Six Years and Counting: Restoration of Photopic Retinal Function and Visual Behavior Following Gene Augmentation Therapy in a Sheep Model of CNGA3 Achromatopsia
Abstract:Achromatopsia causes severely reduced visual acuity, photoaversion, and inability to discern colors due to cone photoreceptor dysfunction. In 2010, we reported on day-blindness in sheep caused by a stop-codon mutation of the ovine CNGA3 gene and began gene augmentation therapy trials in this naturally occurring large animal model of CNGA3 achromatopsia. The purpose of this study was to evaluate long-term efficacy and safety results of treatment, findings that hold great relevance for clinical trials that start… Show more
“…Subsequently, we used these CNGA3 mutation models for successful gene therapy trials, based on subretinal injections of an adeno-associated virus carrying the CNGA3 transgene under the control of a cone-specific promoter 33 , 36 , 37 . Treatment restored cone function and photopic (daytime) vision within a few days in all treated sheep, as demonstrated both behaviorally and electroretinographically (ERG), with the oldest treated sheep still visual after more than 9 years 38 . Based on our results, as well as work of other teams using murine and canine models of the disease 39 – 43 , Phase I/IIa clinical trials have been approved in human ACHM patients (NCT02935517 and NCT02610582), demonstrating the translational relevance of our work with this large animal model of CNGA3 ACHM .…”
Section: Introductionmentioning
confidence: 73%
“…In addition, no significant differences were found between the refractive errors of the left eyes in the treated and untreated day- blind sheep of cohort 1 (P > 0.05). Therefore, in accordance with statistical guidelines recommending that data from both eyes should not be combined 48 , 49 , and because the right eyes of some sheep in Improved Awassi day-blind sheep (cohort 1) were used in a gene therapy study 36 , 38 , the refractive error of only the left eyes was used in subsequent analyses. Figure 1 Refractive errors of day-blind sheep compared to two cohorts of wild type sheep.…”
Section: Resultsmentioning
confidence: 99%
“…Cohort 1 consisted of 21 CNGA3 mutant, day-blind Improved Awassi sheep, of which three were male. Eight animals of cohort 1 previously underwent gene augmentation therapy in their right eye, while their left eye remained untreated as negative control 36,38 Refraction. The refractive error was determined during one experimental session for Improved Awassi dayblind and Afec-Assaf WT (cohorts 1 and 2, respectively), and a second session for WT Local Awassi sheep (cohort 3).…”
Section: Methodsmentioning
confidence: 99%
“…The PLR was evaluated in both eyes of four Improved Awassi day-blind (cohort 1) and four Afec-Assaf WT (cohort 2) sheep. These eight animals were randomly selected from cohorts 1 & 2, respectively, excluding cohort 1 sheep that are part of an ongoing gene therapy study 36,38 . The test was performed on alert animals, manually restrained in dim light (121 lx).…”
Achromatopsia is an inherited retinal disease characterized by loss of cone photoreceptor function. Day blind CNGA3 mutant Improved Awassi sheep provide a large animal model for achromatopsia. This study measured refractive error and axial length parameters of the eye in this model and evaluated chromatic pupillary light reflex (cPLR) testing as a potential screening test for loss of cone function. Twenty-one CNGA3 mutant, Improved Awassi, 12 control Afec-Assaf and 12 control breed-matched wild-type (WT) Awassi sheep were examined using streak retinoscopy and B-mode ocular ultrasonography. Four CNGA3 mutant and four Afec-Assaf control sheep underwent cPLR testing. Statistical tests showed that day-blind sheep are significantly more myopic than both Afec-Assaf and WT Awassi controls. Day-blind sheep had significantly longer vitreous axial length compared to WT Awassi (1.43 ± 0.13 and 1.23 ± 0.06 cm, respectively, p < 0.0002) and no response to bright red light compared to both controls. Lack of response to bright red light is consistent with cone dysfunction, demonstrating that cPLR can be used to diagnose day blindness in sheep. Day-blind sheep were found to exhibit myopia and increased vitreous chamber depth, providing a naturally occurring large animal model of myopia.
“…Subsequently, we used these CNGA3 mutation models for successful gene therapy trials, based on subretinal injections of an adeno-associated virus carrying the CNGA3 transgene under the control of a cone-specific promoter 33 , 36 , 37 . Treatment restored cone function and photopic (daytime) vision within a few days in all treated sheep, as demonstrated both behaviorally and electroretinographically (ERG), with the oldest treated sheep still visual after more than 9 years 38 . Based on our results, as well as work of other teams using murine and canine models of the disease 39 – 43 , Phase I/IIa clinical trials have been approved in human ACHM patients (NCT02935517 and NCT02610582), demonstrating the translational relevance of our work with this large animal model of CNGA3 ACHM .…”
Section: Introductionmentioning
confidence: 73%
“…In addition, no significant differences were found between the refractive errors of the left eyes in the treated and untreated day- blind sheep of cohort 1 (P > 0.05). Therefore, in accordance with statistical guidelines recommending that data from both eyes should not be combined 48 , 49 , and because the right eyes of some sheep in Improved Awassi day-blind sheep (cohort 1) were used in a gene therapy study 36 , 38 , the refractive error of only the left eyes was used in subsequent analyses. Figure 1 Refractive errors of day-blind sheep compared to two cohorts of wild type sheep.…”
Section: Resultsmentioning
confidence: 99%
“…Cohort 1 consisted of 21 CNGA3 mutant, day-blind Improved Awassi sheep, of which three were male. Eight animals of cohort 1 previously underwent gene augmentation therapy in their right eye, while their left eye remained untreated as negative control 36,38 Refraction. The refractive error was determined during one experimental session for Improved Awassi dayblind and Afec-Assaf WT (cohorts 1 and 2, respectively), and a second session for WT Local Awassi sheep (cohort 3).…”
Section: Methodsmentioning
confidence: 99%
“…The PLR was evaluated in both eyes of four Improved Awassi day-blind (cohort 1) and four Afec-Assaf WT (cohort 2) sheep. These eight animals were randomly selected from cohorts 1 & 2, respectively, excluding cohort 1 sheep that are part of an ongoing gene therapy study 36,38 . The test was performed on alert animals, manually restrained in dim light (121 lx).…”
Achromatopsia is an inherited retinal disease characterized by loss of cone photoreceptor function. Day blind CNGA3 mutant Improved Awassi sheep provide a large animal model for achromatopsia. This study measured refractive error and axial length parameters of the eye in this model and evaluated chromatic pupillary light reflex (cPLR) testing as a potential screening test for loss of cone function. Twenty-one CNGA3 mutant, Improved Awassi, 12 control Afec-Assaf and 12 control breed-matched wild-type (WT) Awassi sheep were examined using streak retinoscopy and B-mode ocular ultrasonography. Four CNGA3 mutant and four Afec-Assaf control sheep underwent cPLR testing. Statistical tests showed that day-blind sheep are significantly more myopic than both Afec-Assaf and WT Awassi controls. Day-blind sheep had significantly longer vitreous axial length compared to WT Awassi (1.43 ± 0.13 and 1.23 ± 0.06 cm, respectively, p < 0.0002) and no response to bright red light compared to both controls. Lack of response to bright red light is consistent with cone dysfunction, demonstrating that cPLR can be used to diagnose day blindness in sheep. Day-blind sheep were found to exhibit myopia and increased vitreous chamber depth, providing a naturally occurring large animal model of myopia.
“…11,28 The episomal persistence of the rAAV2 genome and its ability to support prolonged gene expression have been documented for many other tissues as well. 26,[29][30][31] More importantly, the genome of HBoV1 is 18% larger than that of AAV2 (5.54 kb vs. 4.68 kb). 32 Indeed, the HBoV1 capsid comfortably accommodates an oversized rAAV2 genome and is capable of delivering a full-length CFTR coding sequence along with transcriptional elements necessary for efficient CFTR mRNA expression.…”
The genome of recombinant adeno-associated virus 2 (rAAV2) remains a promising candidate for gene therapy for cystic fibrosis (CF) lung disease, but due to limitations in the packaging capacity and the tropism of this virus with respect to the airways, strategies have evolved for packaging an rAAV2 genome (up to 5.8 kb) into the capsid of human bocavirus 1 (HBoV1) to produce a chimeric rAAV2/HBoV1 vector. Although a replication-incompetent HBoV1 genome has been established as a trans helper for capsid complementation, this system remains suboptimal with respect to virion yield. Here, a streamlined production system is described based on knowledge of the involvement of HBoV1 nonstructural (NS) proteins NS1, NS2, NS3, NS4, and NP1 in the process of virion production. The analyses reveal that NS1 and NS2 negatively impact virion production, NP1 is required to prevent premature termination of transcription of the cap mRNA from the native genome, and silent mutations within the polyadenylation sites of the cap coding sequence can eliminate this requirement for NP1. It is further shown that preventing the expression of all NS proteins significantly increases virion yield. Whereas the expression of capsid proteins VP1, VP2, and VP3 from a codon-optimized cap mRNA was highly efficient, optimal virion assembly, and thus potency, required enhanced VP1 expression, entailing a separate VP1 expression cassette. The final NS protein-free production system uses three-plasmid co-transfection of HEK293 cells, with one trans helper plasmid encoding VP1 and the AAV2 Rep proteins, and another encoding VP2-3 and components from adenovirus. This system yielded >16-fold more virions than the prototypic system, without reducing transduction potency. This increase in virion production is expected to facilitate greatly both research on the biology of rAAV2/HBoV1 and preclinical studies testing the effectiveness of this vector for gene therapy of CF lung disease in large animal models.
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