGFP and AAV2/5-CB6-eGFP at the indicated ages. Results are shown as mean ± SD. **p < 0.01 and ***p < 0.001 by Student's t-test. (c) Average distribution of GFP⁺ nuclei within the ONL layer after AAV injection at the indicated ages. As shown in (a), the ONL was divided into three sublayers: A, outermost part of the ONL; B, middle part of the ONL; C, innermost part of the ONL. The percentage of GFP⁺ nuclei/total nuclei (DAPI⁺) was calculated. Scale bars = 25 μm.

Interestingly, the location of GFP⁺ rod nuclei in the ONL correlated with the timing of rod genesis (Fig. 2 and Supplementary Figs. S2b, S4, and S5a). After subretinal injection at PND1, the small percentage of transduced rod nuclei found was primarily located in the most inner row of the ONL where the first-born rods reside, as previously indicated by birth-dating experiments ^{45 –47} (Figs. 1 and 2a and c, and Supplementary Figs. S2b, S4, and S5a). After vector administration at PND5, while the number of transduced rods increased, GFP⁺ rods were still restricted in their location to the four or five most inner rows of the ONL (lower third of ONL; Fig. 2a and c and Supplementary Figs. S4 and S5a), consistent with the observation that newborn rods are stacked on the top of the earlier born ones. ⁴⁵ In contrast, injections after PND10 led to a wide distribution of GFP⁺ nuclei across the ONL (Fig. 2a and c, and Supplementary Figs. S4 and S5a). In line with this finding, the transduction pattern of rod and cone PRs followed the central-to-peripheral (data not shown) and ventral-to-dorsal gradient of PR differentiation ⁴⁸ when the vector was injected at PND3 (Supplementary Fig. S5b). Hence, the order of differentiation of PR cells is clearly correlated with the cells preferentially transduced after subretinal injection of AAV2/5.

Photoreceptor development affects rod transduction, irrespective of AAV serotypes

To determine whether PR development affects PR transduction by other AAV serotypes, neonatal mice were subretinally injected with a panel of 13 scAAV serotypes/variants, including AAV1, -2, -3b, -4, -5, -6, -7, -8, -9, -rh8, -rh10, -rh39, and -rh43, all expressing eGFP under the control of the CB6 promoter (Table 1, Figs. 3 and 4, and Supplementary Figs. S6 and S7). As controls, AAV5, -7, -8, -9, and -rh10 were subretinally injected into PND21 mice (Table 1 and Supplementary Fig. S6), as these vectors are known to exhibit excellent tropism for adult rods. ^14,15,18 Similar to the results observed after PND1 delivery of AAV5, PND1 injections of the all other AAVs resulted in preferential cone transduction when considering the percentage of GFP⁺ cells, with the exception of AAV4, which efficiently transduced RPE cells only (Table 1 and Fig. 3). On average, AAVrh10 exhibited the most efficient transduction of cones (although not statistically different from AAV5, -6, -7, -8, -9, -rh8, and -rh39), whereas AAV3b-mediated transgene expression was limited to sporadic RPE and PRs (Fig. 3b). Notably, no significant difference in the percentage of transduced rods was seen between all the serotypes (Fig. 3). When considering the percentage of GPF⁺ rods, all AAVs led to sparse rod transduction, with most of the GFP⁺ rods localized in the innermost part of the ONL (Fig. 4 and Supplementary Figs. S6 and S7). Additionally, the preferential transduction of rods observed after subretinal injection of AAV2/5 vectors at PND21 was reproduced after injection of AAV7 (Supplementary Fig. S6d and e), -8 (Supplementary Fig. S6a, d, and e), -9 (Supplementary Fig. S6b, d, and e), and -rh10 (Supplementary Fig. S6c–e). Thus, rod PR development is a critical host factor that influences AAV tropism after subretinal delivery independently of the serotype and thus probably independently of the different cell attachments factors utilized by each capsid.

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Figure 3.

Predominant transduction of cone photoreceptors after the injection of 12 different AAV serotypes/variants at PND1. (a) Representative images of retinal flat mounts at 3 wpi of scAAV1, -2, -3b, -4, -6, -7, -8, -9, -rh8, -rh10, -rh39, or –rh43 vectors (all expressing eGFP under the control of the CB6 promoter), as indicated. Labeling with PNA (red) shows expression of eGFP (green) predominantly in cones (asterisks), except for AAV4. (b) Quantification performed on retinal flat mounts and/or cryosections of the percentage of GFP⁺ cones and GFP⁺ rods in the vector exposed area (note: actual transduction efficiency of scAAV4 was 0% for rods and cones). Results are expressed as mean ± SD. Numbers in bars represent the number of retinas analyzed. *p < 0.05; **p < 0.01; ***p < 0.001 by Student's t-test. Scale bar = 25 μm.

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Figure 4.

Comparative analysis of AAV vector transduction in the mouse retina after subretinal or intravitreal injection at PND1. PND1 mice injected either subretinally (upper row) or intravitreally (lower row) with scAAV5, -6, -7, -8, or -9 expressing eGFP under the control of the CB6 promoter, as indicated. Histological analysis was performed at 3 wpi on cryosections stained with CA (red) and counterstained with DAPI (blue). AAV-mediated GFP was detected by its native fluorescence (green). Subretinal injections result in a higher number of transduced cones (asterisks) with most of the transduced rods (arrowheads) located in the inner most part of the ONL (see also Figs. 1 and 2), while intravitreal injections result in scattered rods throughout the ONL and fewer transduced cones. Scale bars = 25 μm. GCL, ganglion cell layer.

Control of AAV transduction by photoreceptor development depends on the injection site

Because a higher number of GFP⁺ rods was observed after administration of higher doses of AAV5-CMV-H2bGFP (9 × 10⁹ vg; Fig. 1d and Table 1) and AAV5-mCAR-H2bGFP (>1 × 10¹⁰ vg) ⁴¹ at PND1, the overall absence of efficient rod transduction at PND1 may relate to the progressive loss of the episomal vector genomes in rods. AAV vectors integrate only at very low frequencies, and thus vector-mediated transgene expression is predicted to diminish over time under conditions of ongoing cellular proliferation. In transduced cones, the number of expressed vector genomes is likely to remain stable over time because cones are already post-mitotic by PND1–PND5. ⁴⁴ In contrast, although the vast majority of rod precursors are already post-mitotic by PND1⁴⁴ and lineage studies using retroviral vectors have revealed that most rod progenitor cells that persist in the rodent retina after birth are terminally dividing, ^{49 –51} rod progenitors may dilute or lose AAV genomes, resulting in consistent decreased transgene expression in mature rods. Alternatively or concomitantly, the overall absence of efficient rod transduction at PND1 may relate to the progressive transcriptional silencing of AAV vector DNA due to developmental epigenetic changes. Indeed, histone modifications have been shown to be important contributors to gene regulation in developing rod PRs. ⁵²

To investigate these two possibilities, AAV5, -6, -7, -8, and -9 were intravitreally injected in PND1 mice. If the factors that determine AAV tropism during PR development are related to AAV vector DNA, the final pattern of transgene expression should globally be similar between the two routes of administration. However, for all five serotypes, it was found that rods were the predominant PR cells transduced when the vectors were injected intravitreally (e.g., for AAV5: 4.2 ± 2.6% of rods vs. 0.9 ± 0.3% of cones near the injection site; n = 3; Fig. 4 and Supplementary Fig. S7). Notably, rod nuclei expressing detectable levels of GFP were evenly distributed across the ONL after intravitreal injection (Fig. 4 and Supplementary Fig. S7), indicating that lack of efficient AAV-mediated transduction of the late-born rods by subretinal injections at PND1 is not due to cell division or differential silencing of the vector genomes. The data suggest that factors affecting the efficiency of AAV-mediated transduction of early and late-born rods are site specific and likely involve the access of AAV particles to the PR cells. Receptors necessary for binding/entry of AAVs while present in PRs may not yet be accessible on late-born cells by AAVs from the subretinal space.

Lack of rod outer segments markedly alters PR tropism of AAV in the adult retina

The timing of rod precursor birth (after E19) is tightly linked to the onset of PR segment formation and rhodopsin expression. ⁵³ To test whether the growth of rod segments and/or the associated matrix (Supplementary Fig. S8a and b) plays a role in the access of AAV particles to PRs, an attempt was made to visualize the distribution of AAV particles after subretinal injection at PND1 and PND21 using anti-AAV capsid antibodies. However, the large number of capsids present in the subretinal space limited the analysis (data not shown). Instead, qPCR was first employed to quantify viral binding on PND1 and PND21 retinas after modification of the rod IPM with neuraminidase (Supplementary Fig. S8). Ex vivo retinal explants were used to limit injection variability related to the surface of the retina exposed to the vector after subretinal delivery at PND1 versus PND21. We found that the rod-associated matrix could specifically interact with AAV particles in vitro (Supplementary Fig. S8d). However, these interactions were not conserved across AAV5, −7, −8, and −9, indicating that binding of AAV to the rod IPM does not govern the common increase in rod transduction observed during PR development after subretinal delivery.

Next, to determine if the development of a normal rod segment plays a role in the access of AAV particles to PRs, rod and cone transduction was quantified in rhodopsin knockout (Rho ^−/−) mice ⁵⁴ after subretinal injection of AAV2/5-CMV-dGFP, AAV2/5-CMV-H2bGFP, and AAV2/5-pQCMV-H2bGFP vectors at PND1 and PND21 (Table 1 and Fig. 5). The Rho ^−/− mouse is a well-characterized model of rod-cone dystrophy that does not develop rod outer segments. ⁴⁰ Importantly, this mouse model does not display any rod degeneration during the first three postnatal weeks. No alterations in the IPM and outer limiting membrane (OLM) ^55,56 were detected in Rho ^−/− mice at PND21 (Supplementary Fig. S9). In PND1-injected mice, the transduction profile was similar between Rho ^−/− and wild-type mice, with a preferential transduction of cone PRs (Fig. 5a and c). However, PND21 injections resulted in a fairly dramatic difference in the final expression patterns. On average, the three AAV vectors tested showed a similarly low transduction of rod PRs in Rho ^−/− retinas, as seen with PND1 injections in healthy retinas (Table 1 and Fig. 5a and c). Rod transduction was mainly observed at the site of injection near the needle tract (data not shown). GFP⁺ rods were located in the outermost part of the ONL (Fig. 5a, arrows) and expressed qualitatively less GFP than cone PRs (Fig. 5a and b). These observations suggest that the presence of a sick rod segment can influence the efficiency rod transduction upon subretinal delivery in the adult retina. Furthermore, they suggest a potential correlation between altered rod transduction and increased cone transduction.

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Figure 5.

Altered of AAV tropism in Rho ^−/− and adult rd1 mice. (a–c) PND1 and PND21 Rho^−/− mice injected with similar doses of AAV2/5-CMV-H2bGFP, AAV2/5-CMV-dGFP, or AAV2/5-pQCMV-H2bGFP. Three weeks after injection, retinas were labeled with PNA or with an antibody raised against CA (red). AAV-mediated GFP was detected by its native fluorescence (green). Representative images after injection of (a) AAV2/5-CMV-H2bGFP and (b) AAV2/5-CMV-dGFP at age indicated. Asterisks indicate GFP⁺ cones and arrowheads indicate GFP⁺ rods. Quantitative analysis of the percentage of GFP⁺ cones (black bars) and rods (white bars) is shown in (c). Error bars represent SD. Numbers in bars represent the number of retina analyzed. **p < 0.01 and ***p < 0.001 by Student's t-test. (d) Retinal flat mounts of rd1 mice injected at PND21with similar doses of AAV2/5-CMV-dGFP, AAV2/5-CMV-H2bGFP, scAAV2/5-CB6-eGFP, or scAAV2/9-CB6-eGFP and stained for CA expression (red) 3 wpi. AAV-mediated GFP was detected by its native fluorescence (green). Percentage of transduced cones for each vector is indicated in corresponding panel as mean ± SD. Scale bars = 25 μm.

To delineate further the influence of rods on the efficiency of cone transduction, the rd1 mouse model was utilized, in which the vast majority of rods are lost by PND21. ⁵⁷ However, in contrast to the Rho ^−/− retinas, the IPM is inevitably altered in this mouse model due to the loss of rods. AAV5 or AAV9 vectors were subretinally injected into PND21 rd1 mice and cone transduction was evaluated on retinal flat mounts at 3 wpi (Table 1 and Fig. 5d). All vectors resulted in dramatically enhanced cone transduction compared to control mice, considering the percentage of cells transduced (Table 1). The consistent finding in two mouse models of rod degeneration that cone transduction increases in the absence of efficient rod transduction or in the absence of rods suggest that rods may negatively affect cone transduction in the adult mouse retina.