To quantify the level of photoreceptor transduction for each capsid mutant vector following intravitreal injection, we employed a transgenic mouse model (B6/Cg
Tg(Nrl-EGFP)1ASW/J, known herein as Nrl-EGFP) that selectively expresses EGFP in rod photoreceptors, which comprise approximately 97% of the photoreceptors in a mouse retina. PW4 Nrl-EGFP mice were injected intravitreally (1.0 × 10
10 vg/eye;
n = 5–7 eyes per group) with rAAV2/2, rAAV2/2[QuadYF+TV], rAAV2/2[7m8], or rAAV2/2[MAX] vector packaging a ubiquitously expressing red fluorescent reporter construct (CBA-mCherry). Four weeks post injection (PW8), infrared reflectance, intrinsic (i.e., transgenic) EGFP, and rAAV-derived mCherry fluorescence was assessed using a custom multiline cSLO instrument that allows fluorophores with green (e.g., EGFP) and red (e.g., mCherry) emission spectra to be imaged independently within the same retina (
Fig. 3; see Methods for specifications). Infrared reflectance imaging was initially employed to align the cSLO with the fundus and locate the ONL, which is identified in vivo as the confocal plane having the highest level of reflectance (
Fig. 3 A–D). cSLO imaging of intrinsic EGFP expression revealed a uniform expression pattern throughout the retina in all groups; the absence of any reduced EGFP signal within the retina in Nrl.EGFP mice following intravitreal injection of rAAV indicates a lack of photoreceptor toxicity arising from vector delivery (
Fig. 3E–H). Consistent with our previous findings (
Supplementary Fig. S1), imaging of mCherry fluorescence revealed poor transduction throughout the retinas of rAAV2/2 vector–injected animals (
Fig. 3I), while increasing levels of pan-retinal expression was detected in animals injected with rAAV2/2[QuadYF+TV], rAAV2/2[7m8], and rAAV2/2[MAX] vectors (
Fig. 3J–L). Following cSLO imaging, retinas were papain-dissociated individually and the number of rod photoreceptor (GFP+ mCherry+) and nonrod photoreceptor (GFP-mCherry+) cells transduced for each capsid mutant serotype quantified by flow cytometry, as previously described.
25,26 Papain dissociation of each retina generated a suspension of single cells of differing size and granularity, as expected, indicating recovery of a heterogeneous population comprising cells from all retinal cell layers (
Fig. 4A). Dissociated retinas from uninjected C57BL/6J wild-type (nonfluorescent) and Nrl-EGFP (GFP+) mice were used as gating controls (
Fig. 4B,
4C). Intravitreally delivered unmodified rAAV2/2 (
n = 7) displayed only modest transduction (3.9% ± 1.5%) of rod photoreceptors (
Fig. 4D,
Supplementary Fig. S3A). By contrast, rAAV2/2[QuadYF+TV] (
n = 5) and rAAV2/2[7m8] (
n = 5) showed dramatically increased levels of rod photoreceptor transduction (11.2% ± 2.5% and 14.0% ± 3.8%, respectively;
Fig. 4E,
4F,
Supplementary S3B, S3C). In line with our in vivo imaging and prior histologic observations, rAAV2/2[MAX] (
n = 5) exhibited significantly higher rod photoreceptor transduction (32.4% ± 10.6%) compared to all other capsid variants tested (
Fig. 4G,
4H,
Supplementary Fig. S3D;
P < 0.001, 1-way ANOVA with Tukey's post hoc test), with a 8.2-fold increase in rod transduction compared to unmodified rAAV2/2. Furthermore, a significantly greater number of nonrod cells in the retina were also transduced by rAAV2/2[MAX] (
Fig. 4I;
P < 0.01, 1-way ANOVA with Tukey's post hoc test,
n = 5–7) with a 6.8-fold increase in transduction compared to rAAV2/2. Taken together, these findings clearly demonstrate that combining capsid mutations with differing modes of action into a single vector results in an additive increase in retinal transduction. The resulting rAAV2/2[MAX] vector is able to mediate effective gene delivery to a significant proportion of photoreceptors following intravitreal delivery.