Functional Study of Two Biochemically Unusual Mutations in GUCY2D Leber Congenital Amaurosis Expressed Via Adenoassociated Virus Vector in Mouse Retinas

Molecular Vision 2016; 22:1342-1351 <http://www.molvis.org/molvis/v22/1342> © 2016 Molecular Vision Received 31 August 2016 | Accepted 8 November 2016 | Published 10 November 2016 Functional study of two biochemically unusual mutations in GUCY2D Leber congenital amaurosis expressed via adenoassociated virus vector in mouse retinas Sanford L. Boye,1 Elena V. Olshevskaya,2 Igor V. Peshenko,2 K. Tyler McCullough,1 Shannon E. Boye,1 Alexander M. Dizhoor2 (The first three authors contributed equally to the study.) 1Department of Ophthalmology, University of Florida, Gainesville, FL; 2Pennsylvania College of Optometry, Salus University, Elkins Park, PA Purpose: To test, in living photoreceptors, two mutations, S248W and R1091x, in the GUCY2D gene linked to Leber congenital amaurosis 1 (LCA1) that fail to inactivate the catalytic activity of a heterologously expressed retinal membrane guanylyl cyclase 1 (RetGC1). Methods: GUC2YD cDNA constructs coding for wild-type human (hWT), R1091x, and S248W GUCY2D under the control of the human rhodopsin kinase promoter were expressed in Gucy2e−/−Gucy2f−/− knockout (GCdKO) mouse retinas, which lack endogenous RetGC activity. The constructs were delivered via subretinally injected adenoassociated virus (AAV) vector in one eye, leaving the opposite eye as the non-injected negative control. After testing with electroretinog- raphy (ERG), the retinas extracted from the AAV-treated and control eyes were used in guanylyl cyclase activity assays, immunoblotting, and anti-RetGC1 immunofluorescence staining. Results: Cyclase activity in retinas treated with either hWT or R1091x GUCY2D transgenes was similar but was un- detectable in the S248W GUCY2D-treated retinas, which starkly contrasts their relative activities when heterologously expressed in human embryonic kidney (HEK293) cells. Rod and cone ERGs, absent in GCdKO, appeared in the hWT and R1091x GUCY2D-injected eyes, while the S248W mutant failed to restore scotopic ERG response and enabled only rudimentary photopic ERG response. The hWT and R1091x GUCY2D immunofluorescence was robust in the rod and cone outer segments, whereas the S248W was detectable only in the sparse cone outer segments and sporadic photoreceptor cell bodies. Robust RetGC1 expression was detected with immunoblotting in the hWT and R1091x-treated retinas but was marginal at best in the S248W GUCY2D retinas, despite the confirmed presence of the S248W GUCY2D transcripts. Conclusions: The phenotype of S248W GUCY2D in living retinas did not correlate with the previously described normal biochemical activity of this mutant when heterologously expressed in non-photoreceptor cell culture. This result suggests that the S248W mutation contributes to LCA1 by hampering the expression, processing, and/or cellular transport of GUCY2D, rather than its enzymatic properties. In contrast, the effective restoration of rod and cone function by R1091x GUCY2D is paradoxical and does not explain the severe loss of vision typical for LCA1 associated with that mutant allele. The GUCY2D gene (Gene ID: 3000; OMIM 600179) RetGC2 (GUCY2F) [5,6], the former catalyzes most of the codes for the human retinal membrane guanylyl cyclase 1 cGMP production in mammalian photoreceptors [7,8]. Note (RetGC1) isozyme and is one of the genes most frequently that the RetGC1 ortholog in mice is coded by the Gucy2e associated with the early onset, inherited recessive blindness, gene [6]; therefore, only the human RetGC1 will be further Leber congenital amaurosis type 1 (LCA1) [1-3]. The acute referred to as GUCY2D (wherever the difference in gene- loss of vision in patients with GUCY2D LCA1 results not based nomenclature between species can cause confusion, from the severe degeneration of rods and cones [3] but from we use the biochemical names, RetGC1 and RetGC2). cGMP the inefficient production in the diseased photoreceptors synthesis becomes decelerated in the dark and accelerated in of the second messenger in phototransduction, cyclic GMP the light by guanylyl cyclase activating proteins (GCAPs) [9]. (cGMP) [4]. Of the two existing retinal membrane guanylyl These proteins are Ca2+-sensor proteins that switch between cyclase isozymes, RetGC1 (GUCY2D in humans) and their Ca2+- and the Mg2+-bound states following the respective increase or decrease of Ca2+ influx through the cGMP gated channels between dark and light [10]. Another photoreceptor Correspondence to: A.M. Dizhoor, Pennsylvania College of Optometry, Research, S-416, Salus University, 8360 Old York Road, protein, retinal degeneration 3 (RD3) [11,12], reduces RetGC1 Elkins Park, PA, 19027; Phone: +1 215-780-1468; FAX: +1-215-780- activity by competing with GCAPs [13] and is essential for 1464; email: [email protected] the normal production of RetGC1 and its transport to the 1342 Molecular Vision 2016; 22:1342-1351 <http://www.molvis.org/molvis/v22/1342> © 2016 Molecular Vision outer segment [14-16]. We previously showed, in vitro, that in GCdKO photoreceptor outer segments and enabled well- different mutations linked to GUCY2D LCA1 inactivate the detectable rod and cone photoresponses. catalytic activity of RetGC1 and/or suppress its ability to bind GCAPs and RD3 [15,17]. METHODS Rod and cone function can be sustainably restored in Animals: The mouse Gucy2e−/− gene knockout line [23] mice that lack endogenous orthologs of RetGC1 (Gucy2e−/−) that originated from the D. Garbers laboratory (University and RetGC2 (Gucy2f−/−; GCdKO) [18] by delivering functional of Texas) and the Gucy2f−/− gene knockout line [18] that GUCY2D cDNA with adenoassociated viral (AAV) vectors originated from the W. Baehr laboratory (University of [19,20]. Because the majority of patients with GUCY2D LCA1 Utah) were bred into a homozygous (Gucy2e−/−Gucy2f−/−) exhibit preservation of the retinal structure, an opportunity state. All experiments were approved by the University of exists to consider a human gene therapy to rescue vision Florida and Salus University’s Institutional Animal Care and in this disease [3,21]. Among the reported recessive alleles Use Committee and were conducted in accordance with the linked to GUCY2D LCA1 [1-3], the properties of two muta- Association for Research in Vision and Ophthalmology’s tions in GUCY2D in vitro would not explain the virtually (ARVO’s) Statement for the Use of Animals in Ophthalmic complete lack of photoresponses in patients with LCA1 [3]. and Vision Research and with National Institutes of Health These mutations include a missense mutation, Ser248→Trp, regulations. Animals were maintained at the University of in the N-terminal extracellular domain of the enzyme (Figure Florida Health Science Center Animal Care Services Facility 1) [3], and a nonsense mutation, R1091x [1,3], that truncates under a 12 h:12 h light-dark cycle. Food and water were avail- a short C-terminal region, downstream from the catalytic able ad libitum. Animals were shipped to Salus University for domain (Figure 1). Unlike other LCA1-linked GUCY2D terminal assays. mutants shown in vitro [3,17,22] to completely inactivate the AAV vector construction and virus packaging: The original enzymatic activity and regulation of the RetGC1 isozyme vector backbone has been previously described in detail 2+ by Ca and GCAPs, the biochemical properties remained [24]. The recombinant AAV vector plasmids contained substantially preserved in R1091x and remained completely flanking AAV2 inverted terminal repeats, human rhodopsin normal in S248W. kinase promoter, an SV40 splice donor-acceptor site, human By using subretinal delivery via AAV in the present GUCY2D cDNA coding for wild-type or LCA1 mutants, study, we expressed in mouse retinas that lacked endogenous and a bovine growth hormone polyadenylation signal. Wild- cyclase activity variants of human GUCY2D cDNA that type GUCY2D cDNA (hWT) was synthesized de novo and harbor the two mutations linked to LCA1 and tested their contained silent mutations that remove several restriction physiologic effects in living photoreceptors. We found that sites to facilitate molecular cloning. The S248W GUCY2D- S248W GUCY2D, despite its maintenance of full catalytic containing vector plasmid was constructed by replacing the capacity as a recombinant protein in vitro, was completely Eco47III-flanked fragment coding for the S248W mutation. unable to sustain rod function and failed to accumulate in The R1091x GUCY2D vector was constructed by replace- rod outer segments in GCdKO mice. S248W GUCY2D ment of the NarI/SalI fragment containing the TGA codon appeared in only a small number of sparse GCdKO cone outer at position 1091. All constructs were verified with DNA segments, thus mediating barely detectable cone responses. sequencing and packaged using a modified AAV8 capsid In contrast, AAV-mediated R1091x GUCY2D accumulated protein containing a single Tyr733Phe substitution. The Figure 1. Schematic representation of the GUCY2D (RetGC1) primary structure [5,6]. The S248W missense mutation is located in the extracellular domain, and the R1091x truncation is located near the C-terminus, downstream of the catalytic domain. LP = leader peptide; ECD = extracellular domain; TM = transmembrane domain; KHD = kinase homology domain; DD = dimerization domain; CAT = catalytic domain. 1343 Molecular Vision 2016; 22:1342-1351 <http://www.molvis.org/molvis/v22/1342> © 2016 Molecular Vision AAV particles were manufactured, purified, and titered GCAP1 were obtained by three rounds of sonication on ice as described elsewhere [25,26]. The titers of the viruses

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