Inner Limiting Membrane Barriers to AAV-mediated Retinal Transduction From the Vitreous

Abstract

Adeno-associated viral gene therapy has shown great promise in treating retinal disorders, with three promising clinical trials in progress. Numerous adeno-associated virus (AAV) serotypes can infect various cells of the retina when administered subretinally, but the retinal detachment accompanying this injection induces changes that negatively impact the microenvironment and survival of retinal neurons. Intravitreal administration could circumvent this problem, but only AAV2 can infect retinal cells from the vitreous, and transduction is limited to the inner retina. We therefore sought to investigate and reduce barriers to transduction from the vitreous. We fluorescently labeled several AAV serotype capsids and followed their retinal distribution after intravitreal injection. AAV2, 8, and 9 accumulate at the vitreoretinal junction. AAV1 and 5 show no accumulation, indicating a lack of appropriate receptors at the inner limiting membrane (ILM). Importantly, mild digestion of the ILM with a nonspecific protease enabled substantially enhanced transduction of multiple retinal cell types from the vitreous, with AAV5 mediating particularly remarkable expression in all retinal layers. This protease treatment has no effect on retinal function as shown by electroretinogram (ERG) and visual cortex cell population responses. These findings may help avoid limitations, risks, and damage associated with subretinal injections currently necessary for clinical gene therapy.

Enhanced gene delivery to the neonatal retina through systemic administration of tyrosine-mutated AAV9

“…Unfortunately, there are a number of formidable barriers impeding gene delivery to the retina from the vitreous such as diffusion in the vitreous cavity and sequestering of viral particles in the inner limiting membrane. Although much progress has been made in the understanding of barriers to retinal transduction from the vitreous for AAV 7,8 as well as improving the limited transduction of the naturally occurring serotypes by rational 9,10 and directed evolution approaches 11,12 major obstacles remain in reaching therapeutically effective levels of gene expression, especially in larger animals where the inner limiting membrane is significantly thicker than in rodents. 13 An alternative approach for transducing large areas of retina is to administer vectors through the ocular vasculature.…”

Long-term restoration of visual function in end-stage retinal degeneration using subretinal human melanopsin gene therapy

“…Virions delivered via intravitreal injection are diluted more in primates compared with mice due to the larger volume of the vitreous, reducing the concentration of vector reaching retinal cells. The inner limiting membrane on the retinal surface is also thicker in primates than in rodents (19), through which virions must pass to reach target cells. The increased risks of an inflammatory response following intravitreal AAV injection (20) may also limit the translational potential of this route of delivery.…”

Leber Congenital Amaurosis Caused by Mutations in GUCY2D

“…Viral serotype choice is dependent on the route of administration, as transduction profiles vary greatly depending on where the vector is placed within the eye (Dalkara et al 2009;Vandenberghe and Auricchio 2012). The route of administration is ideally dictated by the structural integrity of the retina, with subretinal injections considered "safer" under relatively less degenerated tissue that is able to withstand the impact of surgical detachment.…”