Molecular Vision 2018; 24:834-846
Received 25 April 2018 | Accepted 28 December 2018 | Published 30 December 2018
1Department of Ophthalmology, John A. Moran Eye Center, University of Utah Health Science Center, Salt Lake City, UT; 2Department of Ophthalmology, University of Florida, Gainesville, FL; 3Department of Neurobiology and Anatomy, University of Utah Health Science Center, Salt Lake City, UT; 4Department of Biology, University of Utah, Salt Lake City, UT
Correspondence to: Wolfgang Baehr, Department of Ophthalmology and Visual Sciences, University of Utah Health Science Center, Salt Lake City, UT, 84132; Phone: (801) 585-6643; FAX: (801) 587-7686; email: firstname.lastname@example.org.
Dr. Christin Hanke-Gogokhia is now at the Dyson Vision Research Institute, Weill Cornell Medical College, 1300 York Avenue, New York, NY, 10065.
Purpose: Recessive mutations in the human IQCB1/NPHP5 gene are associated with Senior-Løken syndrome (SLS), a ciliopathy presenting with nephronophthisis and Leber congenital amaurosis (LCA). Nphp5-knockout mice develop LCA without nephronophthisis. Mutant rods rapidly degenerate while mutant cones survive for months. The purpose of this study was to reinitiate cone ciliogenesis in a Nphp5−/−; Nrl−/− mouse with viral expression of full-length NPHP5 and rescue function.
Methods: Nphp5−/− mice were mated with Nrl−/− mice to generate Nphp5−/−; Nrl−/− double-knockouts. Nphp5−/−; Nrl−/− mice and Nphp5+/−; Nrl−/− controls were phenotyped with confocal microscopy from postnatal day 10 (P10) until 6 months of age. Nphp5−/−; Nrl−/− mice and Nphp5+/−; Nrl−/− controls were injected at P15 with self-complementary adenoassociated virus 8 (Y733F) (AAV8(Y733F)) expressing GRK1-FL-cNPHP5. Expression of mutant NPHP5 was verified with confocal microscopy and electroretinography (ERG).
Results: In the Nphp5−/− and cone-only Nphp5−/−; Nrl−/− mice, cone outer segments did not form, but mutant cones continued to express cone pigments in the inner segments without obvious signs of cone cell death. The mutant cone outer nuclear layer (ONL) and the inner segments were stable for more than 6 months in the cone-only Nphp5−/−; Nrl−/− retinas. Viral expression of NPHP5 initiated after eye opening showed that connecting cilia and RP1-positive axonemes were formed. Furthermore, cone pigments and other cone outer segment proteins (cone transducin and cone PDE6) were present in the nascent mutant cone outer segments, and rescued mutant cones exhibited a significant photopic b-wave (30% of Nphp5+/−; Nrl−/− controls).
Conclusions: Nphp5−/−; Nrl−/− cones persistently express cone pigments in the inner segments without obvious degeneration, providing an extended duration interval for viral gene expression. Viral expression of full-length NPHP5 initiates ciliogenesis between P15 and P60, and mutant cones are, in part, functional, encouraging future retina gene replacement therapy.
Nephrocystins (NPHP1–18) are a large group of proteins expressed in renal epithelia and other ciliated cells, including photoreceptors. NPHP polypeptides are highly conserved among invertebrate (Chlamydomonas reinhardtii and Caenorhabditis elegans) and mammalian primary cilia and localize to the basal body and transition zones [1-4]. In renal epithelia, NPHP1, -4, -5, -6, and -8 are present in the transition zone (TZ), whereas NPHP2, -3, and -9 localize to the adjacent Inversin (Inv) compartment . Mutations in nephrocystin genes are associated with several syndromic ciliopathies, including Joubert [5-10], Senior-Løken [11-13], Meckel [14-17], Cogan, and Bardet-Biedl syndromes [18-20]. NPHP1 , NPHP4 , IQCB1/NPHP5 , CEP290/NPHP6 [24-26], SDCCAG8/NPHP10 [27,28], and TMEM67/NPHP11 (meckelin)  are known to be associated with retinal degeneration. Mutations in the IQCB1/NPHP5 gene (OMIM 609237) are the most common cause of Senior-Løken syndrome (SLS) in humans [30,31].
SLS is an autosomal recessive retina-renal ciliopathy characterized by photoreceptor dysfunction and progressive degeneration, accompanied by nephronophthisis (NPHP) [32,33]. The retina degeneration usually resembles retinitis pigmentosa (RP)  or Leber congenital amaurosis (LCA) . LCA and RP are characterized by the presence of nonfunctional rods and cones at birth or early in life and progressive loss of rod photoreceptors, respectively . NPHP presents with diminished kidney size, corticomedullary cysts, and tubulointerstitial fibrosis . Most mutations in the human IQCB1 gene are stop codons or frame-shift mutations truncating NPHP5. The retina phenotype of NPHP5-LCA is severe, as the outer nuclear layer (ONL) is barely detectable in young patients . Surprisingly, nonfunctional cones and cone nuclei are retained at the fovea suggesting that the mutant retina may provide a large window for gene therapy. Mutations in NPHP5 have also been identified in patients with non-syndromic LCA [23,31] and in naturally occurring animal models [37,38].
Human, mouse, and canine NPHP5 each consist of 598 amino acids and are 90% identical without gaps. NPHP5 polypeptides contain BBSome interaction sites, IQ calmodulin binding motifs, coiled-coil domains, and a C-terminal CEP290 binding site [32,39,40]. The function of NPHP5 is unknown. Germline Nphp5-knockout mice, deleting exons 5–15 and removing most IQ and coiled-coil domains, are blind at eye opening exhibiting LCA , a phenotype that recapitulates the human pathology of rapid retinal degeneration. Nphp5−/− mice do not develop nephronophthisis even at 1 year of age suggesting non-syndromic ciliopathy, perhaps caused by nephrocystin redundancy in kidneys. Basal bodies in Nphp5−/− photoreceptors dock to the cell membrane, but fully developed transition zones do not form, and outer segments fail to develop. Ultrastructure of postnatal day (P6) and P10 Nphp5−/− photoreceptors reveal aberrant transition zones of reduced diameter.
We generated Nphp5−/−; Nrl−/− double-knockout mice to study the cone degeneration rate and to explore the duration of any window for future gene therapy experiments. We found that although in Nphp5−/−; Nrl−/− mice cone outer segments do not form, the mutant cone outer nuclear layer is stable up to 6 months of age. Mutant cones continued to express cone pigments in the inner segments, axons, and endoplasmic reticulum surrounding nuclei, without recognizable degeneration. Employing self-complementary adenoassociated virus 8 (scAAV8) vectors, we expressed canine NPHP5 (cNPHP5) to test whether ciliogenesis and the formation of the outer segments may be reinitiated postnatally. The results show that functional axonemes and outer segments form in part upon delivery of scAAV8 expressing NPHP5. Moreover, cone phototransduction proteins localize normally to the outer segments, and mutant cones respond to light.
Procedures were approved by the University of Utah Institutional Animal Care and Use Committee and were conducted in compliance with the NIH Guide for Care and Use of Laboratory Animals. The protocol was approved by the University of Utah Animal Care and Use Committee, and all animals were treated according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Nphp5 mutant animals were previously generated in our laboratory  and maintained in a 12 h:12 h light-dark cycle. An Nrl−/− mouse (JAX stock # 021,152) was used to generate the cone-only retina. A transgenic mouse expressing EGFP-CETN2 fusion protein (JAX stock # 008,234) was used to identify the centrioles and transition zones with fluorescence microscopy .
Nphp5−/− male mice were bred with Nrl−/− female mice to generate Nphp5+/−; Nrl+/− double heterozygotes. Male and female Nphp5+/−; Nrl+/− mice were bred to generate double-knockouts. Nphp5−/− mice and Nrl−/− mice were genotyped (Figure 1A) as described [30,42]. Nphp5 mutant animals were genotyped using primers Nphp5F (5’-CCT TTA GGG TGA TAG TAG CCA ATT CC), Nphp5Rw (5’-AGG AAC TAA GCT GTG AAA TGG ACC) and Nphp5mut (5’-CAA CGG GTT CTT CTG TTA GTC C). The WT allele yields an amplicon of 452 bp; mutant allele, 294 bp. The Nrl allele was detected by amplification of a 607-bp fragment of Nrl exon 3 (forward primer, 5′–GCT GGT CTC GAT GTC TGT–3′; reverse, 5′–CAT TCA GCA TGC CAC CTG–3′).
Animals were dark-adapted overnight and euthanized by cervical dislocation under dim red light. Dissection of the eyecups and retina cryosections for confocal microscopy were prepared as described . Briefly, sections were incubated with the following polyclonal primary antibodies: anti-M/L-opsin (1:500; Chemicon, Fisher Scientific, Salt lake City, UT), anti-S-opsin (1:500; Chemicon), anti-RP1 (1:1,000, a generous gift from Drs. Qin Liu and Eric Pierce, Harvard), anti-cone PDE6 (cPDE6, 1:500, a generous gift from Dr. Tiansen Li, NEI), anti-cone-transducin-α (anti-cone Tα, 1:500; Santa Cruz, Dallas, TX), and anti-cone-transducin-γ (anti-cone Tγ, 1:500; Cytosignal). Monoclonal antibodies included anti-Flag (1:100, Sigma). Secondary antibodies Alexa Fluor 555-conjugated goat-anti-rabbit, Alexa Fluor 555-conjugated goat-anti-mouse, and Alexa Fluor 555-conjugated goat-anti-chicken were diluted 1:1,000 in blocking solution (2% bovine serum albumin [BSA], 0.1% Triton X-100, 0.1 M phosphate buffer pH 7.4). Images were acquired using a Zeiss LSM800 confocal microscope.
Photopic electroretinogram (ERG) responses were recorded from 2-month-old Nphp5+/−; Nrl−/− and Nphp5−/−; Nrl−/− mice using a UTAS BigShot Ganzfeld system (LKC Technologies, Gaithersburg, MD). ERGs were measured as described [44,45]. Briefly, mice were light-adapted under a background light of 1.48 log cd s·m−2 for 5–10 min. Single-flash responses of treated and untreated eyes were recorded at stimulus intensities of −1.6 log cd·s·m−2 to 2.4 log cd·s·m−2.
Canine NPHP5 (cNPHP5) expression cassette with an N-terminal Flag tag under the control of the human G-protein-coupled receptor kinase (hGRK1) promoter was packaged into self-complementary AAV2/8 (Y733F;P4211) virus particles as described [46,47]. scAAV8-cNPHP5 virus (1 μl of virus with a titer of 2.27E+12) was injected subretinally shortly after eye opening, between P12 and P15, in the right eye (the treated eye). The left eye was used as the untreated control eye. A total of five of each mutant and control mice were used for virus injection. Eyes were harvested at 2 months of age (approximately 6 weeks post-injection) and evaluated with immunohistochemistry and ERG for retinal rescue.
SigmaPlot12 was used for statistical analysis using Student t test and F test (ANOVA). A p value of 0.05 was considered statistically significant.
Nphp5−/− germline knockout mice recapitulate the human pathology of rapid retinal degeneration mimicking an LCA phenotype . During postnatal development of the photoreceptors, the absence of NPHP5 did not prevent proper anchoring of the basal body to the cell cortex, but the transition zone (the connecting cilium) did not form properly, and rod and cone outer segments were absent . We followed the fate of the cones in the Nphp5−/− retina in more detail (Figure 1B–E). ML-opsin is expressed in Nphp5+/+ controls at P10 (Figure 1B, left panel) and is present in cone outer segments (COS), while ML-opsin accumulates in the IS and the ONL of Nphp5−/− cones (Figure 1B, right panel). At P15, COS are near-normal length in the control mice but absent in the single-knockout mice (Figure 1C). At 1 month of age, the Nphp5−/− ONL is reduced to a single layer of nuclei, presumably cones, as ML-opsin is still present around the nuclei of cell remnants (Figure 1D, right panel). A single nuclear layer is still partly retained at 2 months (Figure 1E, right panel) suggesting that cones may survive longer than rods. The degeneration of Nphp5−/− rod photoreceptors is complete at 1 month of age (Figure 1D,N).
We generated Nphp5−/−; Nrl−/− double-knockout mice. We confirmed that rods and rhodopsin are absent in cone-only retinas (Figure 1F, shown for the heterozygous control). In the Nphp5−/−; Nrl−/− mutant mice and the Nphp5+/+; Nrl−/− control mice, cone outer segments are undetectable at P10, and ML-opsin appears to accumulate around some cone nuclei (Figure 1G) suggesting delayed ML-opsin synthesis compared to the wild type (Figure 1B, left panel). At P15 (Figure 1H, left panel), Nphp5+/+; Nrl−/− COS develop and reach full-length between 1 and 2 months (Figure 1I,J, left panel). In the Nphp5−/−; Nrl−/− double-knockout mutants, cone outer segments never develop, and ML-opsin accumulates in the inner segments and cell bodies (Figure 1H–M, right panels). At 23 months, a significant accumulation of ML-opsin in the cone ONL and the OPL is observed (Figure 1L, right panel). The Nphp5−/−; Nrl−/− cone inner segments and the Nphp5+/+; Nrl−/− cone outer segments survived up to 6 months, and the ONL of cone-only mutants and controls is slightly reduced at this age (Figure 1M, graph Figure 1N). The unexpected survival of mutant cones that are unable to receive and transduce light suggests a relatively long interval to develop AAV vectors expressing NPHP5 to restart cone ciliogenesis and rescue function.
The vector scGRK1-FL-cNPHP5 (Figure 2A) expresses full-length canine NPHP5 (FL-cNPHP5) with an N-terminal Flag-tag (Flag) under the control of the human GRK1 promoter directing expression to rods and cones. Canine NPHP5 is 88% identical with human and mouse NPHP5 (93% sequence similarity), and the three polypeptides are of identical length, suggesting an identical function (Figure 2B,C). The calmodulin-binding IQ motifs are essentially identical (Figure 2B). The vector was packaged into a self-complementary (Y733F;P4211) capsid mutant of AAV2/8 virus that was injected into the subretinal space at P12–P15, and the eyes were harvested 6 weeks post-injection. A total of five each of single-knockout Nphp5+/− and Nphp5-/- and double-knockout Nphp5+/−; Nrl−/− and Nphp5−/−; Nrl−/− mice were injected. The heterozygous controls were comparable to the wild-type controls suggesting haplosufficiency (reported in this and a previous study .
There were no statistically significant differences in the a- and b-wave responses in the photopic ERGs of the untreated and treated Nphp5+/−; Nrl−/− retinas at two different light intensities (1.4 Log cd·s·m−2 and 2.4 Log cd·s·m−2), indicating an intact retina/RPE after the subretinal virus injection (Figure 3A,B). The uninjected Nphp5−/−; Nrl−/− double-knockout retina shows no photopic response even at high light intensity (2.4 Log cd·s·m−2) indicating nonfunctional cone photoreceptors (Figure 3A–D). Treatment with FL-cNPHP5 AAV (AAV+) rescues cone function in mutant animals as there is a 30% increase in photopic ERG (at light intensities of 0.9 Log cd·s·m−2 and higher) relative to that for the untreated mice (Figure 3A–D). The statistically significant increase in the photopic b-wave amplitude (Figure 3C) and the absence of the scotopic a-wave are consistent with a cone-only retina.
In the virus-injected Nphp5+/− retina containing a stable ONL and exhibiting no degeneration at 6 weeks post-injection, Flag-tagged FL-cNPHP5 (red, Figure 4A, right panel) is detectable in the proximal outer segments. The inset in the figure shows photoreceptor cells in which centrioles and a connecting cilium (CC), identified with transgenic EGFP-CETN2 (eCetn2+) expression, are recognized (green). In the treated Nphp5−/− retina (Figure 4B, right panel), expression of Flag-tagged FL-cNPHP5 is detectable, and the mutant ONL has recovered slightly to two layers of nuclei (or is not as degenerated as the 2-month-old untreated Nphp5−/− retina; Figure 4B, left panel). At 2 months, the Nphp5−/− photoreceptors are completely degenerated (Figure 4B, left panel; Figure 1E, right panel).
The uninjected and treated (AAV+) Nphp5+/−; Nrl−/− retina shows normal ciliogenesis (Figure 4C), while the treated heterozygotes show strong expression of Flag-tagged FL-cNPHP5 (red) in the proximal COS. In the uninjected Nphp5−/−; Nrl−/− retina, a connecting cilium does not develop (Figure 4D, left panels), while in the treated retina, Flag-tagged FL-cNPHP5 accumulates in the proximal outer segments (Figure 4D, right panels), resembling the localization of Flag-cNPHP5 in the control retina (Figure 4C, right panels) and suggesting the initiation of normal ciliogenesis. This effect is reproducible in four additional animals. The results indicate the presence of a connecting cilium and a rudimentary outer segment in the Nphp5−/−; Nrl−/− retina formed upon injection of a virus expressing full-length cNPHP5.
We examined the spread of the injected virus by analyzing the expression of cone PDE6 (cPDE) in Nphp5−/−; Nrl−/− whole transverse sections. Uninjected heterozygous controls on the Nrl−/− background show uniform cone PDE6 expression in the cone outer segments, both nasally and temporally (Figure 4E). In the untreated Nphp5−/−; Nrl−/− retina (Figure 4F), in contrast, cone PDE6 is undetectable (the few red dots of Figure 4F are RPE artifacts), as no outer segments form, and cone PDE6 is degraded. In the treated heterozygous controls, cone PDE6 is present in the OS layer, both nasally and temporally (Figure 4G). In the treated double-knockouts, cone PDE6 can be detected only on the nasal side near the point of injection (white asterisk, Figure 4H).
As a measure of the formation of outer segments, we selected an antibody directed against retinitis pigmentosa protein 1 (RP1), a microtubule-associated protein that interacts with proximal microtubules of the axoneme, but not the doublet microtubule array of the transition zone (the connecting cilium) or basal body . In the untreated and treated Nphp5+/− retinas with normally developed photoreceptors (Figure 5A), RP1 (red) labels the proximal OS of rods and cones, nicely visible in the enlarged insets of Figure 5A. In the treated Nphp5−/− retina (Figure 5B, right panel), a functional CC and a proximal OS are absent as the degeneration is fast.
The RP1 antibody labels the axoneme in the treated and untreated undegenerated Nphp5+/−; Nrl−/− retinas (Figure 5C). In the untreated Nphp5−/−; Nrl−/− retina, the axoneme and the outer segments are absent (Figure 5D, left panel). Treatment with scAAV8-cNPHP5 rebuilds the transition zone and the axoneme of mutant mice (Figure 5D, right panel). Enlargements of the treated Nphp5−/−; Nrl−/− retina (Figure 5D, lower panel) reveal that each cone basal body extends a transition zone (white arrows) and an axoneme (red). Lower panels of the untreated Nphp5−/−; Nrl−/− retina (Figure 5D, left panel) show only basal bodies and daughter centrioles with eCetn2+ dispersion within the distal inner segment. Accumulation of soluble eCetn2+ in the Nphp5−/−; Nrl−/− inner segments is characteristic of other mouse models that are unable to form axonemes and transition zones .
Cone pigments (red) localize normally to relatively disorganized membranes of the treated and untreated Nphp5+/−; Nrl−/− outer segments (Figure 6A,C, and lower panel enlargements) and do not colocalize with eCetn2+ (green). In the untreated Nphp5−/−; Nrl−/− retina, S-opsin and ML-opsin mislocalize within the inner segments and the ONL due to the total absence of outer segments (Figure 6B,D, left and lower panels). Treatment of the Nphp5−/−; Nrl−/− retina with Flag-tagged FL-cNPHP5 enables formation of outer segments and therefore, allows correct trafficking of cone pigments to the outer segments (Figure 6B,D, right and lower panels).
Cone PDE6 and cone transducin (cTα and cTβγ) traffic normally to the untreated and treated Nphp5+/−; Nrl−/− cone outer segments where phototransduction occurs (Figure 6, E,G,I). In the untreated Nphp5−/−; Nrl−/− retina, phototransduction proteins mislocalize to the inner segments and the ONL, as outer segments fail to form, and are degraded (Figure 6F,H,J, left panels). Treatment with scAAV8 expressing FL-cNPHP5 reinitiates development of the CC and formation of rudimentary outer segments, such that cPDE6, cTα, and cTγ relocate to the outer segments (Figure 6F,H,J, right panels).
Nphp5−/− mice are blind at eye opening (P12), a phenotype resembling Leber congenital amaurosis. Photoreceptors deficient in NPHP5 are unable to form connecting cilia and outer segments, the site of phototransduction. Mutant rods degenerate rapidly although a single row of nuclei, presumably those of cones, survives much longer (Figure 1E, right panel) and . We showed that in Nphp5−/−; Nrl−/− (cone-only) mice, cone nuclei and inner segments are able to survive for more than 6 months (Figure 1N). The thickness of the cone double-knockout ONL persists, similarly as observed in the Cep290/Nphp6rd16/rd16/Nrl double-knockout retina . Nphp5−/−; Nrl−/− cones continue to express cone pigments without triggering cone degeneration (Figure 1G–M). With the application of AAV gene replacement therapy with a vector expressing canine NPHP5 (Figure 2), transition zones and cilia in Nphp5−/−; Nrl−/− cones were reestablished (Figures 4–6), and cone outer segment proteins trafficked normally to the outer segment membranes (Figure 6). Six weeks postinjection, ERG of the AAV-treated retina showed a photopic b-wave roughly one third of the control (Figure 3), suggesting a significant number of cones had been rescued and were functional.
Photoreceptor ciliogenesis in Nphp5 germline knockouts was arrested at a stage when basal bodies are docked to the cortex of the inner segments but are unable to generate an axoneme and functional transition zones . The most obvious defect is the formation of a few stunted transition zones to which amorphous bags lacking discernible disc structures are attached . As shown in Figure 4D and Figure 5D, the association of CETN2 with centrioles is impaired resulting in solubilization and dispersion of CETN2 to the inner segments. Similar results were observed in the Arl3−/− mouse which is unable to form an axoneme . In wild-type mice, docking of the basal body and generation of the transition zone consisting of nine doublet microtubules takes place around P3 and P4. Around P6 and P7, an axoneme emerges, and the first stacks of discs are synthesized. The formation of the outer segments is complete at P21 [49,50]. Inability to form transition zones has been observed in various retina-specific knockouts, including KIF3a, the obligatory subunit of heterotrimeric kinesin-2  and the small GTPases, ARL3 and ARL13b [45,52]. Absence of ARL3-GTP has been linked to defects in photoreceptor intraflagellar transport (IFT), a pathway essential for the formation of the transition zone and axonemes. The nearly identical photoreceptor phenotypes of Nphp5−/−, retKif3a−/−, and retArl3−/− mice raise the possibility that NPHP5 participates in some aspect of IFT.
Based on tissue culture experiments with IMCD3, HEK293, and RPE cell lines, NPHP5 and Cep290/NPHP6 are known to form a complex localizing to the transition zone, possibly being part of a gate complex. With the use of transmission electron microscopy of the mouse retina, NPHP6 was shown to be part of the Y linkers connecting the microtubule doublets with the ciliary membrane . Cep290−/− photoreceptor basal bodies formed but did not dock to the apical cell membrane and therefore, never formed a transition zone. According to immunohistochemistry, NPHP5 and NPHP6 also locate to the proximal outer segments [30,32]. Cep290 germline knockouts with deletions of exons 1–4 died before weaning due to the development of syndromic ciliopathy and hydrocephalus , a phenotype much stronger than that seen with germline Nphp5 deletions. The homozygous mutants exhibit a Joubert syndrome (JBTS)/ciliopathy phenotype, including retinal degeneration, cerebral abnormalities, and progressive cystic kidney disease consistent with the human phenotype. Obviously, the in vivo functions of NPHP5 and NPHP6 are distinct and not complementary.
In other mammalian species, Nphp5−/− ciliary phenotypes differ, perhaps because stable truncated proteins are generated. In a Crd2 canine animal model (American pit bull terrier) with a p.S319fsX12 premature truncation of NPHP5, one of the two IQ calmodulin binding motifs and the two central coiled-coil motifs are present in the truncated protein. The mutant photoreceptors form a sensory cilium but are nearly nonfunctional. At 6 weeks, the ONL looks normal, and the rod ERG is absent, but mutant dogs show a weak photopic b-wave suggesting that mutant cones survive longer [38,54]. A 2 bp deletion in exon 13 of NPHP5 in an African black-footed cat (Felix nigripes, Fnig) truncates NPHP5 (p.L428*), removing 170 amino acids at the C-terminal of NPHP5, including Cep290 interaction sites. The mutation is associated with early onset non-syndromic progressive retinal atrophy (PRA) where the ERG is non-recordable. It is unclear at which point ciliogenesis is interrupted in the cat model.
Gene replacement therapies for patients with Senior-Løken syndrome or other syndromic ciliopathies are currently not available, mostly because gene delivery to internal organs like kidneys is challenging, and AAV gene delivery is restricted by size. Furthermore, the application window is often short as organs degenerate before the gene of interest can be expressed. Fortunately, this laboratory-generated mouse model and naturally occurring canine and cat models harboring NPHP5 gene defects do not develop nephronophthisis, allowing researchers to test retina gene replacement strategies in an otherwise healthy and fertile animal. These results show that Nphp5−/− cones are initially viable, and the cone-only mutant mice provide an extended time interval during which mutant cones can be rescued with viral expression of NPHP5. As cones are most important for daylight vision, these results provide hope for patients with NPHP5-SLS that visual defects may be ameliorated in the future with gene-based therapies.
This work was supported in part by NIH grants R01EY008123 (WB), R01EY019298 (WB), P30EY014800 (NEI core grant); unrestricted grants to the University of Utah Department of Ophthalmology from Research to Prevent Blindness (RPB; New York). WB is the recipient of an RPB Senior Investigator award, a RPB Nelson Trust Award, and an award from the Retina Research Foundation (Alice McPherson, MD), Houston, TX.