Choroideremia (CHM) is an X-linked retinal degeneration leading to loss of the photoreceptors, retinal pigment epithelium (RPE), and choroid. Adaptive optics optoretinography is an emerging technique ...for noninvasive, objective assessment of photoreceptor function. Here, we investigate parafoveal cone function in CHM using adaptive optics optoretinography and compare with cone structure and clinical assessments of vision. Parafoveal cone mosaics of 10 CHM and four normal-sighted participants were imaged with an adaptive optics scanning light ophthalmoscope. While acquiring video sequences, a 2 s 550Δ10 nm, 450 nW/deg
stimulus was presented. Videos were registered and the intensity of each cone in each frame was extracted, normalized, standardized, and aggregated to generate the population optoretinogram (ORG) over time. A gamma-pdf was fit to the ORG and the peak was extracted as ORG amplitude. CHM ORG amplitudes were compared to normal and were correlated with bound cone density, ellipsoid zone to RPE/Bruch's membrane (EZ-to-RPE/BrM) distance, and foveal sensitivity using Pearson correlation analysis. ORG amplitude was significantly reduced in CHM compared to normal (0.22 ± 0.15 vs. 1.34 ± 0.31). In addition, CHM ORG amplitude was positively correlated with cone density, EZ-to-RPE/BrM distance, and foveal sensitivity. Our results demonstrate promise for using ORG as a biomarker of photoreceptor function.
IMPORTANCE: Subretinal injection for gene augmentation in retinal degenerations forcefully detaches the neural retina from the retinal pigment epithelium, potentially damaging photoreceptors and/or ...retinal pigment epithelium cells. OBJECTIVE: To use adaptive optics scanning light ophthalmoscopy (AOSLO) to assess the short-term integrity of the cone mosaic following subretinal injections of adeno-associated virus vector designed to deliver a functional version of the CHM gene (AAV2-hCHM) in patients with choroideremia. DESIGN, SETTING, AND PARTICIPANTS: This longitudinal case series study enrolled adult patients with choroideremia from February 2015 to January 2016 in the US. To be included in the study, study participants must have received uniocular subfoveal injections of low-dose (5 × 1010 vector genome per eye) or high-dose (1 × 1011 vector genome per eye) AAV2-hCHM. Analysis began February 2015. MAIN OUTCOMES AND MEASURES: The macular regions of both eyes were imaged before and 1 month after injection using a custom-built multimodal AOSLO. Postinjection cone inner segment mosaics were compared with preinjection mosaics at multiple regions of interest. Colocalized spectral-domain optical coherence tomography and dark-adapted cone sensitivity was also acquired at each time point. RESULTS: Nine study participants ranged in age from 26 to 50 years at the time of enrollment, and all were White men. Postinjection AOSLO images showed preservation of the cone mosaic in all 9 AAV2-hCHM–injected eyes. Mosaics appeared intact and contiguous 1 month postinjection, with the exception of foveal disruption in 1 patient. Optical coherence tomography showed foveal cone outer segment shortening postinjection. Cone-mediated sensitivities were unchanged in 8 of 9 injected and 9 of 9 uninjected eyes. One participant showed acute loss of foveal optical coherence tomography cone outer segment–related signals along with cone sensitivity loss that colocalized with disruption of the mosaic on AOSLO. CONCLUSIONS AND RELEVANCE: Integrity of the cone mosaic is maintained following subretinal delivery of AAV2-hCHM, providing strong evidence in support of the safety of the injections. Minor foveal thinning observed following surgery corresponds with short-term cone outer segment shortening rather than cone cell loss. Foveal cone loss in 1 participant raises the possibility of individual vulnerability to the subretinal injection.
Within the next decade, we will see many gene therapy clinical trials for eye diseases, which may lead to treatments for thousands of visually impaired people around the world. To target retinal ...diseases that affect specific cell types, several recombinant adeno-associated virus (AAV) serotypes have been generated and used successfully in preclinical mouse studies. Because there are numerous anatomic and physiologic differences between the eyes of mice and "men" and because surgical delivery approaches and immunologic responses also differ between these species, this study evaluated the transduction characteristics of two promising new serotypes, AAV7m8 and AAV8BP2, in the retinas of animals that are most similar to those of humans: non-human primates (NHPs). We report that while AAV7m8 efficiently targets a variety of cell types by subretinal injection in NHPs, transduction after intravitreal delivery was mostly restricted to the inner retina at lower doses that did not induce an immune response. AAV8BP2 targets the cone photoreceptors efficiently but bipolar cells inefficiently by subretinal injection. Additionally, transduction by both serotypes in the anterior chamber of the eye and the optic pathway of the brain was observed post-intravitreal delivery. Finally, we assessed immunogenicity, keeping in mind that these AAV capsids may be used in future clinical trials. We found that AAV8BP2 had a better safety profile compared with AAV7m8, even at the highest doses administered. These studies underscore the differences in AAV transduction between mice and primates, highlighting the importance of careful evaluation of therapeutic vectors in NHPs prior to moving to clinical trials.
Choroideremia (CHM) is an X-linked inherited retinal degeneration causing loss of the photoreceptors, retinal pigment epithelium, and choriocapillaris, although patients typically retain a central ...island of relatively preserved, functioning retina until late-stage disease. Here, we investigate cone photoreceptor morphology within the retained retinal island by examining cone inner segment area, density, circularity, and intercone space.
Using a custom-built, multimodal adaptive optics scanning light ophthalmoscope, nonconfocal split-detection images of the photoreceptor mosaic were collected at 1°, 2°, and 4° temporal to the fovea from 13 CHM and 12 control subjects. Cone centers were manually identified, and cone borders were segmented. A custom MATLAB script was used to extract area and circularity for each cone and calculate the percentage of intercone space in each region of interest. Bound cone density was also calculated. An unbalanced two-way ANOVA and Bonferroni post hoc tests were used to assess statistical differences between the CHM and control groups and along retinal eccentricity.
Cone density was lower in the CHM group than in the control group (P < 0.001) and decreased with eccentricity from the fovea (P < 0.001). CHM cone inner segments were larger in area (P < 0.001) and more circular (P = 0.042) than those of the controls. Intercone space in CHM was also higher than in the controls (P < 0.001).
Cone morphology is altered in CHM compared to control, even within the centrally retained, functioning retinal area. Further studies are required to determine whether such morphology is a precursor to cone degeneration.
Photoreceptors mediate the first step of vision, transducing light and passing signals to retinal neurons that ultimately send signals along the optic nerve to the brain. A functional deficiency in ...the photoreceptors, due to either congenital or acquired disease, can significantly affect an individual's sight and quality of life. Methods for quantifying the health and function of photoreceptors are essential for understanding both the progression of disease and the efficacy of treatment. Given that emerging treatments such as gene, stem cell, and small molecule therapy are designed to operate at the cellular scale, it is desirable to monitor function at the commensurate resolution of individual photoreceptors. Previously, non-invasive imaging methods for visualizing photoreceptor mosaic structure have been used to infer photoreceptor health, but these methods do not assess function directly. Conversely, most functional techniques, such as ERG and conventional microperimetry, measure function by aggregating the effects of signals from many photoreceptors. We have previously shown that stimulus-evoked intrinsic changes in intensity can be measured reliably in populations of cone photoreceptors in the intact human eye, a measurement we refer to more generally as the cone optoretinogram. Here we report that we can resolve the intensity optoretinogram at the level of individual cones. Moreover, we show that the individual cone optoretinogram exhibits two key signatures expected of a functional measure. First, responses in individual cones increase systematically as a function of stimulus irradiance. Second, we can use the amplitude of the functional response to middle wavelength (545 nm) light to separate the population of short-wavelength-sensitive (S) cones from the population of middle- and long-wavelength-sensitive (L and M) cones. Our results demonstrate the promise of optoretinography as a direct diagnostic measure of individual cone function in the living human eye.
Summary Background Gene therapy has the potential to reverse disease or prevent further deterioration of vision in patients with incurable inherited retinal degeneration. We therefore did a phase 1 ...trial to assess the effect of gene therapy on retinal and visual function in children and adults with Leber's congenital amaurosis. Methods We assessed the retinal and visual function in 12 patients (aged 8–44 years) with RPE65 -associated Leber's congenital amaurosis given one subretinal injection of adeno-associated virus (AAV) containing a gene encoding a protein needed for the isomerohydrolase activity of the retinal pigment epithelium (AAV2-hRPE65v2) in the worst eye at low (1·5×1010 vector genomes), medium (4·8×1010 vector genomes), or high dose (1·5×1011 vector genomes) for up to 2 years. Findings AAV2-hRPE65v2 was well tolerated and all patients showed sustained improvement in subjective and objective measurements of vision (ie, dark adaptometry, pupillometry, electroretinography, nystagmus, and ambulatory behaviour). Patients had at least a 2 log unit increase in pupillary light responses, and an 8-year-old child had nearly the same level of light sensitivity as that in age-matched normal-sighted individuals. The greatest improvement was noted in children, all of whom gained ambulatory vision. The study is registered with ClinicalTrials.gov , number NCT00516477. Interpretation The safety, extent, and stability of improvement in vision in all patients support the use of AAV-mediated gene therapy for treatment of inherited retinal diseases, with early intervention resulting in the best potential gain. Funding Center for Cellular and Molecular Therapeutics at the Children's Hospital of Philadelphia, Foundation Fighting Blindness, Telethon, Research to Prevent Blindness, F M Kirby Foundation, Mackall Foundation Trust, Regione Campania Convenzione, European Union, Associazione Italiana Amaurosi Congenita di Leber, Fund for Scientific Research, Fund for Research in Ophthalmology, and National Center for Research Resources.
Retinal pigment epithelial (RPE) cells are critical for the health of the retina, especially the photoreceptors. A recent study demonstrated that individual RPE cells could be imaged in macaque in ...vivo by detecting autofluorescence with an adaptive optics scanning laser ophthalmoscope (AOSLO). The current study extended this method to image RPE cells in fixating humans in vivo and to quantify the RPE mosaic characteristics in the central retina of normal humans and macaques.
The retina was imaged simultaneously with two light channels in a fluorescence AOSLO; one channel was used for reflectance imaging of the cones while the other detected RPE autofluorescence. The excitation light was 568 nm, and emission was detected over a 40-nm range centered at 624 nm. Reflectance frames were registered to determine interframe eye motion, the motion was corrected in the simultaneously recorded autofluorescence frames, and the autofluorescence frames were averaged to give the final RPE mosaic image.
In vivo imaging demonstrated that with increasing eccentricity, RPE cell density, and mosaic regularity decreased, whereas RPE cell size and spacing increased. Repeat measurements of the same retinal location 42 days apart showed the same RPE cells and distribution.
The RPE cell mosaic has been resolved for the first time in alert fixating human subjects in vivo using AOSLO. Mosaic analysis provides a quantitative database for studying normal and diseased RPE in vivo. This technique will allow longitudinal studies to track disease progression and assess treatment efficacy in patients and animal models of retinal disease.
We characterized retinal structure in patients and carriers of choroideremia using adaptive optics and other high resolution modalities.
A total of 57 patients and 18 carriers of choroideremia were ...imaged using adaptive optics scanning light ophthalmoscopy (AOSLO), optical coherence tomography (OCT), autofluorescence (AF), and scanning light ophthalmoscopy (SLO). Cone density was measured in 59 eyes of 34 patients where the full cone mosaic was observed.
The SLO imaging revealed scalloped edges of RPE atrophy and large choroidal vessels. The AF imaging showed hypo-AF in areas of degeneration, while central AF remained present. OCT images showed outer retinal tubulations and thinned RPE/interdigitation layers. The AOSLO imaging revealed the cone mosaic in central relatively intact retina, and cone density was either reduced or normal at 0.5 mm eccentricity. The border of RPE atrophy showed abrupt loss of the cone mosaic at the same location. The AF imaging in comparison with AOSLO showed RPE health may be compromised before cone degeneration. Other disease features, including visualization of choroidal vessels, hyper-reflective clumps of cones, and unique retinal findings, were tabulated to show the frequency of occurrence and model disease progression.
The data support the RPE being one primary site of degeneration in patients with choroideremia. Photoreceptors also may degenerate independently. High resolution imaging, particularly AOSLO in combination with OCT, allows single cell analysis of disease in choroideremia. These modalities promise to be useful in monitoring disease progression, and in documenting the efficacy of gene and cell-based therapies for choroideremia and other diseases as these therapies emerge. (ClinicalTrials.gov number, NCT01866371.).
Strong magnetic fields from magnetic resonance (MR) scanners induce a Lorentz force that contributes to vertigo and persistent nystagmus. Prior studies have reported a predominantly horizontal ...direction for healthy subjects in a 7 Tesla (T) MR scanner, with slow phase velocity (SPV) dependent on head orientation. Less is known about vestibular signal behavior for subjects in a weaker, 3T magnetic field, the standard strength used in the Human Connectome Project (HCP). The purpose of this study is to characterize the form and magnitude of nystagmus induced at 3T.
Forty-two subjects were studied after being introduced head-first, supine into a Siemens Prisma 3T scanner. Eye movements were recorded in four separate acquisitions over 20 min. A biometric eye model was fitted to the recordings to derive rotational eye position and then SPV. An anatomical template of the semi-circular canals was fitted to the T2 anatomical image from each subject, and used to derive the angle of the B0 magnetic field with respect to the vestibular apparatus.
Recordings from 37 subjects yielded valid measures of eye movements. The population-mean SPV ± SD for the horizontal component was -1.38 ± 1.27 deg/sec, and vertical component was -0.93 ± 1.44 deg/sec, corresponding to drift movement in the rightward and downward direction. Although there was substantial inter-subject variability, persistent nystagmus was present in half of subjects with no significant adaptation over the 20 min scanning period. The amplitude of vertical drift was correlated with the roll angle of the vestibular system, with a non-zero vertical SPV present at a 0 degree roll.
Non-habituating vestibular signals of varying amplitude are present in resting state data collected at 3T.
The photoreceptor/RPE complex must maintain a delicate balance between maximizing the absorption of photons for vision and retinal image quality while simultaneously minimizing the risk of ...photodamage when exposed to bright light. We review the recent discovery of two new effects of light exposure on the photoreceptor/RPE complex in the context of current thinking about the causes of retinal phototoxicity. These effects are autofluorescence photobleaching in which exposure to bright light reduces lipofuscin autofluorescence and, at higher light levels, RPE disruption in which the pattern of autofluorescence is permanently altered following light exposure. Both effects occur following exposure to visible light at irradiances that were previously thought to be safe. Photopigment, retinoids involved in the visual cycle, and bisretinoids in lipofuscin have been implicated as possible photosensitizers for photochemical damage. The mechanism of RPE disruption may follow either of these paths. On the other hand, autofluorescence photobleaching is likely an indicator of photooxidation of lipofuscin. The permanent changes inherent in RPE disruption might require modification of the light safety standards. AF photobleaching recovers after several hours although the mechanisms by which this occurs are not yet clear. Understanding the mechanisms of phototoxicity is all the more important given the potential for increased susceptibility in the presence of ocular diseases that affect either the visual cycle and/or lipofuscin accumulation. In addition, knowledge of photochemical mechanisms can improve our understanding of some disease processes that may be influenced by light exposure, such as some forms of Leber's congenital amaurosis, and aid in the development of new therapies. Such treatment prior to intentional light exposures, as in ophthalmic examinations or surgeries, could provide an effective preventative strategy.
PDF
