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

Abstract

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.

Noninvasive gene delivery to foveal cones for vision restoration

“…Three weeks after subretinal injections, retinal cross-sections were stained with cone arrestin, and GFP expression was examined ( Figure 1, A-C). We found GFP expression in both rod and cone photoreceptors with mCAR promoter, while PR2.1 and PR1.7 led to strong expression mostly in cones, as reported previously (18,19). Using the same vectors, we obtained strikingly different expression patterns after intravitreal delivery (Figure 1, D-F).…”

“…Interestingly, we found a chicken ovalbumin upstream promoter-transcription factor I (COUP-TFI) binding site within this 337-bp sequence (Supplemental Table 1). COUP-TFI has been shown to suppress green opsin gene (Opn1mw) expression in the mouse retina (22) and might thus be accountable for lower expression with the PR2.1 promoter in macaque cones when AAV is delivered subretinally as previously shown (18). Within the same specific 337-bp region, we also found multiple binding sites for generic, ubiquitous activator TFs ( Figure 2B and Supplemental Table 1), such as CCAAT/enhancer binding protein β (CEBPB) and general transcription factor II-I (GTF2I).…”

“…Specific targeting of cone cells has never been attempted using vitreally administered AAV. In order to find suitable promoter sequences for restricted gene expression in cones applicable in the clinic, we focused on promoters that have previously been validated in either nonhuman primate (NHP) (18) or human tissue (4). We generated AAV2-7m8 vectors encoding GFP under the control of mouse cone arrestin (mCAR), PR2.1 and PR1.7 promoters (synthetic promoters based on the human red opsin gene enhancer and promoter sequences -their size is equal to 2.1 and 1.7 kilobases, respectively) and injected them at equal titers into eyes of 6-week-old WT mice.…”

“…To do so, we analyzed transcription factor (TF) binding sites within each promoter sequence using bioinformatics (Supplemental Tables 1 and 2). The present analysis aimed to answer the following questions: (i) why is PR1.7 more efficient than PR2.1 in cones (18), and (ii) why do PR2.1 and mCAR promoters lead to off-target expression after intravitreal administration? We hypothesized that the differential expression patterns observed between PR1.7 and PR2.1 are due to additional TF binding sites found in the 337-bp sequence located in the 5′ region of the PR2.1 promoter but not in the PR1.7 promoter (Figure 2, A and B).…”

Safety and Efficacy Evaluation of rAAV2tYF-PR1.7-hCNGA3 Vector Delivered by Subretinal Injection in CNGA3 Mutant Achromatopsia Sheep

“…[6][7][8] Studies in mouse, sheep, and dog models of achromatopsia caused by mutations in the CNGA3 and CNGB3 genes indicate that gene augmentation therapy using a recombinant adeno-associated virus (AAV) vector expressing a normal CNGA3 or CNGB3 gene can produce the functional CNGA3 or CNGB3 proteins and restore cone photoreceptor function. [9][10][11][12][13] As part of the efforts to develop a product candidate for treatment of humans with ACHM caused by mutations in the CNGA3 gene, an Investigational New Drug (IND)-enabling safety and efficacy study of AGTC-402, an AAV vector containing a conespecific promotor (PR1.7), 14 a codon-optimized human CNGA3 cDNA, and a SV40 polyadenylation sequence packaged in an AAV2 capsid containing three tyrosine to phenylalanine (YF) mutations, was conducted in CNGA3-deficient sheep.…”