Novel Adeno-Associated Virus Serotypes Efficiently Transduce Murine Photoreceptors

Abstract

Severe inherited retinal diseases, such as retinitis pigmentosa and Leber congenital amaurosis, are caused by mutations in genes preferentially expressed in photoreceptors. While adeno-associated virus (AAV)-mediated gene transfer can correct retinal pigment epithelium (RPE) defects in animal models, approaches for the correction of photoreceptor-specific diseases are less efficient. We evaluated the ability of novel AAV serotypes (AAV2/7, AAV2/8, AAV2/9, AAV2rh.43, AAV2rh.64R1, and AAV2hu.29R) in combination with constitutive or photoreceptor-specific promoters to improve photoreceptor transduction, a limiting step in photoreceptor rescue. Based on a qualitative analysis, all AAV serotypes tested efficiently transduce the RPE as well as rod and cone photoreceptors after subretinal administration in mice. Interestingly, AAV2/9 efficiently transduces Müller cells. To compare photoreceptor transduction from different AAVs and promoters in both a qualitative and quantitative manner, we designed a strategy based on the use of a bicistronic construct expressing both enhanced green fluorescent protein and luciferase. We found that AAV2/8 and AAV2/7 mediate six-to eightfold higher levels of in vivo photoreceptor transduction than AAV2/5, considered so far the most efficient AAV serotype for photoreceptor targeting. In addition, following subretinal administration of AAV, the rhodopsin promoter allows significantly higher levels of photoreceptor expression than the other ubiquitous or photoreceptor-specific promoters tested. Finally, we show that AAV2/7, AAV2/8, and AAV2/9 outperform AAV2/5 following ex vivo transduction of retinal progenitor cells differentiated into photoreceptors. We conclude that AAV2/7 or AAV2/8 and the rhodopsin promoter provide the highest levels of photoreceptor transduction both in and ex vivo and that this may overcome the limitation to therapeutic success observed so far in models of inherited severe photoreceptor diseases.

Adult Human Müller Glia Cells Are a Highly Efficient Source of Rod Photoreceptors

“…Discrimination of MGCs from the feeder PA6 cells, required their labeling using either the nuclear dye Syto13 (Supporting Information Fig. S6A-S6C) or a lentivirus expressing the GFP under the control of the rhodopsin promoter [29] (see Materials and Methods section), showing that only the recoverinþ MGCs activate the GFP transgene (Supporting Information Fig. S6D-S6F").…”

Gene supplementation therapy for recessive forms of inherited retinal dystrophies

“…Efficient AAV2-mediated transduction of both rod and cone photoreceptors, as well as the retinal pigment epithelium (RPE), suggests that the great majority of IRDs might be targeted effectively using AAV2, 21,22 although the relatively slow rate of onset of transgene expression limits the utility of AAV2 in mouse models with rapidly progressive degeneration. 23 Pseudotyping of the AAV2 genome using capsid proteins of other AAV serotypes has led to the development of several vectors, including AAV5, 24,25 AAV8 26,27 and AAV9, 26 that have higher transduction efficiencies and mediate more rapid onset of transgene expression. A further development of AAV as a vector for photoreceptor cell gene targeting has involved mutagenesis of the viral capsid proteins to increase the efficiency of transduction and penetration of the vector across the layers of the neurosensory retina.…”

“…The reason for the apparent discrepancy between the efficacy of intervention in the Rd1 mouse and the Aipl1 À/À mouse is not yet clear, and it seems unlikely to result from a substantial difference between AAV5 and AAV2/8, as both the vectors transduce photoreceptor cells rapidly and efficiently. 26 More probable is a fundamental difference in the functions of the proteins involved. PDE is directly involved in the phototransduction cascade; the amount of protein is likely to be tightly controlled and relatively small deviations from physiological levels may have significant effects on photoreceptor cell health.…”

Gene-based therapies for dominantly inherited retinopathies

“…29,30 In addition, AAV2/9 has been found to enable gene delivery after systemic administration to many tissues including brain and retina. 31,32 Another attractive feature of AAV as a vector is its predilection to remain episomal particularly in the context of postmitotic cells such as photoreceptors thereby reducing the risk of insertional mutagenesis. Results from recent human clinical trials for a severe recessively inherited retinopathy, retinal pigment epithelium-65-linked Leber congenital amaurosis, have served to highlight the attractive features of AAV as a delivery vector for the human eye.…”