Molecular Vision 2007; 13:1701-1710 <>
Received 23 May 2007 | Accepted 15 September 2007 | Published 18 September 2007

Electroretinographic analyses of Rpe65-mutant rd12 mice: developing an in vivo bioassay for human gene therapy trials of Leber congenital amaurosis

Alejandro J. Roman,1 Sanford L. Boye,2 Tomas S. Aleman,1 Ji-jing Pang,2 J. Hugh McDowell,2 Shannon E. Boye,2 Artur V. Cideciyan,1 Samuel G. Jacobson1, William W. Hauswirth2

1Department of Ophthalmology, Scheie Eye Institute, Philadelphia, PA, 2Department of Ophthalmology, University of Florida, Gainesville, FL

Correspondence to: Samuel G Jacobson, MD 51 N 39th Street, Philadelphia, PA 19104. Phone: (215) 662-9981; FAX: (215) 662-9388; email:


Purpose: Dramatic restoration of retinal function has followed subretinal viral-mediated gene therapy in RPE65-deficient animal models of human Leber congenital amaurosis (LCA) caused by RPE65 mutations. Progress in early-phase clinical trials of RPE65-LCA prompted us to begin development of an in vivo bioassay of clinical grade vector stability for later-phase trials.

Methods: Naturally-occurring Rpe65-mutant rd12 mice (2-4 mo of age) were studied with full-field electroretinograms (ERGs). Flash stimuli (range, -4.1 to 3.6 log scot-cd.s.m-2) were used to evoke ERGs in anesthetized, dark-adapted mice. B-wave amplitudes were measured conventionally and luminance-response functions were fit. Leading edges of photoresponses were analyzed with a model of rod phototransduction activation. A unilateral subretinal injection of AAV2-CBSB-hRPE65 vector was delivered and therapeutic efficacy of 4 doses spanning a 2 log unit range was studied with ERGs performed about 6 weeks after injection. Uninjected rd12 eyes and wild-type (wt) mice served as controls.

Results: Rd12 mice showed substantially smaller amplitudes and lower sensitivities than wt mice for all measured ERG b-wave and photoresponse parameters. For the dose-response study, there was no difference between 0.01X-dosed mice and untreated mutants. Improved receptoral and post-receptoral function was evident for 0.1X, 0.3X, 1X doses: b-wave semi-saturation constants decreased, b-wave amplitudes increased with dose; photoresponses showed faster kinetics and higher maximum amplitudes. ERG b-wave amplitude to a selected stimulus light intensity could provide evidence of biologic activity of the vector; interocular differences in b-wave amplitude comparing treated versus untreated eyes in the same animal also revealed vector efficacy.

Conclusions: We have taken the first steps toward developing an ERG assay of biologic activity of human grade vector for future clinical trials of RPE65-LCA. Faithful murine models of treatable human disease tested with specific ERG protocols may emerge as valuable in vivo bioassays for future human clinical trials of therapy in many retinal degenerative diseases.


RPE65 (retinal pigment epithelium-specific 65 kDa protein) is the isomerohydrolase of the visual (retinoid) cycle [1-3], the pathway that regenerates visual pigment after light is absorbed [4]. Mutations in the RPE65 gene are known to cause Leber congenital amaurosis (LCA), a severe early-onset blinding human disease [5,6]. Longstanding scientific interest in details of the visual cycle, the availability of naturally-occurring and genetically-engineered animals with RPE65 deficiency, and relevance to human blindness has accelerated scientific and medical activity toward initiating gene therapy clinical trials in RPE65-LCA. Systematic steps have been taken toward human trials. Proof-of-concept studies with viral gene transfer of RPE65 have occurred in dogs [7-11], and in mice [12-17]. Dose-response and safety studies have been performed in dogs and non-human primates [18,19]. Human studies have inquired whether the successfully-treated animals with RPE65 deficiency sufficiently model the human disease to warrant translation to the clinic [20], and what outcome measures would accommodate the severe visual loss and nystagmus in the RPE65-LCA patients [21-23].

The steady progress toward early-phase human clinical trials of RPE65-LCA prompted us to begin considering the needs of later-phase trials. One such need will be an analytical method to determine whether the clinical grade gene therapy agent is biologically active [24,25]. Building upon the foundation of studies to date, we chose to explore an in vivo assay of biologic activity using a surrogate measure of gene expression in an available murine model with Rpe65 deficiency. The measurement tool is the electroretinogram (ERG), the time-honored non-invasive retinal function test used in the clinic [26,27], in the laboratory to determine retinal phenotype in mice [28-30], and in proof-of-concept studies of vector-mediated gene therapy in Rpe65-deficient mice [12-17].

First, we defined the ERG abnormality of the naturally-occurring rd12 mouse model of Rpe65 deficiency [12-17,31-38]. Then, we quantified ERG parameters at different doses of AAV2 vector containing human RPE65 cDNA delivered by subretinal injection [18,19]. The results lead to suggestions for bioassays of clinical grade vector for future late-phase clinical trials of RPE65-LCA.



Rd12 (N=39; 16 female and 23 male) and normal wildtype (wt, N=10; 1 female; 9 male) C57BL/6J mice were used in this study. Mice were generated from breeding pairs obtained from the Jackson Laboratories (Bar Harbor, ME) and were 2 to 4 mo of age at the time of testing. Animals were kept in cages (average 2-3 animals per cage) under 12 h-on/12-h-off cyclic lighting (ambient illumination 75 lx), with lights on at 7 am. Access to food (LM-485, Harlan Teklad, Madison, WI) and water was ad libitum. Procedures were conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Visual Research and with institutional approval.


Full field bilateral ERGs were recorded using a custom-built ganzfeld, a computer-based system (EPIC-XL, LKC Technologies, Gaithersburg, MD) and specially-made contact lens electrodes (Hansen Ophthalmics, Iowa City, IA). Animals were dark-adapted (>12 h) and anesthetized with a mixture of ketamine HCl (65 mg/kg) and xylazine (5 mg/kg) intramuscularly under dim red light. Corneas were anesthesized with proparacaine HCl, and pupils were dilated with tropicamide (1%) and phenylephrine (2.5%). Medium energy (10 μs duration) and high energy (1 ms duration) flash stimulators with unattenuated luminances of 0.8 and 3.6 log scot-cd.s.m-2, respectively, were used. Neutral density (Wratten 96) and blue (Wratten 47A) filters served to attenuate and spectrally-shape the stimuli. The signals evoked by medium energy flashes were amplified, filtered (-3 dB cutoff at 0.3 and 300 Hz) and digitized (2 kHz) with an 12-bit analog-to-digital converter. Signals evoked by higher energy flashes were recorded with higher bandwidth (1500 Hz filtering and 3.33 kHz sampling).

First, dark-adapted ERGs were obtained with increasing intensities of blue light flashes from -4.2 to 0.1 log scot-cd.s.m-2. Dimmer intensities were presented at 0.5 Hz and 10 waveforms were averaged; brighter intensities were presented at 0.1 Hz and 2 waveforms were averaged. Intensity increments were in 0.3-0.5 log unit steps. Next, an ERG photoresponse was evoked with a single blue 2.2 log scot-cd.s.m-2 flash. A 2-min wait served to permit complete recovery of the photoresponse. Then a single white 3.6 log scot-cd.s.m-2 flash was used to evoke the maximal photoresponse.

B-wave amplitudes were measured conventionally, from baseline or a-wave trough to positive peak, and fit with a Naka-Rushton function [39,40] to obtain estimates of maximum amplitude (Vmax) and sensitivity (semisaturation intensity, log K). A derived parameter, log(Vmax/K), was used to succinctly represent overall post-receptoral function. Leading edges (4 to 20 ms, depending on the response) of the two photoresponses were fitted as an ensemble with a model of rod phototransduction activation [12,32,39,41] and maximum amplitude (Rmax) and sensitivity (log S) parameters were derived. A derived parameter, log(Rmax*S), was used to represent overall photoreceptor function.

Efficacy of the treatment was statistically evaluated considering the derived ERG parameters, log(Rmax*S) and log(Vmax/K), in injected and control eyes. A one-way analysis of variance (ANOVA) with repeated measures was employed to adjust for the possible correlation between the eyes of each animal. When the overall difference among 5 groups of eyes was statistically significant, post-hoc pair-wise comparisons were performed between injected eyes at each dose level with control eyes. Additionally, test of linear trend was performed for each variable. Computations were executed on the statistical software SAS (version 9.1, SAS Institute, Inc., Cary, NC).

Efficacy of the treatment was alternatively evaluated by estimating the fraction of injected eyes (at each dose) that show an ERG parameter substantially better than what would be expected from uninjected eyes. The four ERG parameters (Rmax, log S, Vmax, and log K) were individually evaluated using a conservative criterion of mean±3SD derived from control eyes [9]. Additionally, a parameter consisting of the interocular difference (IOD) of b-wave amplitude evoked by a 0.1 log scot-cd.s.m-2 blue flash was considered as a simpler and shorter methodology for a potential bioassay. For this analysis, IOD limits (mean+3SD) were first defined for smaller amplitude rd12 records (21 μV IOD at a mean amplitude of 16 μV) and larger amplitude wt records (284 μV IOD at a mean amplitude of 356 μV) in animals with bilateral ERG recordings and no treatment. Treatment efficacy in the injected rd12 eyes was determined using a criterion that was dependent on the amplitude of the uninjected eye and was derived from the linear interpolation of IOD limits between the rd12 and wt results.

Vector and injection

A single 1 μl subretinal injection of a serotype 2 AAV vector containing the human RPE65 cDNA (rAAV2-CBSB-hRPE65) was delivered to one eye (N=34) of rd12 mice at an average age of 3.3 weeks (range, 2 to 5.3). The construct and dosage of vector was based on previous studies [18,19]. Four vector doses were studied over a two log unit range: 0.01X (N=5), 0.1X (N=10), 0.3X (N=11) and 1X (N=8) where 1X corresponded to 1010 vg/ μl. The remaining five rd12 mice were not treated. All uninjected rd12 eyes (N=44) served as controls.


Rpe65-deficient rd12 mouse electroretinography parameters

Waveforms recorded to increasing intensities of light stimuli from two-month-old normal wt (left) and rd12 (right) mice are illustrated (Figure 1A). ERG b-waves of the rd12 mouse were not detectable below an intensity of -0.8 log scot-cd.s.m-2, but intensities at and above 0.1 log scot-cd.s.m-2 can evoke recordable and sometimes sizeable responses. Of note, there were small and slow but detectable a-waves in mutant eyes at higher intensities [32]. Leading edges of these a-waves evoked by 2.2 and 3.6 log scot-cd.s.m-2 intensities directly assess photoreceptor activation kinetics and were fit with a model of phototransduction activation (Figure 1B). Compared to wt, rd12 mice showed a-waves (also called ERG photoresponses) with a significant reduction of maximum amplitude and slower response kinetics. The prominent retinal function abnormalities were consistent with previous reports in rd12 [14,17,37,38] and Rpe65-/- mice [12,13,31-36].

A summary of the phototransduction activation parameters, maximum amplitude (Rmax) and sensitivity (log S), obtained by modeling of the leading edges of the ERG photoresponses is shown (Figure 1C). Mean (±SD) Rmax was 320 (±94) μV for wt and 40 (±15) μV for rd12; log S values were 3.62 (±0.36) and 1.32 (±0.27) log scot-cd-1.m2.s-3 for wt and rd12, respectively (p<0.001 for both). Overall photoreceptor function as estimated by log(Rmax*S) was significantly depressed by 3.2 log units in rd12 mice compared to wt.

Dark-adapted ERG b-waves originate primarily from the activity of bipolar cells driven by photoreceptor cells [42,43]. Luminance-response functions obtained from ERG b-wave series can thus parameterize the post-receptoral function to a good first approximation [39]. Even though b-wave amplitudes in some rd12 mice could be sizeable at the higher intensity stimuli (Figure 1A), there were significant differences between rd12 and wt mice (Figure 1D). Average b-wave luminance-response function fit with a Naka-Rushton equation (Figure 1D, gray lines) was significantly right-shifted in rd12 compared to wt mice. Log K values were -1.45±0.43 and 1.86±0.38 log scot-cd.s.m-2, for wt and rd12, respectively, corresponding to a 3.3 log unit loss of light sensitivity due to the Rpe65 deficiency (Figure 1D). Vmax was 518±176 μV in wt and 137±42 μV in rd12. Rd12 mice showed pronounced impairment of overall post-receptoral function as estimated by log(Vmax/K) showing a 3.8 log unit difference from wt.

Dose-response functions in rd12 mice with subretinal injections of human grade AAV2-RPE65 vector

Representative rd12 mice evaluated 6.5 (range, 5.3 to 7.6) weeks after treatment demonstrate the effect of vector dose on ERG responses in the AAV2-hRPE65 injected eyes (Figure 2A, colored traces) as compared to contralateral control eyes (Figure 2A, gray traces). At the lowest vector dose (0.01X) used in these studies, b-wave and photoresponse waveforms from the two eyes were indistinguishable and were not different from untreated rd12 eyes. At higher doses, however, there were notable asymmetries between the eyes. Treated eyes had responses with greater b-wave amplitude and faster photoresponses with larger amplitudes.

Statistical analysis (one-way ANOVA with repeated measures) of the overall photoreceptor and post-receptoral ERG function showed highly significant differences among the groups of injected and control eyes (p<0.0001 for log(Rmax*S) and p=0.0003 for log(Vmax/K)). Pairwise post-hoc comparison between the eyes at each dose level and control eyes showed significant improvement in both parameters at 0.1X, 0.3X and 1X (Table 1). Furthermore, there was a statistically significant dose response relationship for both parameters (P for linear trend, 0.0002 and 0.0003 for log(Rmax*S) and log(Vmax/K), respectively).

To understand better the physiologic basis of the treatment effect, photoreceptor activation parameters were compared between treated and untreated rd12 eyes (Figure 2B). Photoreceptor activation of eyes treated with 0.01X dose (blue symbols) fell within the 99% confidence interval of untreated eyes (gray symbols). With increasing treatment dose between 0.1X and 1X, there was a gradual increase in photoresponse sensitivity (log S) such that some of the 0.3X and 1X eyes approached values from wt eyes. Additionally, many eyes receiving the highest doses showed a large increase in maximum amplitude (Figure 2B).

Post-receptoral function parameters derived from ERG b-wave series in treated rd12 eyes also showed an orderly relationship with treatment dose (Figure 2C). At 0.01X dose, both sensitivity (log K) and maximum amplitude (Vmax) parameters clustered within the 99% confidence interval of values obtained from untreated eyes. With increasing treatment dose, there was a gradual increase in log K. A fraction of rd12 eyes treated with the 1X dose showed log K and Vmax values approaching those of wt eyes.

Estimating treatment efficacy with electroretinography parameters

ERG data from individual treated eyes (Figure 2B,C) suggested that increasing dose was associated with increasing fraction of eyes showing a detectable treatment effect. To quantify this impression, the proportion of vector-injected eyes with ERG treatment efficacy are shown for each of the 4 doses tested (Figure 3A,B). A conservative criterion for efficacy was established from the limits (mean±3SD) of data from untreated eyes. Both photoresponse and b-wave sensitivity measures (log S and log K) demonstrated similar treatment efficacies, rising from 0% at 0.01X to 88% at 1X dose (Figure 3A,B). Maximum amplitude measures (Rmax and Vmax) on the other hand, showed little or no treatment efficacy at 0.01X, 0.1X and 0.3X doses but did rise to 40%-50% at 1X dose.

The dose-related increases in ERG sensitivity parameters suggested the possibility of using ERGs evoked by a flash carefully chosen to be near b-wave threshold in untreated rd12 eyes. Based on the luminance-response function (Figure 1D), a stimulus intensity of 0.1 log scot-cd.s.m-2 was chosen. A left-shift of the luminance-response function due to efficacious treatment would be expected to increase the amplitude of this response potentially simplifying and shortening the experimental protocol. ERG b-wave amplitudes of treated eyes were plotted against untreated eyes and treatment efficacy was defined by expected interocular differences (IOD) of this amplitude in untreated rd12 and wt eyes (Figure 3C). The resulting treatment efficacy curve (Figure 3D) was similar to that obtained from the log S parameter whether using IOD analysis (Figure 3D) or considering all untreated eyes as a control group (Figure 3A). IOD analysis of the other sensitivity parameter, log K, showed identical results (data not shown).


Naturally-occurring rd12 mice with Rpe65 deficiency showed ERG b-wave features of severely reduced responsiveness to light, in concurrence with earlier studies [14,17,37,38]. Novel to the current study was the analysis of ERG photoresponses which are dominated by the phototransduction activation in photoreceptors [44,45]. Rd12 mice showed substantially reduced photoresponse maximum amplitude and sensitivity parameters; the severity of dysfunction in rd12 mice appeared to be slightly greater than that reported in Rpe65-/- mice [12,20,32]. Assuming isorhodopsin is responsible for remnant rod retinal function in these models [35,46], reduced photoresponse sensivity could imply a difference in endogenous 9-cis-retinal production between the two mutant mice either due to genetic background [46] or due to differences in laboratory lighting conditions [35]. The differences in maximum amplitude of the photoresponses in the mutants could be due to differences in spontaneous activity of opsin or its constitutive phosphorylation [47-49]. Alternatively or additionally, the reduction in photoresponse maximum amplitude could be caused by a greater underlying degenerative component in rd12 mice. More rapidly advancing degeneration in a mouse model may have particular value for testing therapies intended for translation to man, considering human degeneration in RPE65-LCA is severe compared to the models [20]. When we previously sought to determine efficacy in advanced disease using Rpe65-/- mice, we reared mice to nearly two years of age [20]. A shorter natural history for severe degeneration in rd12 mice could facilitate and expedite studies that would be relevant to more severely affected humans with RPE65-LCA.

ERG data presentations in most reports of Rpe65-deficient mice illustrate small or non-detectable ERG b-waves to conventional stimuli [12-14,17,31-38]. With higher intensity stimuli, however, sizeable ERG b-waves are recordable in both models. We previously reported relatively large but insensitive photoreceptor responses using higher intensity light stimuli in Rpe65-/- mice [12,32]. These signals were also observed in rd12 mice in the current study. On the other hand, detectable but insensitive ERG photoreceptor responses have not been reported in human RPE65-LCA to date [32,50-56]. Assuming the functional reserve in mice is due to endogenous 9-cis-retinal production [35,46], the apparent lack of such signals in human RPE65-LCA may point to differences in systemic and/or ocular retinoid metabolism between the two species [57].

A relationship between dose and treatment efficacy for AAV2-hRPE65 vector has previously been reported only in the RPE65 mutant dog [18]. In those experiments, two conventional ERG stimuli were used and b-waves were quantified at 8 levels of vector doses in 16 dogs. Of interest, 3 of these dogs were tested with the same human vector (at relative doses 0.1X, 0.3X and 1X) used in the present rd12 mouse study. Results of the ERG experiments in the rd12 mice confirm and extend the dog studies. Like the canine results, the mice showed dose-dependent improvement in ERG: 0.01X dose was ineffective whereas doses over the range of 0.1X to 1X showed an increasingly larger proportion of injected eyes with a statistically significant treatment effect.

ERG photoresponses provide a non-invasive estimate of the photoreceptor circulating current averaged across the retina [39,41,44,45]. The photoresponse sensitivity parameter assays the amplification gain of the photoreceptors and, all else being equal, it tracks changes in rhodopsin concentration [58]. Thus, increased photoresponse sensitivity (log S) following successful treatment, as observed in the current work, would have been the predictable consequence of increased rhodopsin production [12]. Improvements in photoresponse maximum amplitude observed at higher doses, on the other hand, may have more complex origins including increases in dark current due to reduction of the concentration of unliganded opsin causing spontaneous activation of phototransduction [32,34,49] and elongation of photoreceptor outer segments. Evidence to date suggests that both types of improvement in rod photoreceptor function are not only transmitted to secondary retinal neurons, as evidenced by b-waves, but also to higher visual centers [7,11,14,21,38,59].

Laboratory progress of ocular gene therapy in treating Rpe65 deficiency is now sufficient to begin developing analytical methods for products intended for human RPE65-LCA clinical trials [25]. We are unaware of any established potency assays for this specific purpose, so we started to fill this void by using a naturally-occurring animal model of the human disease, subretinal gene delivery, different doses of human grade vector product and an ERG assay for activity of the expressed gene. The ERG results are promising at this initial stage, but further work is needed. Variations in ERG responding to different doses of vector, for example, should be addressed. This is not a trivial issue because of the difficulties associated with subretinal injections in these small eyes, including uncertainty of degree of retinal detachment, amount of transduced RPE and the confounding effects of potential vector toxicity or retinal injury during this type of surgery. Vehicle only or sham controls would be worth performing in subsequent studies with the goal of teasing apart the contributions of retinal injury and degree of retinal detachment, for example, from degree of vector potency or vector toxicity in eyes with this retinal disease. With further advances of this bioassay, experiments should be conducted to quantify retinal detachment at surgery and determine the relation to retinal injury and amount of transduced RPE.

Biologic assays, and particularly in vivo ones, are recognized to be challenging [25]. Parallel exploration of other directions is thus warranted. For example, in vitro cell function assays involving quantitation of retinoid isomerase activity have been developed to reproduce the visual cycle and express mutant proteins. Results from these assays have the potential to help understand the pathogenicity of different RPE65 mutations found in LCA [3,60,61]. It may be possible to adapt such cell systems to act as a surrogate potency assay for human grade vector, but there will be issues relating to the efficiency of vector transduction of cells in culture versus transduction of RPE in vivo that may complicate this approach. As another example, assays of biochemical phenotype in an in vivo experimental paradigm such as we used in the current work could also be of value. The mouse models are incapable of synthesizing requisite levels of 11-cis-retinal, the chromophore of visual pigments, and they accumulate retinyl esters, a substrate for Rpe65. Restoration of Rpe65 activity by AAV-mediated gene therapy reverses these results [4,14,31]. Although any assay of retinal tissue after gene therapy in the small mouse eye necessarily has the surgical uncertainties noted above, it may be desirable in the future to validate more than one in vivo potency assay of the Rpe65-deficient murine retinal response to therapy.


Supported by Macula Vision Research Foundation, Foundation Fighting Blindness NIH/NEI (EY11123, EY13729, EY17280, EY08571, NS36302, P30EY001583), Research to Prevent Blindness, Hope for Vision, Ruth and Milton Steinbach Foundation, and the Alcon Research Institute. The authors gratefully acknowledge the help with statistics from Dr. Gui-Shuang Ying, and with data processing and coordination from Ms. Sharon Schwartz, Ms. Anjani Naidu, Ms. Mary Nguyen, and Ms. Elaine Smilko.


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