Reversal of Blindness in Animal Models of Leber Congenital Amaurosis Using Optimized AAV2-mediated Gene Transfer

Abstract

We evaluated the safety and efficacy of an optimized adeno-associated virus (AAV; AAV2.RPE65) in animal models of the RPE65 form of Leber congenital amaurosis (LCA). Protein expression was optimized by addition of a modified Kozak sequence at the translational start site of hRPE65. Modifications in AAV production and delivery included use of a long stuffer sequence to prevent reverse packaging from the AAV inverted-terminal repeats, and co-injection with a surfactant. The latter allows consistent and predictable delivery of a given dose of vector. We observed improved electroretinograms (ERGs) and visual acuity in Rpe65 mutant mice. This has not been reported previously using AAV2 vectors. Subretinal delivery of 8.25 x 10(10) vector genomes in affected dogs was well tolerated both locally and systemically, and treated animals showed improved visual behavior and pupillary responses, and reduced nystagmus within 2 weeks of injection. ERG responses confirmed the reversal of visual deficit. Immunohistochemistry confirmed transduction of retinal pigment epithelium cells and there was minimal toxicity to the retina as judged by histopathologic analysis. The data demonstrate that AAV2.RPE65 delivers the RPE65 transgene efficiently and quickly to the appropriate target cells in vivo in animal models. This vector holds great promise for treatment of LCA due to RPE65 mutations.

Gene therapy in the second eye of RPE65-deficient dogs improves retinal function

“…Previous studies have documented the histologic appearance of lesions resulting from subretinal injections in dogs and include damage at the injection site, retinal thinning and focal defects in reattachment resulting in retinal 'ripples' . 10,11,33 The small areas of tapetal hyper-reflectivity that we detected in 3 of the 18 eyes did not preclude successful recovery of rod and cone function in these eyes. Reasons for the more diffuse tapetal hyper-reflectivity and presumed retinal thinning in the injected area of eye 2b are not clear but may reflect the potential detrimental effects of the creation of a retinal detachment.…”

“…Consistent with this, we have previously observed transgene expression in both eyes of Rd12 mice when a lower concentration (1Â10 11 vg ml À1 ) of vector construct was used, whereas a higher concentration (5Â10 11 vg ml À1 ) resulted in higher NAb titers and variable transgene expression in the second eye. 27 In the larger canine eye, we have achieved successful photoreceptor rescue at both high and low concentrations (1Â10 11 and 1Â10 12 vg ml À1 ) in both first and second treated eyes without evidence of any significant variation in NAb response. Similarly, administration of a larger total dose of vector did not have a major effect on immune responses or degree of rescue in the first or second treated eyes.…”

“…6 A number of studies have shown rod and cone photoreceptor rescue using rAAV vectors to deliver a normal copy of the RPE65 gene via a subretinal injection in the RPE65 mutant Briard. [6][7][8][9][10][11][12][13] On the basis of the great success of the canine trials, phase I/II clinical trials of rAAV-RPE65 gene replacement therapy in human LCA patients have started with the first reported results showing great promise. [14][15][16] Thus far in all human patients only one eye has been treated.…”

“…Acland et al 8 reported bilateral subretinal rAAV2-RPE65 injections in nine dogs, of these only one dog (BR29 in Table 1) had the second eye injected at a later date and outcome for this eye was not shown. Bennicelli et al 11 reported bilateral subretinal injections of rAAV-RPE65 in two dogs, but both eyes were treated at the same time.…”

Cellular tropism and transduction properties of seven adeno-associated viral vector serotypes in adult retina after intravitreal injection

“…2 In the eye, rAAV has been used in experimental studies aimed at treating retinal degenerative diseases or for cell rescue following trauma. [3][4][5][6][7][8][9][10][11][12][13][14] On the basis of the success of some of these animal studies, clinical trials with AAV vectors have recently been initiated in humans with genetically linked ophthalmic diseases. 15,16 Human AAV serotype 2 was originally found as a contaminant in adenovirus preparations 17 and has not been associated with any human pathogenicity.…”