Gene Augmentation Therapy Restores Retinal Function and Visual Behavior in a Sheep Model of CNGA3 Achromatopsia

Abstract

Achromatopsia is a hereditary form of day blindness caused by cone photoreceptor dysfunction. Affected patients suffer from congenital color blindness, photosensitivity, and low visual acuity. Mutations in the CNGA3 gene are a major cause of achromatopsia, and a sheep model of this disease was recently characterized by our group. Here, we report that unilateral subretinal delivery of an adeno-associated virus serotype 5 (AAV5) vector carrying either the mouse or the human intact CNGA3 gene under the control of the red/green opsin promoter results in long-term recovery of visual function in CNGA3-mutant sheep. Treated animals demonstrated shorter maze passage times and a reduced number of collisions with obstacles compared with their pretreatment status, with values close to those of unaffected sheep. This effect was abolished when the treated eye was patched. Electroretinography (ERG) showed marked improvement in cone function. Retinal expression of the transfected human and mouse CNGA3 genes at the mRNA level was shown by polymerase chain reaction (PCR), and cone-specific expression of CNGA3 protein was demonstrated by immunohistochemisrty. The rescue effect has so far been maintained for over 3 years in the first-treated animals, with no obvious ocular or systemic side effects. The results support future application of subretinal AAV5-mediated gene-augmentation therapy in CNGA3 achromatopsia patients.

Safety and Efficacy Evaluation of rAAV2tYF-PR1.7-hCNGA3 Vector Delivered by Subretinal Injection in CNGA3 Mutant Achromatopsia Sheep

“…15 This large animal model has been used successfully in gene augmentation therapy studies in which treatment with an AAV5 vector carrying either a mouse or human CNGA3 gene under the control of the 2.1 kb red/green opsin promoter (PR2.1) resulted in long-term recovery of visual function. 9 The current study design is summarized in Table 1. A total of 13 animals were divided into three groups of four or five affected sheep. The age of the tested animals at the time of treatment ranged from 8 to 35 months, and animals were followed for 13 weeks post treatment.…”

“…Sheep in group 1 that served as an efficacy positive control received an AAV5-PR2.1-hCNGA3 vector (6 · 10 11 vg/eye) with the same design but from a different production batch of the one that was shown to be effective in a previous study. 9 The left eye of each animal received a subretinal injection of 500 lL of vehicle (four animals distributed across the three experimental groups) or was untreated (nine animals). The original protocol included 12 sheep but was amended to add one additional sheep (animal ID #7965) to group 3 to accommodate the possible loss of animal ID #7070 in this group, which was found to have considerable subconjunctival hemorrhage immediately after its surgical procedure and substantial inflammation in the posterior segment (retina and vitreous) at the 2-week evaluation.…”

“…9 A similar vector containing the CNGB3 gene has been used successfully to restore vision in a canine model of CNGB3-related ACHM. 13 Histopathological findings.…”

“…Similar findings had not been observed in the previous study in which different batches of this vector had been administered by the same investigators. 9 Because determination of vector concentration can vary widely between different laboratories, 17,18 the vector concentration for the batch of AAV5-PR2.1-hCNGA3 vector used to treat the first four animals in the previous published study was tested using the quantitative polymerase chain reaction (qPCR) method used to determine the vector concentration in the present study. Based on the vector concentration determined by …”

“…[6][7][8] Studies in mouse, sheep, and dog models of achromatopsia caused by mutations in the CNGA3 and CNGB3 genes indicate that gene augmentation therapy using a recombinant adeno-associated virus (AAV) vector expressing a normal CNGA3 or CNGB3 gene can produce the functional CNGA3 or CNGB3 proteins and restore cone photoreceptor function. [9][10][11][12][13] As part of the efforts to develop a product candidate for treatment of humans with ACHM caused by mutations in the CNGA3 gene, an Investigational New Drug (IND)-enabling safety and efficacy study of AGTC-402, an AAV vector containing a conespecific promotor (PR1.7), 14 a codon-optimized human CNGA3 cDNA, and a SV40 polyadenylation sequence packaged in an AAV2 capsid containing three tyrosine to phenylalanine (YF) mutations, was conducted in CNGA3-deficient sheep.…”