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Treatment Potential for LCA5-Associated Leber Congenital Amaurosis

Purpose To determine the therapeutic window for gene augmentation for Leber congenital amaurosis (LCA) associated with mutations in LCA5. Methods Five patients (ages 6–31) with LCA and biallelic LCA5 mutations underwent an ophthalmic examination including optical coherence tomography (SD-OCT), full-field stimulus testing (FST), and pupillometry. The time course of photoreceptor degeneration in the Lca5gt/gt mouse model and the efficacy of subretinal gene augmentation therapy with AAV8-hLCA5 delivered at postnatal day 5 (P5) (early, n = 11 eyes), P15 (mid, n = 14), and P30 (late, n = 13) were assessed using SD-OCT, histologic study, electroretinography (ERG), and pupillometry. Comparisons were made with the human disease. Results Patients with LCA5-LCA showed a maculopathy with detectable outer nuclear layer (ONL) in the pericentral retina and at least 4 log units of dark-adapted sensitivity loss. The Lca5gt/gt mouse has a similarly severe and rapid photoreceptor degeneration. The ONL became progressively thinner and was undetectable by P60. Rod- and cone-mediated ERGs were severely reduced in amplitudes at P30 and became nondetectable by P60. Subretinal AAV8-hLCA5 administered to Lca5gt/gt mice at P5 and P15, but not at P30, resulted in structural and functional rescue. Conclusions LCA5-LCA is a particularly severe form of LCA that was recapitulated in the Lca5gt/gt mouse. Gene augmentation resulted in structural and functional rescue in the Lca5gt/gt mouse if delivered before P30. Retained photoreceptors were visible within the central retina in all patients with LCA5-LCA, at a level equivalent to that observed in rescued Lca5gt/gt mice, suggesting a window of opportunity for the treatment of patients with LCA5-LCA.

L eber congenital amaurosis (LCA) is a molecularly heterogeneous, early-onset, severe inherited retinal degeneration that typically presents within the first year of life with nystagmus and profound vision loss. LCA has been associated with mutations in more than 25 genes [Retnet: https://sph.uth.edu/retnet/] that are essential for photoreceptor and retinal pigment epithelium (RPE) function. One such gene, Lebercilin (LCA5), accounts for approximately 2% of LCA cases and results in a particularly severe form of the disease. [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15] The LCA5 gene (also known as C6ORF152) located in chromosome 6q14.1 encodes an 80 kDa protein expressed in the connecting cilium of photoreceptors. 3 Lebercilin interacts with other intraflagellar transport proteins required for the bidirectional movement of proteins from the inner to outer segments of photoreceptors and is necessary for photoreceptor outer segment formation. 7,16,17 In the Lca5 gt/gt mouse model of the disease, homozygous null Lca5 gene trap (gt) insertions cause a severe retinal degeneration within a few weeks of birth. 16,18 At postnatal day (P) 12, the outer nuclear layer (ONL) is normal in thickness, but the photoreceptor inner and outer segments (POS) are already shorter than normal. 16 By P28 the ONL thins to about one third of normal with only 3 or 4 rows of nuclei. At P60 only a single row of ONL remains, which is no longer observable by 4 months. 16 Both scotopic and photopic electroretinogram (ERG) responses have been reported as undetectable by P25. 16 The remarkable success of Luxturna (voretigene neparvovec-rzyl; Spark Therapeutics, Philadelphia, PA, USA), the first gene therapy product approved by the Food and Drug Administration for an inherited retinal degeneration (IRD) (RPE65-LCA), has energized the search for treatments for other molecular forms of IRDs. 19,20 We have previously shown that a recombinant adeno-associated viral vector (AAV7m8) containing human LCA5 cDNA can rescued ONL thickness, pupillary responses, and water maze performance when delivered to the subretinal space of neonatal Lca5 gt/gt mice. 16,18 Of note, these injections were performed in P4-P6 mice before retinal degeneration had started and thus is not translatable to the early phases of eventual human clinical trials. Patients with LCA5-LCA typically show a moderate to severe retinal degeneration by the time they are diagnosed early in life. In this work we performed AAV-mediated rescue experiments in Lca5 gt/gt mice at time points when photoreceptor degeneration is well under way, better modeling the structural and functional phenotype of patients who may be considered for enrollment in the early phases of future human clinical trials.

Human Studies
Five patients ages 6 to 31 years (hereafter referred to as Patients 1-5, P1-P5), diagnosed with LCA caused by biallelic null mutations in LCA5 underwent a comprehensive eye examination. The data were obtained from the patients' clinical records. Retinal imaging was performed with spectral domain optical coherence tomography (SD-OCT) and when possible with near-infrared reflectance (NIR-REF) and fundus autofluorescence (FAF) with NIR and shortwavelength (SW) excitation lights (Spectralis, Heidelberg Engineering, Carlsbad, CA). Wide-field retinal imaging was performed (Optos, Inc., Marlborough, MA) in one patient. SD-OCT imaging was performed with 9 mm-long horizontal sections crossing the anatomic fovea; our scanning protocol, analyses, and normative data have been published. [21][22][23] In brief, segmentation of SD-OCT images was done both with the built-in segmentation software of the Spectralis OCT system and with ImageJ analysis software (http://imagej. nih.gov/ij/links.html) using longitudinal reflectivity profile (LRP) analyses. Retinal thickness was defined as the distance between the signal peak at the vitreoretinal interface (the internal limiting membrane, ILM) and the posterior boundary of the major signal peak that corresponds to the basal retinal pigment epithelium/Bruch's membrane (RPE/BrM). In normal subjects, the RPE/BrM is the last reflectivity within the four to six signals that are identifiable in the outer retina. In patients, the presumed RPE signal was often the only signal in the outer retina merging with signals from the anterior choroid. To avoid ambiguities in the determination of the external limiting membrane in severely degenerated regions, the term ''outer retinal thickness'' was adopted to define the thickness between the outer plexiform layer (OPL) and RPE/BrM. Inner retinal thickness was defined as the distance between the ILM and the OPL. Visual sensitivity was measured in dilated, dark-adapted patients using full-field stimulus testing (FST). 24,25 All patients were able to complete FST testing except patient 1 (P1, 6 years old). Results in P3 were highly variable and considered unreliable and thus were excluded from analysis. The direct transient pupillary light reflex (TPLR) was elicited by full-field, 100 ms chromatic stimuli delivered by a Ganzfeld simulator of a full-field electroretinography system (ColorDome, Espion E 3 ; Diagnosys, Lowell, MA, USA) connected to an eyetracker (EyeFrame; Arrington Research, Scottsdale, AZ, USA) and software (VoltagePoint; Arrington Research) as previously described. 21 TPLR luminance response functions were recorded with increasing intensities of scotopically matched blue (467 nm) and red (637 nm) lights presented at 0.5 log unit increments in the dark-adapted state. Four-second video clips were recorded after each stimulus, which were used to manually measure pupil diameter in these patients with large-amplitude nystagmus that interfered with automated tracking. Informed consent was obtained; all procedures followed institutional guidelines protocol numbers 815348 and 832468) and complied with the Declaration of Helsinki.

Murine Studies
Husbandry and Study Design. All animals were bred and maintained in the animal facilities of The University of Pennsylvania under a 12-hour light (993 lux)/12-hour dark cycle with free access to food and water. Experiments were conducted in accordance with federal and institutional regulations on IACUC protocol no. 805890 and with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Animals were anesthetized with intraperitoneal ketamine (87.5 mg/kg) and xylazine (10 mg/kg). Corneas were anesthetized with topical 0.5% proparacaine hydrochloride (Akron, Lake Forest, IL, USA), and pupils were dilated with topical 0.5% tropicamide/phenylephrine hydrochloride combination drops (Mydrephrine P; Nitto Medic Co., Ltd., Japan). Media opacities were minimized by ocular eye shields 26  Subretinal Injections. AAV vectors were generated and purified in the research vector core of the Center for Advanced Retinal and Ocular Therapeutics (CAROT) at the University of Pennsylvania. Because of decreased expression and increased toxicity of the AAV7m8 vector in the nonhuman primate retina, wild-type LCA5 was delivered using a well-characterized AAV8 serotype that has a more favorable safety and expression profile in both mouse and primate retina. [28][29][30] The AAV8-hLCA5 vector contains a full-length human native LCA5 cDNA under the control of the hybrid CMV-chicken β-actin promoter to drive ubiquitous expression in transduced retinal cells. 3,18 Vectors were generated by triple transfection and delivered in excipient containing phosphate-buffered saline solution (PBS) with 0.001% Pluronic F68 (PF68). 31 Bilateral subretinal injections were performed in P5, P15, and P30 Lca5 gt/gt mice using previously described methods with modification. 32 Briefly, an incision was created just posterior to the temporal limbus with a 30 -gauge disposable needle. A 33-gauge, blunt, unbeveled needle on a 5-μL Hamilton syringe (Hamilton Co, Reno, NV, USA) was introduced through the incision and advanced posterior to the lens into the subretinal space. One microliter of a solution containing AAV8-hLCA5 was injected into the subretinal space, delivering 1.0E+09 viral genomes (vg) per eye, an optimal dose based on previous expression and toxicity profiling studies. 18 Control injections were performed with excipient only. Pred-G ophthalmic ointment (Allergan) was applied to the eyes immediately after the procedure. Surgical complications included retinal hemorrhage, retinal detachment, and cataract, as identified by indirect ophthalmoscopy and optical coherence tomography (OCT). Retinal hemorrhages were small and resolved spontaneously. Retinal detachments persisted throughout the study (to P60) but did not interfere with testing. Eyes with dense cataracts that interfered with imaging and functional studies (one eye in the P5-treated cohort and two eyes from the P30-treated cohort) were excluded from further analysis.

Retinal
Imaging. An SD-OCT system (Bioptigen Model R2200 Envisu; Leica Microsystems, Buffalo Grove, IL, USA) was used to obtain orthogonal radial volume scans (1.4-mm; 1000 A/scans × 2 B-scans × 15 frames @ 0°and 90°o rientation) and averaged B-scans (15 frames) of the retina from the central (optic nerve centrally positioned within the en-face image), temporal, nasal, superior, and inferior quadrants. After acquisition, images underwent coregistration and averaging using Bioptigen Envisu Software v2.1. Images were exported as bitmaps to imaging analysis software (ImageJ). The ONL was defined as the hyporeflective band between the OPL and the external limiting membrane. ONL thickness was measured at two locations per eye in uninjected Lca5 gt/gt and wild-type mice (200 μm nasal and temporal to the optic nerve) and at two locations within regions with detectable ONL in the nasal retina of treated animals (at the site of the subretinal injection), at a location that corresponded to the nasal measurements of uninjected Lca5 gt/gt and wild-type animals.

Electroretinography and Pupillometry. Animals
were dark-adapted overnight before testing. At 30 or 60 days after treatment, animals were anesthetized and dilated as above and placed on a 37-degree stage. Custom-made clear plastic contact lens electrodes were placed over each eye, and a platinum wire loop reference electrode was placed in the animal's mouth. Full-field ERGs were recorded using a custom-built Ganzfeld dome and a computer-based system (Espion E2; Diagnosys) in a climate-controlled, electrically isolated dark room under dim red illumination. Band-pass filter cutoff frequencies were 0.3 Hz and 300 Hz. Experiments were started with dark-adapted ERGs elicited with increasing intensities of blue (467 nm; −4.5 log scot-cd.s.m −2 to +1.9 log scot-cd.s.m −2 in 0.5 log unit steps) to single flash stimuli averaged at 0.5 Hz, as well as with 4-Hz and 10-Hz flicker stimuli. 33 Separate intensity series were recorded, starting with the single flash (0.5 Hz) series, followed by the 4-Hz and then the 10-Hz series; 10 to 20 responses were averaged for each of the intensity steps. Amplitudes and timings of the a-and b-wave amplitudes, as well as of the peak amplitude of the flicker ERGs were measured conventionally. 33,34 Small ERGs were anticipated in Lca5 gt/gt mice, and thus the starting intensity for each of the series was set at ∼2 log units above the threshold response of wild-type mice. 18 Three mice developed corneal opacities after ERG testing and were excluded from subsequent pupillometry analyses. Transient pupillary light reflexes were recorded using a NeurOptics A-2000 (NeurOptics, Irvine, CA, USA) small animal pupillometry system as previously described. 18 The subset of mice receiving bilateral injections (AAV-hLCA5 or excipient) were used for TPLR analysis, to avoid confounding of the treated eye causing a contralateral pupil constriction in the untreated eye. The amplitude of pupillary constriction was measured in response to a series of five 40-ms 1000 and 3000 lux flashes per eye in anesthetized, dark-adapted mice and was defined as the difference between the baseline pupil diameter and the diameter obtained 0.6 to 1.2 seconds after stimulus onset.
Histology and Immunofluorescence. Eyes were enucleated, fixed in 4% paraformaldehyde, sectioned, and stained with hematoxylin and eosin using standard protocols. Slides were scanned using an Aperio AT2 Digital Pathology Slide Scanner at original magnification × 20 under brightfield illumination (Leica Microsystems) and analyzed using Aperio ImageScope software (Leica Microsystems). Images were exported as TIF files into image analysis software (http://imagej.nih.gov/ij/links.html) for retinal layer segmentation and quantification. ONL thickness was defined as the distance between the outer limiting membrane and the outer plexiform layer and was measured at locations that corresponded with the locations used for the OCT measurements. Measurements were averaged and plotted as mean values ±2 SD. For immunofluorescence studies, 12μm frozen sections were incubated with anti-LCA5 (1:200; Proteintech, Rosemont, IL, USA), and anti-Rhodopsin antibodies (1:1000; Abcam, Cambridge, MA, USA), and ProLong Gold Antifade Mountant with DAPI (Thermo Fisher Scientific, Waltham, MA, USA). Slides were imaged with an Axio Imager M2 microscope (Zeiss, Oberkochen, Germany) equipped with epifluorescence and Axio-Vision 4.6 software.
Statistical Analysis. Statistical analysis was performed using GraphPad Prism 8.2.0 (GraphPad Software, Inc; La Jolla, CA, USA). Data distribution was assessed using the D'Agostino-Pears test. Differences in retinal layer thickness, ERG testing, and pupillary responses between AAV8-hLCA5injected eyes and control eyes at P5, P15, and P30 were analyzed by one-way analysis of variance with a Bonferroni correction. A P value <.05 was considered significant (indicated in the figures by *P < 0.05, **P < 0.01, and ***P < 0.001).

LCA5-LCA: One of the Most Severe Early-Onset Inherited Retinopathies
Five patients carrying homozygous null mutations in LCA5 were included in the study (Table). Patients presented with reduced vision and nystagmus within the first year of life. Visual acuity was limited to light perception (LP) in three patients, 20/300 in one patient, and 20/500 in the remaining patient, in each eye. When measured, refractive errors were hyperopic (Table). To the best of our knowledge this level of vision has been present since diagnosis. All patients had prior ERG testing that was reported as nondetectable. En face imaging on patient 5 (P5), the oldest patient in the cohort, illustrates the abnormalities (Fig. 1A). There were pigmentary changes within the macula, but the pericentral and peripapillary retina had a better coloration (Fig. 1A). Buried optic nerve drusen were evidenced by their hyperautofluorescence on SW-FAF (Fig. 1A, white arrow). The better-preserved coloration of the RPE in these regions colocalizes with better preserved melanin-originating signals on NIR-FAF (Fig. 1A, yellow arrows). A range of pigmentary changes with better preservation of the pericentral and peripapillary retina were documented in the remainder of the patients (Fig. 1B). A yellow coloration of the fovea, more pronounced than expected for the normal macular pigment, was observed in at least one patient (Fig. 1B, P3). Figure 1C illustrates and expands the reported spectrum of central SD-OCT structural abnormalities in LCA5-LCA, ranging from the least severe (P5, age 31) with a clearly detectable, albeit thin ONL band extending throughout the central scan, to the most severe (P3, age 21) with foveal atrophy and interruption of the RPE band (Fig. 1C, asterisk) with posterior displacement of the ocular layers. 6,11,12 The ONL could be detected at the fovea and in the peripapillary retina of the most affected patient. Individual OCT scans from at least one eye of each patient demonstrated  Fig. 1). We were unable to capture an image through the fovea in the youngest patient (P1; age 6) because of nystagmus and limited patient cooperation. A vertical scan in this patient clearly shows a thinned continuous ONL extending from the pericentral to the midperipheral retina ( Supplementary  Fig. 1). Signals superficial to the RPE that may correspond to abnormal but preserved photoreceptor inner segments, outer segments, or both, are visualized at the fovea with the help of LRPs in P5. The inner retina appears normal or thickened. To confirm detection of residual photoreceptors, LRPs were generated from the four patients (P2-P5) who had scans crossing the fovea (Fig. 1D). LRPs from a location 1.5 mm nasal to the foveal center, which showed possible presence of photoreceptors in all patients, were aligned vertically by the RPE signal peak and compared with a representative normal subject at that location (Fig. 1D).
In the normal LRP the ONL can be identified as a trough between the signal peak of the OPL and RPE (Fig. 1D, blue segment). In all patients a clear trough in the reflectivity signal was present bracketed by signal peaks from the RPE and the OPL. P2 and P3 showed an additional small signal peak that may correspond to remnant signals from the inner (EZ band) or outer segments or both (Fig. 1D, asterisks). Of note, clear outer retinal sublaminae (EZ, IZ) present at the foveal center in P5 were not detectable in pericentral nasal retina. Dark-adapted FST sensitivities to chromatic stimuli in patients who could reliably complete testing (P2, P4, P5) were reduced by at least 4.5 log units (range 4.5 to 5.5 log units; 467 nm) compared with normal values (Fig. 1E). Sensitivity differences between the red and blue stimulus were used to determine photoreceptor mediation. 21 Despite severely reduced sensitivities there was evidence of rod detection of the stimuli in the youngest patient in which measurements were possible (P2). The other two patients show evidence of cone-mediated detection. TPLR objectively confirmed the FST thresholds and provided a sensitivity estimate in a patient who could not reliably perform FST. TPLR thresholds were elevated on average by 5.1 log units (3.6 to 7.1 log units, with 467 nm light stimuli). Of the 2 patients who had both FST and TPLR (P2 and P4), thresholds were within 0.5 log units of each modality.

AAV8-hLCA5 Gene Therapy Arrests Photoreceptor Loss in Early but not Late Stage Retinal Degeneration in the Lca5 gt/gt mouse
To assess the window for therapeutic intervention for LCA5-LCA, we injected Lca5 gt/gt mice subretinally at early-(P5, n = 12 eyes), mid-(P15, n = 14), and late-stage (P30, n = 15) photoreceptor degeneration with either an AAV8 vector containing the full-length wild-type native human LCA5 cDNA or an injection of excipient-only (PBS with 0.001% Pluronic F68) control (P5, P15, P30, n = 30). OCT imaging could not be performed in eyes with lens opacities, including one eye injected at P5 and two eyes injected at P30 (Supplementary Table 1). The longevity of the treatment was assessed by examining these mice after treatment at P30 (for those treated before P30) and P60 (Figs. 3A, 3B). Failed resolution of the therapeutic retinal detachments led to persistent, localized, retinal detachments, which occurred more frequently when subretinal injections were performed in neonatal (P5) mice compared with older mice: 82% of P5-treated mice (9/11 eyes), 14% of P15-treated mice (2/14 eyes), and 8% of P30-treated mice (1/13 eyes). The retinal detachments visualized on OCT at P30 remained detached at P60 but did not interfere with subsequent imaging or testing. Figure 3B shows representative horizontal OCT crosssections through the optic nerve in Lca5 gt/gt mice treated at P5, P15, or P30 and imaged after treatment at P30 and P60. Wild-type, uninjected Lca5 gt/gt and excipient-injected eyes are shown at the same time-points for comparison (Figs. 3A, 3B). Inspection of the OCT scans at P30 demonstrates a variably thinned but clearly detectable ONL in all groups. There are no obvious differences in thickness of the ONL between the treated Lca5 gt/gt animals and the uninjected Lca5 gt/gt or excipient-injected controls. At P60, however, eyes treated with AAV8-hLCA5 at P5 or P15 show an obvious ONL band, whereas animals injected at P30, excipient-injected, and uninjected Lca5 gt/gt controls have no detectable ONL (Fig. 3B).
To ensure that the structural retinal preservation seen in the P5-and P15-treated Lca5 gt/gt mice corresponded to regions of expression of the human LCA5 protein (Lebercilin), we performed immunofluorescence staining using antibodies specific to human LCA5. In control (excipient only) Lca5 gt/gt animals, there was a single remaining row of ONL, no detectable LCA5 staining, and a thin band of rhodopsin expression just distal to the ONL (Fig. 4, upper  panel), contrasting with the strong LCA5 immunostaining pattern reported in wild-type mice. 18 In P5-and P15-treated animals there were 3 to 6 layers of nuclei in the ONL, distinct regions of human LCA5 expression in both inner and outer retinal cells, and rhodopsin staining in photoreceptor outer segments of LCA5-positive and adjacent LCA5-negative photoreceptors (Fig. 4, middle panels; LCA5 expression in green, outer segments in red). P30-treated animals had a rudimentary layer of ONL similar to control animals, patchy expression of LCA5 in primarily outer retinal but not inner retinal cells, and distinct but scattered rhodopsin staining in a few cells distal to the ONL and in close proximity to the RPE (Fig. 4, lower panel).
ERGs were performed to determine whether the relatively preserved retinal structure documented in the P5-and P15treated animals resulted in improved retinal function. When tested at P30, both P5-and P15-treated mice had measurable dark-adapted ERG responses to both single flash (+1.9 log scot-cd.s.m −2 ) and flicker (4Hz and 10Hz; −1.0 log scotcd.s.m −2 ) stimuli (Fig. 5A). Untreated Lca5 gt/gt mice, however, can have small detectable ERGs at P30 but are always nondetectable by P60 (Fig. 5A). When evaluated at P60, two of seven P5-treated mice with detectable ONL and one of eight P15-treated mice with detectable ONL had measurable, albeit reduced ERG signals. In contrast, P30-treated mice had no reliably detectable ERGs to any stimulus condition. Quantification of the average b-wave amplitude, however, did not show a statistically significant difference between the treated and untreated groups at P60, suggesting that the degree or extent of photoreceptor rescue in P5-and P15-treated animals was not sufficient in extent or magnitude to cause a significant change in retina-wide measures of function such as the full-field ERG (Fig. 5B). Locally efficacious treatment effects may thus be diluted by the combination of fullfield ERG signals from responsive and unresponsive retina, whereas signal integration over large retinal areas by the pupillary light reflect allows detection of small changes in function of even small retinal regions. 35 With this in mind we measured the TPLR in treated and untreated Lca5 gt/gt mice in response to 1000 and 3000 lux stimuli and found that P5-and P15-treated mice had larger TPLR responses compared with P30-treated, untreated, and excipient-only (excipient pooled from all injection time points) mice ( Fig. 5C and 5D). TPLR constriction amplitudes expressed as a fraction of the baseline pupil diameter for each of the two light stimuli showed a graded TPLR response, with the greatest response in P5treated animals (15%-17%), a moderately preserved response in P15-treated animals (∼12%), and minimally detectable pupillary responses in P30-treated (<3%) and uninjected Lca5 gt/gt control animals (<5%) (Figs. 5C, 5D).  We next sought to extrapolate results of these murine data to determine whether there is a window of opportunity for treatment in human LCA5-LCA patients. ONL thickness measured in vivo with SD-OCT in treated Lca5 gt/gt mice and expressed as a fraction of the location-specific mean normal thickness demonstrates that unequivocal treatment success in P15-injected (or earlier) corresponds to retinas with a peripapillary ONL thickness that is at least ∼85% of normal thickness at the time of injection (and presumably less at the time of AAV gene expression ∼5 days later) (Fig. 6, green symbols). P30-treated animals with questionable or no signs of rescue at P60 by TPLR (Fig. 5C) showed ONL thicknesses that were reduced to at least 30% of normal (Fig. 6, black symbols). If photoreceptor loss as measured is the main determinant of treatment potential, and assuming the fast photoreceptor degeneration in Lca5 gt/gt mice follows a linear decay between the two time points of treatment (P15 and P30), then a therapeutic window may still exist for animals with severe photoreceptor degeneration as long as their residual ONL thickness is greater than 30% of normal (Fig. 6). It follows that treatment efficacy may be achievable in some LCA5-LCA patients in regions where degeneration still spares photoreceptors to at least 30% of their normal complement. Residual islands of photoreceptors in the pericentral retina of LCA5-LCA patients in this study were about half the normal thickness (Fig. 6, red symbols), equivalent to levels of photoreceptor degeneration in the Lca5 gt/gt mouse before P30.

DISCUSSION
In the spectrum of severe early-onset retinal degenerative conditions, LCA5-LCA ranks as a particularly severe disease with profound vision loss and central structural abnormalities detected from early childhood. [1][2][3][4][5][6][7][8][9][10][12][13][14][15]36 Consistent with previous reports we found a severe photoreceptor degeneration with areas of relatively preserved outer pericentral retina and variable foveal abnormalities in all patients. 2,11,37 There was no clear relationship between the specific genotype or age and the severity of phenotype in our patients, analogous to several other forms of LCA. [38][39][40] Although our youngest patient (6 years old) showed the largest expanse (into the midperiphery) of detectable photoreceptors in LCA5-LCA reported to date, our oldest patient (age 31) also showed remarkable central photoreceptor preservation, equivalent to what has been reported with OCT for this disease. 6,12 Ages sampled between these two extremes in this work showed more severe examples of degeneration. Detecting a structural-functional dissociation, a reliable indicator of treatment potential in inherited retinal degenerations is challenging in LCA5-LCA. Unlike RPE65-LCA, for example, in LCA5-LCA there may be both severe retinal dysfunction and structural loss even in the youngest patients, which challenges treatment potential. 6,[40][41][42] The relatively preserved retinas of some patients in this study (for example, P1 and P5) showed ONL thicknesses that were between 50% to 75% of the normal thickness values for the most preserved locations, whereas FST sensitivity losses exceeded 4.5 log units. This supports a pattern of structural-functional dissociation previously hinted by pupillometric thresholds in this disease and raises hopes that gene augmentation may be able to restore some level of vision in these patients. 6 The presence of regions of preserved RPE and photoreceptors well into adulthood also provides hope that this disease may be amenable to treatment in some patients into adulthood. Both rod-and cone-mediated sensitivities were documented by FST early in the disease in one of our youngest patients in this study, as well as residual cone vision late in the disease, a pattern of degeneration involving both rods and cone photoreceptors reminiscent of that observed in the Lca5 gt/gt mouse model. 18 Measurable cone function in late-stage disease suggests relative resilience of cone photoreceptors to degeneration and the potential for their rescue by gene therapy. On the other hand, quantifiable visual function by FST and TPLR despite the severity of the disease supports the use of FIGURE 6. ONL thickness expressed as a percentage of the measures in wild-type mice at the same location for Lca5 gt/gt mice treated at P15 and P30. ONL thickness declines dramatically in the Lca5 gt/gt mouse, from 85% of wild-type at P15, to 37% of wild-type by P30; diagonal green dashed line models linear ONL loss between the two time points. Horizontal dashed lines represent the ONL mean thickness for each P15 and P30 group, solid lines represent ± SD. The colored background represents a theoretical gradient of functional improvement after gene therapy relating to the percentage of remaining ONL at the time of treatment, with green indicating preserved ONL and improved functional outcome, and red indicating loss of ONL and limited therapeutic potential. ONL thickness from best preserved regions in the pericentral retina of four LCA5-LCA patients expressed as a percentage of the location-specific normal mean value (red symbols). P2, P3, and P5 had similar ONL thickness (54%, 52%, 54%, respectively) whereas P4 had a slightly thinner ONL (28%) similar to that of a P30 Lca5 gt/gt mouse. these measurements as outcome measures of the safety and efficacy of future interventions for this particularly severe disease, as has been the case for other forms of LCA, such as RPE65-LCA. 6,21,35,[41][42][43][44] Future studies in this particular group of patients are needed to determine their performance in terms of reliability and reproducibility before the tests are fully endorsed as outcome measures for future clinical trials. It is possible that objective measures of structural change may take a dominant role in this scenario, a picture quite different from that expected at the initiation of the RPE65-LCA gene therapy trials.
We sought to characterize the natural history of photoreceptor degeneration in the mouse model of LCA5-LCA to draw parallels with the human disease with the ultimate goal of assessing the potential efficacy and time windows of treatments in patients. We report the first quantitative temporal analysis of retinal degeneration in the Lca5 gt/gt mouse model using noninvasive retinal imaging (OCT), a technique widely used in ophthalmology clinics. We show that OCT provides an accurate measurement of ONL thickness and that the Lca5 gt/gt mouse model replicates the fast and early photoreceptor degeneration observed in patients. ONL quantification by OCT revealed a fast-progressing retinal degeneration with outer segment loss at the earliest time-point (Fig. 2B,  P15), detectable ONL thinning between P15 and P30, and total loss of photoreceptors within the sampled regions by P60. These measurements were verified by quantification of ONL thickness on histologic sections. The ONL thickness of Lca5 gt/gt mice was decreased by 15% compared with wild-type animals at P15 and decreased by almost 70% of wild-type thickness at P30. Interestingly, residual rod-and cone-mediated ERG signals were barely detectable at this stage despite the fact that one third of the photoreceptors were clearly detectable, suggesting a disproportionate loss of retinal function compared with measurable structure, an expected phenotype in some forms of LCA. 40,42,45 Admittedly, sampling the peripapillary retina by OCT in this model assumes homogenous loss across the entire retina and may, in fact, overestimate or underestimate the level of photoreceptor preservation contributing to the full-field ERG. The pattern of loss involving rod and cone photoreceptors, as well as the possible dissociation between photoreceptor structure and function in the Lca5 gt/gt mice, is also analogous with what we describe in the patients included in this study.
There is evidence that subretinal delivery of an AAV7m8 viral vector containing human LCA5 cDNA can rescue the retinal structure and function when administered to Lca5 gt/gt mice at P4 to P6. 18 Because of innate differences in the size, anatomy, and physiology of the rodent eye compared to the human eye, however, not all viral vectors that are effective in preclinical mouse studies can be directly translated to nonhuman primates (NHP) or to human clinical trials. Testing of AAV7m8 in the NHP eye, which has a cone-rich macula and most closely resembles the human eye, revealed decreased cellular expression and increased toxicity when compared to the safety and expression profile of this vector in the mouse. 30 Therefore we delivered wild-type LCA5 using a well-characterized AAV8 serotype that has a more favorable safety and expression profile in both the mouse and NHP and thus may be more suitable for translation into human clinical studies. 28,29,46 Precipitous degeneration in a period of 1 month between P30 and P60 in the Lca5 gt/gt mouse allowed us to unequivocally confirm partial rescue of the phenotype at P60 for treatments delivered before P30. At the fast rate of photoreceptor degeneration described for this model, it is still conceivable that lack of rescue for animals injected at ages >P30 may be a consequence of the added delay in initiation of protein expression after the delivery of gene therapy (approximately 5 days for AAV8), placing the potential beneficial effect beyond P30. 47 The longitudinal assessment of the TPLR and of the structural preservation with SD-OCT in Lca5 gt/gt mice treated before P30 will help define both the longevity of treatment and its dependency on the level of photoreceptor degeneration, questions of critical importance for future clinical trials. 18,48 Photoreceptor loss at P30 in the Lca5 gt/gt mice is comparable to the level of photoreceptor loss observed in pockets of residual photoreceptors in the patients with the most severe disease described herein (P2, P3, P4), whereas locally milder disease in other patients (P1 and P5) suggests a level of preservation similar to that of the younger mice (P15) in which gene therapy-mediated rescue was possible. The findings suggest that treatment efficacy in patients will be related to the level of photoreceptor loss rather the age of the patients, although earlier intervention may be desirable to achieve efficacy as well as to prevent interference of the disease with retinal and retinocortical development, oculomotor stability, and emmetropization. The presence of detectable signals by SD-OCT that may correspond to preserved inner segments and proximal outer segments in this and a previous study raises hope that correction of the molecular defect in this ciliopathy may lead to restoration of the outer segment physiology and to improvements in function as has been recently documented for CEP290-LCA, a similarly severe ciliopathy. 49 Evaluations of larger groups of patients with LCA5-LCA both in cross-section and longitudinally are needed to confirm if these observations are generalizable. Overall, these findings suggest that LCA5-LCA patients may benefit from gene augmentation therapies despite early-onset and uniquely severe retinal degeneration.