Jonathan B. Jacobs

Safety and Efficacy of Gene Transfer for Leber’s Congenital Amaurosis

Lebers congenital amaurosis (LCA) is a group of inherited blinding diseases with onset during childhood. One form of the disease, LCA2, is caused by mutations in the retinal pigment epithelium-specific 65-kDa protein gene (RPE65). We investigated the safety of subretinal delivery of a recombinant adeno-associated virus (AAV) carrying RPE65 complementary DNA (cDNA) (ClinicalTrials.gov number, NCT00516477 [ClinicalTrials.gov]). Three patients with LCA2 had an acceptable local and systemic adverse-event profile after delivery of AAV2.hRPE65v2. Each patient had a modest improvement in measures of retinal function on subjective tests of visual acuity. In one patient, an asymptomatic macular hole developed, and although the occurrence was considered to be an adverse event, the patient had some return of retinal function. Although the follow-up was very short and normal vision was not achieved, this study provides the basis for further gene therapy studies in patients with LCA.

Reversal of Blindness in Animal Models of Leber Congenital Amaurosis Using Optimized AAV2-mediated Gene Transfer

We evaluated the safety and efficacy of an optimized adeno-associated virus (AAV; AAV2.RPE65) in animal models of the RPE65 form of Leber congenital amaurosis (LCA). Protein expression was optimized by addition of a modified Kozak sequence at the translational start site of hRPE65. Modifications in AAV production and delivery included use of a long stuffer sequence to prevent reverse packaging from the AAV inverted-terminal repeats, and co-injection with a surfactant. The latter allows consistent and predictable delivery of a given dose of vector. We observed improved electroretinograms (ERGs) and visual acuity in Rpe65 mutant mice. This has not been reported previously using AAV2 vectors. Subretinal delivery of 8.25 x 10(10) vector genomes in affected dogs was well tolerated both locally and systemically, and treated animals showed improved visual behavior and pupillary responses, and reduced nystagmus within 2 weeks of injection. ERG responses confirmed the reversal of visual deficit. Immunohistochemistry confirmed transduction of retinal pigment epithelium cells and there was minimal toxicity to the retina as judged by histopathologic analysis. The data demonstrate that AAV2.RPE65 delivers the RPE65 transgene efficiently and quickly to the appropriate target cells in vivo in animal models. This vector holds great promise for treatment of LCA due to RPE65 mutations.

A new surgery for congenital nystagmus: Effects of tenotomy on an achiasmatic canine and the role of extraocular proprioception

PURPOSE Human eye-movement recordings have documented that surgical treatment of congenital nystagmus (CN) also produces a broadening of the null zone and changes in foveation that allow increased acuity. We used the achiasmatic Belgian sheepdog, a spontaneously occurring animal model of human CN and see-saw nystagmus (SSN), to test the hypothesis that changes induced by surgical interruption of the extraocular muscle afference without a change in muscle-length tension could damp both oscillations. METHODS An achiasmatic dog with CN and SSN underwent videotaping and infrared oculography in a sling apparatus and head restraints before and after all extraocular muscles (stage 1: 4 horizontal rectus muscles and stage 2 [4 months later]: 4 vertical rectus muscles and 4 oblique muscles) were surgically tenotomized and immediately reattached at their original insertions. RESULTS The dog had immediate and persistent visible, behavioral, and oculographic changes after each stage of this new procedure. These included damped CN and SSN, increased ability to maintain fixation, and increased periods of maintaining the target image on the area centralis over a broad range of gaze angles. CONCLUSIONS Severing and reattaching the tendons of the extraocular muscles affect some as-yet-unknown combination of central nervous system processes producing the above results. This new procedure may prove effective in patients with CN with either no null, a null at primary position, or a time-varying null (due to asymmetric, (a)periodic, alternating nystagmus). We infer from our results in an achiasmatic dog that tenotomy is the probable cause of the damping documented in human CN after Anderson-Kestenbaum procedures and should also damp CN and SSN in achiasma in humans. It may also prove useful in acquired nystagmus to reduce oscillopsia. The success of tenotomy in damping nystagmus in this animal suggests that the proprioceptive feedback loop has a more important role in ocular-motor control than has been appreciated. Finally, we propose a modified bimedial recession procedure, on the basis of the damping effects of tenotomy.

Congenital nystagmus: hypotheses for its genesis and complex waveforms within a behavioral ocular motor system model.

Attempts to simulate dysfunction within ocular motor system (OMS) models capable of exhibiting known ocular motor behavior have provided valuable insight into the structure of the OMS required for normal visual function. The pendular waveforms of congenital nystagmus (CN) appear to be quite complex, composed of a sustained sinusoidal oscillation punctuated by braking saccades and foveating saccades followed by periods of extended foveation. Previously, we verified that these quick phases are generated by the same mechanism as voluntary saccades. We propose a computer model of the ocular motor system that simulates the responses of individuals with pendular CN (including its variable waveforms) based on the instability exhibited by the normal pursuit subsystem and its interaction with other components of the normal ocular motor control system. Fixation data from subjects with CN using both infrared and magnetic search coil oculography were used as templates for our simulations. Our OMS model simulates data from individuals with CN during fixation and in response to complex stimuli. The use of position and velocity efference copy to suppress oscillopsia is the key element in allowing for normal ocular motor behavior. The models responses to target steps, pulse-steps, ramps, and step-ramps support the hypothetical explanation for the conditions that result in sustained pendular oscillation and the rules for the corrective saccadic responses that shape this underlying oscillation into the well-known family of pendular CN waveforms: pendular (P), pseudopendular (PP), pendular with foveating saccades (Pfs), and pseudopendular with foveating saccades (PPfs). Position error determined the saccadic amplitudes of foveating saccades, whereas stereotypical braking saccades were not dependent on visual information. Additionally, we propose a structure and method of operation for the fixation subsystem, and use it to prolong the low-velocity intervals immediately following foveating saccades. The models robustness supports the hypothesis that the pendular nystagmus seen in CN is due to a loss of damping of the normal pursuit-system velocity oscillation (functionally, it is pursuit-system nystagmus--PSN).

The congenital and see-saw nystagmus in the prototypical achiasma of canines: comparison to the human achiasmatic prototype

We applied new methods for canine eye-movement recording to the study of achiasmatic mutant Belgian Sheepdogs, documenting their nystagmus waveforms and comparing them to humans with either congenital nystagmus (CN) alone or in conjunction with achiasma. A sling apparatus with head restraints and infrared reflection with either earth- or head-mounted sensors were used. Data were digitized for later evaluation. The horizontal nystagmus (1-6 Hz) was similar to that of human CN. Uniocular and disconjugate nystagmus and saccades were recorded. See-saw nystagmus (SSN), not normally seen with human CN, was present in all mutants (0.5-6 Hz) and in the one human achiasmat studied thus far. This pedigree is an animal model of CN and the SSN caused by achiasma or uniocular decussation. Given the finding of SSN in all mutant dogs and in a human, achiasma may be sufficient for the development of congenital SSN and, in human infants, SSN should alert the clinician to the possibility of either achiasma or uniocular decussation. Finally, the interplay of conjugacy and disconjugacy suggests independent ocular motor control of each eye with variable yoking in the dog.

Latent and congenital nystagmus in Down syndrome.

OBJECTIVES Although nystagmus has been reported in Down syndrome (DS), it has been poorly characterized, because most investigators have relied on clinical observations rather than on eye movement recordings. This study was conducted to investigate nystagmus in DS, using quantitative measurements of eye movements. METHODS Ocular motility and visual functions were examined in 26 unselected adults with DS and compared with those in an age-matched group of 35 subjects with other causes of mental retardation. The eye movements of those with clinically evident nystagmus were recorded with the infrared technique. We also recorded the eye movements of a child with DS and nystagmus. RESULTS Nystagmus was identified in six (23%) adults with DS and in none in the control group. All six patients showed latent/manifest latent nystagmus (LMLN), prominent with the covering of one eye, and esodeviations of 10 to 30 prism diopters. Eye movement recordings confirmed LMLN with its exponentially decaying waveform. Frequencies ranged from 2 to 5 Hz and amplitudes from 5 degrees to 20 degrees. While attempting to fixate straight ahead in the absence of visual cues, three subjects exhibited shifts in the mean eye position. In contrast with the findings in adults, the only child with DS examined had both congenital nystagmus and LMLN waveforms. CONCLUSIONS The predominant type of nystagmus in the study subjects with DS is LMLN. The high prevalence of LMLN may reflect abnormal integration of visuospatial information that is typical of DS. The concurrent presence of congenital nystagmus in a child but only LMLN in the adults with DS raises the possibility of age-related waveform changes or could reflect sample variation.

A normal ocular motor system model that simulates the dual-mode fast phases of latent/manifest latent nystagmus

Abstract. The fast phases of latent/manifest latent nystagmus (LMLN) may either cause the target image to fall within (foveating) or outside (defoveating) the foveal area. We previously verified that both types are generated by the same mechanism as voluntary saccades and propose a hypothetical, dual-mode mechanism (computer model) for LMLN that utilizes normal ocular-motor control functions. Fixation data recorded during the past 30 years from 97 subjects with LMLN using both infrared and magnetic search coil oculography were used as a basis for our simulations. The MATLAB/Simulink software was used to construct a robust, modular, ocular motor system model, capable of simulating LMLN. Fast-phase amplitude versus both peak velocity and duration of simulated saccades were equivalent to those of saccades in normal subjects. Based on our LMLN studies, we constructed a hypothetical model in which the slow-phase velocity acted to trigger the change between foveating and defoveating LMLN fast phases. Foveating fast phases were generated during lower slow-phase velocities whereas defoveating fast phases occurred during higher slow-phase velocities. The bidirectional model simulated Alexanders law behavior under all viewing and fixation conditions. Our ocular-motor model accurately simulates LMLN patient ocular motility data and provides a hypothetical explanation for the conditions that result in both foveating and defoveating fast phases. As is the case for normal physiological saccades, the position error determined the saccadic amplitudes for foveating fast phases. However, the final slow-phase velocity determined the amplitudes of defoveating fast phases. In addition, we suggest that individuals with LMLN use their fixation subsystem to further decrease the slow-phase velocity as the target image approaches the foveal center.

The Torsional Component of "Horizontal" Congenital Nystagmus

Objectives To study the relationship between the major horizontal and minor torsional components of congenital nystagmus to elucidate the diagnostic importance, effects on vision, and pathogenetic implications of the torsional components. Methods We recorded the eye movements of 13 subjects with congenital nystagmus using a three-dimensional magnetic search coil technique over a 15-year period. The subjects fixated on stationary targets straight ahead and along the horizontal and vertical meridians. Six of the 10 subjects with horizontal congenital nystagmus were asymptomatic; the remaining 4 (plus two with a vertical component to their congenital nystagmus) had adult-onset symptoms. An additional subject without symptoms had a vertical congenital nystagmus component plus seesaw nystagmus; one of the symptomatic subjects also had seesaw nystagmus. Results In all 13 subjects, the horizontal and torsional cycles were phase-locked, and positive horizontal (rightward), vertical (upward, if any), and torsional (clockwise) motion coincided in 10 subjects. That is, rightward horizontal eye rotation coincided with clockwise curvilinear motion (rightward and downward) of the upper pole of each eye. During the horizontal foveation periods, torsional motion was also of low velocity. In 2 of 13 subjects, the torsional waveforms differed from those in the horizontal plane; in others, the direction or the variation with gaze angle differed from that predicted by Listing. In each of the 13 subjects, the torsional components ranged from 8.16% to 94.42% (median, 32.94%) of the peak-to-peak magnitudes of the congenital nystagmus. In most cases, the measured torsion was far greater than that predicted by Listings law for a worst-case analysis (range, 0.69–11.83%; median, 4.91%). The torsional components of the two subjects with seesaw nystagmus were 60.48% and 264.02%. Conclusions The manner in which the horizontal and torsional components of “horizontal” congenital nystagmus were phase-locked made clinical detection of the torsional component difficult. Most “horizontal” congenital nystagmus is actually horizontal-torsional congenital nystagmus. Visual acuity during horizontal foveation periods is not significantly diminished by torsional motion. In only one subject did the torsional component of the congenital nystagmus have an amplitude equivalent to Listing torsion; in the other 12 subjects, torsion exceeded our estimate of what Listings law would predict. The torsional components of the seesaw nystagmus in two subjects also greatly exceeded the torsion predicted by Listing torsion. The most parsimonious explanation for our data is that the cyclic torsion in congenital nystagmus was generated centrally and not a result of Listing torsion, mechanical crosstalk, or normal or abnormal extraocular-muscle (plant) dynamics. Further measurements are needed to confirm this hypothesis.

Using the NAFX to Measure the Effectiveness over Time of Gene Therapy in Canine LCA

PURPOSE To use ocular motility recordings to determine the changes over time of infantile nystagmus syndrome (INS) in RPE65-deficient canines with Leber Congenital Amaurosis (LCA) and assess the time course of the recalibration of the ocular motor system (OMS). METHODS Nine dogs were treated bilaterally with AAV-RPE65. A second cohort of four dogs was treated with AAV2.RPE65, an optimized vector. Their fixation eye movements were recorded before treatment and at 4-week intervals for 3 months, by using high-speed (500 Hz) digital videography. The dogs were suspended in a sling and encouraged to fixate on distant (57 inches) targets at gaze angles varying between +/-15 degrees horizontally and +/-10 degrees vertically. The records for each eye were examined for qualitative changes in waveform and for quantitative changes in centralisation with the expanded nystagmus acuity function (NAFX) and compared with ERG results for restoration of receptor function. RESULTS First group: Before treatment, five of the dogs had clinically apparent INS with jerk, pendular, or both waveforms and with peak-to-peak amplitudes as great as 15 degrees . One dog had intermittent nystagmus. At the 1- and 2-month examinations, no change in nystagmus waveform or NAFX was observed in any of the initial dogs, while at 10 weeks, one dog treated bilaterally with the standard dosage showed reduced nystagmus in only one eye. The other eye did not respond to treatment, as confirmed by ERG. This result was unexpected since it was previously documented that unilateral treatment leads to bilateral reduction of INS. The other dog treated with the standard dosage showed no reduction of its small-amplitude, high-frequency pendular nystagmus despite positive ERG responses. Second group: Only one dog of the four had clinically detectable INS, similar in characteristics to that seen in the affected dogs of the first group. Unlike any previous dog studied, this one showed a damping of the nystagmus within the first 4 weeks after treatment. CONCLUSIONS In all but one of the cases in which OMS recalibration occurred, as measured by the clinical appearance of nystagmus and by quantitative measurement using the NAFX, the improvement was apparent no sooner than 10 weeks after treatment. Longer term, dose-related studies are needed to determine the minimum necessary degree of restored receptor functionality, the duration after rescue for recalibration of the OMS, and the conditions under which recalibration information can successfully affect the contralateral eye.

Characteristics of braking saccades in congenital nystagmus.

Several of the characteristic waveforms of congenital nystagmus (CN) contain braking saccades. We test the hypothesis that braking (including foveating) saccades, while not always satisfying the standard relationships for saccades, are normal; any differences are due to the presence of high-velocity, slow-phase eye movements. Better measurements of saccadic properties, including position- and velocity-based measures and skewness, can eliminate some of this apparent distortion. We also evoked an analogous effect in normal subjects by use of a ramp-step-ramp stimulus. Finally, we used a model to further demonstrate this distortion in the saccades of normals, deviating from their intended magnitude as a function of the magnitude of the opposing velocity. The saccadic analysis methods developed herein are applicable to all saccades made during ongoing eye movements, whether normal or pathological. The above findings support the hypothesis that the braking saccades integral to many CN waveforms have normal characteristics and are the result of a normal saccadic systems responses to a slow-eye-movement oscillation.