Evaluation of Photoreceptor Transduction Efficacy of Capsid-Modified Adeno-Associated Viral Vectors Following Intravitreal and Subretinal Delivery in Sheep.

Gene augmentation therapy based on subretinal delivery of adeno-associated viral (AAV) vectors is proving to be highly efficient in treating several inherited retinal degenerations. However, due to potential complications and drawbacks posed by subretinal injections, there is a great impetus to find alternative methods of delivering the desired genetic inserts to the retina. One such method is an intravitreal delivery of the vector. Our aim was to evaluate the efficacy of two capsid-modified vectors that are less susceptible to cellular degradation, AAV8 (doubleY-F) and AAV2 (quadY-F+T-V), as well as a third, chimeric vector AAV[max], to transduce photoreceptor cells following intravitreal injection in sheep. We further tested whether saturation of inner limiting membrane (ILM) viral binding sites using a nonmodified vector, before the intravitreal injection, would enhance the efficacy of photoreceptor transduction. Only AAV[max] resulted in moderate photoreceptor transduction following intravitreal injection. Intravitreal injection of the two other vectors did not result in photoreceptor transduction nor did the saturation of the ILM before the intravitreal injection. However, two of the vectors efficiently transduced photoreceptor cells following subretinal injection in positive control eyes. Previous trials with the same vectors in both murine and canine models resulted in robust and moderate transduction efficacy, respectively, of photoreceptors following intravitreal delivery, demonstrating the importance of utilizing as many animal models as possible when evaluating new strategies for retinal gene therapy. The successful photoreceptor transduction of AAV[max] injected intravitreally makes it a potential candidate for intravitreal delivery, but further trials are warranted to determine whether the transduction efficacy is sufficient for a clinical outcome.

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

Applied Genetic Technologies Corporation (AGTC) is developing a recombinant adeno-associated virus (rAAV) vector expressing the human CNGA3 gene designated AGTC-402 (rAAV2tYF-PR1.7-hCNGA3) for the treatment of achromatopsia, an inherited retinal disorder characterized by markedly reduced visual acuity, extreme light sensitivity, and absence of color discrimination. The results are herein reported of a study evaluating safety and efficacy of AGTC-402 in CNGA3-deficient sheep. Thirteen day-blind sheep divided into three groups of four or five animals each received a subretinal injection of an AAV vector expressing a CNGA3 gene in a volume of 500 µL in the right eye. Two groups (n = 9) received either a lower or higher dose of the AGTC-402 vector, and one efficacy control group (n = 4) received a vector similar in design to one previously shown to rescue cone photoreceptor responses in the day-blind sheep model (rAAV5-PR2.1-hCNGA3). The left eye of each animal received a subretinal injection of 500 µL of vehicle (n = 4) or was untreated (n = 9). Subretinal injections were generally well tolerated and not associated with systemic toxicity. Most animals had mild to moderate conjunctival hyperemia, chemosis, and subconjunctival hemorrhage immediately after surgery that generally resolved by postoperative day 7. Two animals treated with the higher dose of AGTC-402 and three of the efficacy control group animals had microscopic findings of outer retinal atrophy with or without inflammatory cells in the retina and choroid that were procedural and/or test-article related. All vector-treated eyes showed improved cone-mediated electroretinography responses with no change in rod-mediated electroretinography responses. Behavioral maze testing under photopic conditions showed significantly improved navigation times and reduced numbers of obstacle collisions in all vector-treated eyes compared to their contralateral control eyes or pre-dose results in the treated eyes. These results support the use of AGTC-402 in clinical studies in patients with achromatopsia caused by CNGA3 mutations, with careful evaluation for possible inflammatory and/or toxic effects.

Gene Augmentation Therapy for a Missense Substitution in the cGMP-Binding Domain of Ovine CNGA3 Gene Restores Vision in Day-Blind Sheep.

Purpose: Applying CNGA3 gene augmentation therapy to cure a novel causative mutation underlying achromatopsia (ACHM) in sheep. Methods: Impaired vision that spontaneously appeared in newborn lambs was characterized by behavioral, electroretinographic (ERG), and histologic techniques. Deep-sequencing reads of an affected lamb and an unaffected lamb were compared within conserved genomic regions orthologous to human genes involved in similar visual impairment. Observed nonsynonymous amino acid substitutions were classified by their deleteriousness score. The putative causative mutation was assessed by producing compound CNGA3 heterozygotes and applying gene augmentation therapy using the orthologous human cDNA. Results: Behavioral assessment revealed day blindness, and subsequent ERG examination showed attenuated photopic responses. Histologic and immunohistochemical examination of affected sheep eyes did not reveal degeneration, and cone photoreceptors expressing CNGA3 were present. Bioinformatics and sequencing analyses suggested a c.1618G>A, p.Gly540Ser substitution in the GMP-binding domain of CNGA3 as the causative mutation. This was confirmed by genetic concordance test and by genetic complementation experiment: All five compound CNGA3 heterozygotes, carrying both p.Arg236* and p.Gly540Ser mutations in CNGA3, were day-blind. Furthermore, subretinal delivery of the intact human CNGA3 gene using an adeno-associated viral vector (AAV) restored photopic vision in two affected p.Gly540Ser homozygous rams. Conclusions: The c.1618G>A, p.Gly540Ser substitution in CNGA3 was identified as the causative mutation for a novel form of ACHM in Awassi sheep. Gene augmentation therapy restored vision in the affected sheep. This novel mutation provides a large-animal model that is valid for most human CNGA3 ACHM patients; the majority of them carry missense rather than premature-termination mutations.

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

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.