The Role of the Human Visual Cortex in Assessment of the Long-Term Durability of Retinal Gene Therapy in Follow-on RPE65 Clinical Trial Patients.

PURPOSE: Gene therapy (GT) has offered immense hope to individuals who are visually impaired because of RPE65 mutations. Although GT has shown great success in clinical trials enrolling these individuals, evidence for stability and durability of this treatment over time is still unknown. Herein we explored the value of functional magnetic resonance imaging (fMRI) as an objective measure to assess independently the longevity of retinal GT. DESIGN: Individuals with RPE65 mutations who underwent GT in their worse-seeing eye in a phase 1 clinical trial received a second subretinal injection in their contralateral eye in a follow-on clinical trial. Functional magnetic resonance imaging (MRI) was performed longitudinally to assess brain responses of patients with RPE65 mutations after stimulation of their most recently treated eye before and 1 to 3 years after GT. PARTICIPANTS: Seven participants with RPE65 mutations who were part of the follow-on clinical trial gave informed consent to participate in a longitudinal neuroimaging fMRI study. METHODS: All participants underwent fMRI using a 3-Tesla MRI system and a 32-channel head coil. Participants' cortical activations were assessed using a block design paradigm of contrast reversing checkerboard stimuli delivered using an MRI-compatible video system. MAIN OUTCOME MEASURES: The primary parameters being measured in this study were the qualitative and quantitative fMRI cortical activations produced by our population in response to the visual task. RESULTS: Functional MRI results showed minimal or no cortical responses before GT. Significant increase in cortical activation lasting at least 3 years after GT was observed for all participants. Repeated measures analysis showed significant associations between cortical activations and clinical measures such as full-field light sensitivity threshold for white, red, and blue colors; visual field; and pupillary light reflex. CONCLUSIONS: Participants with RPE65 mutations showed intact visual pathways, which became responsive and strengthened after treatment. Functional MRI results independently revealed the efficacy and durability of a 1-time subretinal injection. The fMRI results paralleled those recently reported during the long-term clinical evaluations of the same patients. Results from this study demonstrated that fMRI may play an important role in providing complementary information to patients' ophthalmic clinical evaluation and has usefulness as an outcome measure for future retinal intervention studies.

Cones respond to light in the absence of transducin ß subunit.

Mammalian cones respond to light by closing a cGMP-gated channel via a cascade that includes a heterotrimeric G-protein, cone transducin, comprising Gαt2, Gß3 and Gγt2 subunits. The function of Gßγ in this cascade has not been examined. Here, we investigate the role of Gß3 by assessing cone structure and function in Gß3-null mouse (Gnb3(-/-)). We found that Gß3 is required for the normal expression of its partners, because in the Gnb3(-/-) cone outer segments, the levels of Gαt2 and Gγt2 are reduced by fourfold to sixfold, whereas other components of the cascade remain unaltered. Surprisingly, Gnb3(-/-) cones produce stable responses with normal kinetics and saturating response amplitudes similar to that of the wild-type, suggesting that cone phototransduction can function efficiently without a Gß subunit. However, light sensitivity was reduced by approximately fourfold in the knock-out cones. Because the reduction in sensitivity was similar in magnitude to the reduction in Gαt2 level in the cone outer segment, we conclude that activation of Gαt2 in Gnb3(-/-) cones proceeds at a rate approximately proportional to its outer segment concentration, and that activation of phosphodiesterase and downstream cascade components is normal. These results suggest that the main role of Gß3 in cones is to establish optimal levels of transducin heteromer in the outer segment, thereby indirectly contributing to robust response properties.