Adenovirus Vectors Targeting Distinct Cell Types in the Retina

Loss of any one of the multiple cell types found in the retina can lead to blindness, the second most feared condition after cancer among Americans. The human retina is a laminated tissue composed of light-sensitive photoreceptors in the outer retina that modulate current with a complex array of inner retinal neurons. Photoreceptors rely on the juxtaposing retinal pigment epithelium (RPE) for generation and recycling of retinal (vitamin A), the chromophore that absorbs light to initiate the phototransduction cascade that is eventually perceived as vision. Retinal neurons are supported by Muller glia, which provide critical support for the maintenance and functioning of the retina. Loss of RPE cells is characteristic of age-related macular degeneration (AMD), the most common cause of blindness in elderly individuals in the United States.1 Loss of photoreceptors may lead to retinitis pigmentosa (RP), the most common cause of inherited blindness in the working population, affecting approximately 1 in 3000 individuals.2 The rare hereditary disease referred to as Muller cell sheen dystrophy is caused by primary Muller cell dysfunction.3 Hence, there is a substantial need for gene transfer vectors that effectively transduce each of the different cell types in the retina. To date, all gene transfer vectors injected in the outer retina, in a region referred to as the subretinal space, primarily infect the RPE. However, through vector modifications, such as pseudotyping or deletion of integrin binding motifs in the viral capsids, other cell types in the retina, such as photoreceptors, may also be transduced, though still not as effectively as the RPE. Human adenovirus (Ad) has been previously used in two clinical trials for eye diseases without any serious adverse events.4,5 Short-term transgene expression typically observed from adenovirus vectors may be overcome by the use of the helper-dependent adenovirus vectors.6–8 Therefore, a clear understanding of the tropism of different adenovirus serotypes in the retina would greatly enhance the progression of adenovirus-mediated ocular gene therapy studies. Adenovirus serotype 5 (Ad5) infects cells by initial binding of the adenovirus fiber protein to the membrane-associated coxsackie adenovirus receptor (CAR), followed by internalization of the virus through an interaction between the RGD motif in adenovirus penton base with αvβ3/5 integrins.9–11 In contrast, Ad35 and Ad37 fibers have been shown to interact with CD46 and sialic acid, respectively.12,13 Previously, it had been shown that the transduction of photoreceptors can be enhanced either by deletion of the RGD motif in the penton base or by replacement of the Ad5 fiber with that of Ad35 or Ad37.14–16 Although each of these studies described improved photoreceptor transduction, the data from these studies cannot be directly compared because of the different genes, promoters, or transgene detection methods used. Hence, the relative performance between these vectors is still an unanswered and relevant question for the advancement of Ad-mediated retinal gene therapy. One aim of the present study was to compare the performance of these different adenovirus vectors under conditions that permit their direct comparison in the retina, i.e., the use of the same transgene and promoter combination and a similar detection method for the presence of the gene product. A second aim of this study was to use the most potent vector identified herein to attempt photoreceptor-specific transgene expression in the retina through the use of cell-specific promoters. Significant photoreceptor-specific transgene expression has not thus far been described using adenovirus vectors. Our results and conclusions differ significantly from those of previous studies and, hence, highlight the need for continued evaluation of adenovirus tropism in the retina before their advancement into the clinic.