Virus-mediated transduction of murine retina with adeno-associated virus: effects of viral capsid and genome size

Abstract

Gene therapy vectors based on adeno-associated viruses (AAVs) show promise for the treatment of retinal degenerative diseases. In prior work, subretinal injections of AAV2, AAV5, and AAV2 pseudotyped with AAV5 capsids (AAV2/5) showed variable retinal pigmented epithelium (RPE) and photoreceptor cell transduction, while AAV2/1 predominantly transduced the RPE. To more thoroughly compare the efficiencies of gene transfer of AAV2, AAV3, AAV5, and AAV6, we quantified, using stereological methods, the kinetics and efficiency of AAV transduction to mouse photoreceptor cells. We observed persistent photoreceptor and RPE transduction by AAV5 and AAV2 up to 31 weeks and found that AAV5 transduced a greater volume than AAV2. AAV5 containing full-length or half-length genomes and AAV2/5 transduced comparable numbers of photoreceptor cells with similar rates of onset of expression. Compared to AAV2, AAV5 transduced significantly greater numbers of photoreceptor cells at 5 and 15 weeks after surgery (greater than 1,000 times and up to 400 times more, respectively). Also, there were 30 times more genome copies in eyes injected with AAV2/5 than in eyes injected with AAV2. Comparing AAVs with half-length genomes, AAV5 transduced only four times more photoreceptor cells than AAV2 at 5 weeks and nearly equivalent numbers at 15 weeks. The enhancement of transduction was seen at the DNA level, with 50 times more viral genome copies in retinas injected with AAV having short genomes than in retinas injected with AAV containing full-length ones. Subretinal injection of AAV2/6 showed only RPE transduction at 5 and 15 weeks, while AAV2/3 did not transduce retinal cells. We conclude that varying genome length and AAV capsids may allow for improved expression and/or gene transfer to specific cell types in the retina.

FIG. 1.

Cartoon of the AAV vectors used. CMV, CMV promoter and enhancer; nls eGFP, nucleus-targeted eGFP; dsRed, coding sequence for red fluorescent protein; short, total length of vector is 2.3 kb; long, total length of vector is equivalent to that of wild type (4.6 to 5.0 kb). ∗, eGFP or dsRed coding sequence plus stuffer sequences (see Materials and Methods).

FIG. 2.

Stereological analysis utilizing the optical fractionator was performed as schematically depicted to evaluate the efficiency of AAV transduction of murine photoreceptor cells. Eyes were serially sectioned (A) and oriented in such a way that each section contained the anterior and posterior parts of the eye (B). (C) Enlarged area of the retina, depicting the area of interest (blue, photoreceptor cells) and the overlying grid. Within the grid the counting box is shown. To determine the number of transgene-positive photoreceptor cells, the counting box was translated over grid intersections and cells were counted as described in Materials and Methods.

FIG. 3.

Murine retina coinfected with AAV5.nlsGFP.Long and AAV2.dsRed.Long 31 weeks after subretinal delivery. (A) nucleus-targeted GFP-positive nuclei and dsRed-positive nuclei and cytoplasm of photoreceptor cells. (B) DAPI (4′,6′-diamidino-2-phenylindole)-positive nuclei in the layers of murine retina. (C) Superimposed images of panels A and B, showing positive nuclei in the ONL. (D) Extent of AAV5- versus AAV2-mediated reporter expression in the ONL and RPE. GL, ganglion layer. Photomicrographs are similar to those taken from retinas harvested 15 weeks after surgery. Bars, 50 μm.

FIG. 4.

Stereological assessment of transgene-positive cells in murine retina coinfected with AAV5.nlsGFP.Long and AAV2.dsRed.Long at 5, 15, and 31 weeks. Transgene-positive cells were determined as shown in Fig. 2 and as described in Materials and Methods. Numbers of eyes examined per group are given in parentheses. NS, not significant.

FIG. 5.

Evaluation of transduction volume of murine retinas coinfected with AAV5.nlsGFP.Long and AAV2.dsRed.Long at 15 (A) and 31 (B) weeks. Serial sections of the entire eye were systematically sampled, and volumes were determined as described in Materials and Methods.

FIG. 6.

Murine retinas infected with AAV5.GFP.Short 5 weeks after subretinal delivery. (A) GFP-positive RPE and photoreceptor cells (ONL and OS [outer segment]). (B) DAPI fluorescence depicting the nuclei within the ONL and INL. (C and D) Superimposed images demonstrating GFP-positive nuclei in the ONL. GL, ganglion layer. The photomicrographs are similar to those taken from retinas infected with AAV5.GFP.Short 15 weeks after injection and from retinas infected with AAV2.GFP.Short and AAV2/5.GFP.Long 5 and 15 weeks after surgery. Bars, 50 μm.

FIG. 7.

Stereological assessment of transgene-positive cells in murine retinas infected with AAV5.GFP.Short and AAV2.GFP.Short at 5 and 15 weeks. NS, not significant.

FIG. 8.

Comparison of numbers of photoreceptor cells transduced with AAV2 carrying a half-length genome versus the number transduced with AAV2 carrying a wild-type length genome (see Fig. 1). NS, not significant.

FIG. 9.

Stereological assessment of transgene-positive cells in murine retinas infected with AAV2/5.GFP.Long and AAV2.GFP.Long at 5 and 15 weeks. NS, not significant.

FIG. 10.

Quantification of viral genomes extracted from murine retinas 14 weeks after subretinal delivery of AAV2/5.GFP.Long, AAV2.GFP.Long, and AAV2.GFP.Short using real-time PCR.

FIG. 11.

Murine retina infected with AAV2/6.GFP 5 weeks after subretinal delivery. (A) Light microscopy of retina using differential interference contrast optics. (B) DAPI-positive nuclei. The area near the RPE has been lightened to allow visualization of DAPI staining. (C) GFP-positive RPE. (D) Superimposed images of panels A to C. Photomicrographs are similar to those taken from retinas harvested 15 weeks after surgery. Bar, 50 μm.