Long-term doxycycline-regulated transgene expression in the retina of nonhuman primates following subretinal injection of recombinant AAV vectors

Abstract

Adeno-associated viral gene therapy has shown promise for the treatment of inherited and acquired retinal disorders. In most applications, regulation of expression is a critical concern for both safety and efficacy. The purpose of our study was to evaluate the ability of the tetracycline-regulatable system to establish long-term transgene regulation in the retina of nonhuman primates. Three rAAV vectors expressing the tetracycline-dependent transactivator (rtTA) under the control of either the ubiquitous CAG promoter or the specific RPE65 promoter (AAV2/5.CAG.TetOn.epo, AAV2/4.CAG.TetOn.epo, and AAV2/4.RPE65.TetOn.epo) were generated and administered subretinally to seven macaques. We demonstrated that repeated inductions of transgene expression in the nonhuman primate retina can be achieved using a Tet-inducible system via rAAV vector administration over a long period (2.5 years). Maximum erythropoietin (EPO) secretion in the anterior chamber depends upon the rAAV serotype and the nature of the promoter driving rtTA expression. We observed that the EPO isoforms produced in the retina differ from one another based on the transduced cell type of origin within the retina and also differ from both the physiological EPO isoforms and the isoforms produced by AAV-transduced skeletal muscle.

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

“…[34][35][36] This should also be taken into account when choosing promoters that may increase the specificity of transgene expression [37][38][39] and when designing vector systems that allow the temporal regulation of transgene expression. [40][41][42][43] Although comparable in terms of overall transduction efficiency, the tropism profile between rAAV2/2 and rAAV2/6 was considerably different. AAV2 and AAV6 share 82% homology and differ by 53 amino acids in the variable region of the Cap gene.…”

Gene supplementation therapy for recessive forms of inherited retinal dystrophies

“…Manipulation of transgene expression by inducible promoters has been described but to date is largely restricted to the expression of reporter genes. 68,96 Doxycyclin-inducible transgene expression in the RPE65-deficient dog enabled regulation of RPE65 expression, but failed to improve vision. 69 Further studies are therefore required to determine whether the expression levels that can currently be achieved using inducible promoters are sufficient to rescue photoreceptor function and prevent photoreceptor loss.…”

An update on bone substitutes for spinal fusion

“…Various improvements have been implemented to ensure such safety [82,97,114], and gene therapy has been validated as a safe technique in preclinical animal experiments [13,30,43,64,70,99,101]. However, long-term results have not yet been elucidated, and further studies are required before these vectors can be used in clinical practice.…”