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Guo-jie Ye, Sanford L Boye, James Peterson, William W Hauswirth, Savitri Mandapati, Jeffrey D Chulay; Rescue of Cone ERG Function by Treatment with AAV-hCNGB3 Vectors in CNGB3 Knockout Mice. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5477.
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© ARVO (1962-2015); The Authors (2016-present)
To evaluate the efficacy of an AAV vector expressing codon-optimized human CNGB3 cDNA driven by a PR1.7 promoter compared to other cone-specific promoters driving codon-optimized or non-codon-optimized cDNA for rescue of cone function in CNGB3 KO mice.
PR1.7 is a shorter version of the 2.1 kb human red cone opsin promoter (PR2.1). IRBP/GNAT2 is a hybrid promoter consisting of a 277-bp human GNAT2 promoter and a 214-bp IRBP enhancer (Ying et al.,1998). In dogs, PR2.1 directs specific expression of GFP in red/green (L/M) cones but not blue (S) cones (Komaromy et al., 2008), and IRBP/GNAT2 directs specific expression of GFP in both L/M cones and S cones (Komaromy et al., 2013). In nonhuman primates (NHPs) PR1.7 is more efficient than PR2.1 in directing specific expression of GFP in L, M and S cones, while IRBP/GNAT2 is unable to support expression of GFP in cones (Ye et al., 2014). In the present study, the efficiency of functional improvement in cone mediated ERG by AAV vectors expressing hCNGB3 or codon-optimized hCNGB3, driven by PR1.7, PR2.1 or IRBP/GNAT2 promoters, packaged in AAV5 capsids and delivered by subretinal injection, was evaluated in CNGB3 KO mice. Five groups of 10 animals each received an AAV vector (1 µL at a concentration of 5 × 10^12 vg/mL) in one eye by subretinal injection. The contralateral eye received either vehicle or no treatment. Within each group, maximum photopic b-wave amplitudes generated at 10 dB from treated and control eyes were averaged and used for comparison.
Statistically significant improvements in cone ERG responses were observed in mice treated with AAV vectors containing codon-optimized or non-codon-optimized hCNGB3 cDNA driven by IRBP/GNAT2, and with AAV vectors containing non-codon optimized hCNGB3 cDNA driven by PR2.1 or PR1.7, but not with AAV vectors containing codon optimized hCNGB3 cDNA driven by PR2.1. Between-group comparisons demonstrated greater cone ERG responses with non-codon optimized than codon-optimized hCNGB3 cDNA and with the IRBP/GNAT2 promoter than with PR2.1 or PR1.7 promoters.
Although the AAV vector designed for maximal efficiency in primates was not as effective in CNGB3 KO mice, critically this study confirms that the hCNGB3 cDNA codon optimized based on human codon usage and the PR1.7 promoter optimized for expression efficiency in NHPs are functional and able to improve cone function in this model of ACHM.
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