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. 2016 Jan;27(1):72-82.
doi: 10.1089/hum.2015.130.

Cone-Specific Promoters for Gene Therapy of Achromatopsia and Other Retinal Diseases

Free PMC article

Cone-Specific Promoters for Gene Therapy of Achromatopsia and Other Retinal Diseases

Guo-Jie Ye et al. Hum Gene Ther. .
Free PMC article


Adeno-associated viral (AAV) vectors containing cone-specific promoters have rescued cone photoreceptor function in mouse and dog models of achromatopsia, but cone-specific promoters have not been optimized for use in primates. Using AAV vectors administered by subretinal injection, we evaluated a series of promoters based on the human L-opsin promoter, or a chimeric human cone transducin promoter, for their ability to drive gene expression of green fluorescent protein (GFP) in mice and nonhuman primates. Each of these promoters directed high-level GFP expression in mouse photoreceptors. In primates, subretinal injection of an AAV-GFP vector containing a 1.7-kb L-opsin promoter (PR1.7) achieved strong and specific GFP expression in all cone photoreceptors and was more efficient than a vector containing the 2.1-kb L-opsin promoter that was used in AAV vectors that rescued cone function in mouse and dog models of achromatopsia. A chimeric cone transducin promoter that directed strong GFP expression in mouse and dog cone photoreceptors was unable to drive GFP expression in primate cones. An AAV vector expressing a human CNGB3 gene driven by the PR1.7 promoter rescued cone function in the mouse model of achromatopsia. These results have informed the design of an AAV vector for treatment of patients with achromatopsia.


<b>Figure 1.</b>
Figure 1.
Design of AAV-GFP vectors containing promoters based on the L-opsin promoter. The checkerboard-patterned portion of the four promoters represents nucleotides immediately upstream of the L-opsin coding sequence, and the stippled portion of the PR1.1 promoter represents the cytomegalovirus immediate-early enhancer. Nucleotide sequences of the PR1.7, PR1.5, and PR1.1 promoters are available in GenBank (accession numbers KT886395, KT886396, and KT886397). ITR, inverted terminal repeat; SV40, simian virus 40; SD/SA, splice donor/splice acceptor; polyA, polyadenylation sequence; GFP, green fluorescent protein cDNA; LCR, locus control region.
<b>Figure 2.</b>
Figure 2.
Evaluation of GFP expression in mouse retinas. Representative images from eyes injected with AAV5-GFP vectors containing the PR1.1 promoter (A), PR1.5 promoter (B), PR1.7 promoter (C), or PR2.1 promoter (D) were taken at identical exposure settings at an original magnification of ×20. GFP labeling (green) was seen in the outer nuclear layer (i.e., photoreceptor cell bodies), inner and outer segments, and to a lesser extent in the retinal pigment epithelium. GC, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; IS/OS, photoreceptor inner segment–outer segment junction; RPE, retinal pigment epithelium. Scale bar: 45 μm.
<b>Figure 3.</b>
Figure 3.
Evaluation of GFP expression in primate retinas by fundus fluorescence imaging. AAV-GFP vectors containing the PR1.7 promoter (A), PR2.1 promoter (B), or IRBP/GNAT2 promoter (C) were administered by subretinal injection. Areas with GFP expression appear white on a dark background. The camera automatically adjusts the exposure time; the image in (A), with the greatest intensity of GFP expression, has the darkest background color, but for the image in (C), with the weakest intensity of GFP expression, there is little difference between the area of the subretinal bleb and the background.
<b>Figure 4.</b>
Figure 4.
Evaluation of GFP expression in primate retinas by immunohistochemical staining. Retinal sections were obtained after subretinal injection of an AAV2tYF-GFP vector containing the IRBP/GNAT2 promoter (B), PR2.1 promoter (C), or PR1.7 promoter (DF), or from a control uninjected eye (A). Sections were stained with antibodies to GFP (green) and with an antibody to L/M opsin [red in (AD) and (F)] or an antibody to S opsin [red in (E)] and counterstained with DAPI (blue). (A) Background green fluorescence in the retinal pigment epithelium layer. No cone-specific GFP expression was seen in (B) and limited GFP expression was seen in (C). Strong GFP expression was seen in essentially all cones in (D) and (E). (F) An overexposed image to show strong GFP staining in periphery cones without GFP staining in rods. Only the cones are positive for GFP. Arrows in (E) indicate individual S cones. Animal identification numbers are indicated at the bottom left of each panel. OD, right eye; OS, left eye. Scale bars: (AD) 50 μm; (E and F) 20 μm.
<b>Figure 5.</b>
Figure 5.
Representative photopic electroretinography (ERG) tracings 3 months after subretinal injection of AAV vectors in CNGB3-deficient mice. Red tracings are from the treated eye and black tracings are from the untreated eye, except that the wild-type control red tracings are from the right eye and black tracings are from the left eye.

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