Trans-ocular Electric Current In Vivo Enhances AAV-Mediated Retinal Gene Transduction after Intravitreal Vector Administration

Abstract

Adeno-associated virus (AAV) vector-mediated gene delivery is a promising approach for therapy, but implementation in the eye currently is hampered by the need for delivering the vector underneath the retina, using surgical application into the subretinal space. This limits the extent of the retina that is treated and may cause surgical injury. Vector delivery into the vitreous cavity would be preferable because it is surgically less invasive and would reach more of the retina. Unfortunately, most conventional, non-modified AAV vector serotypes penetrate the retina poorly from the vitreous; this limits efficient transduction and expression by target cells (retinal pigment epithelium and photoreceptors). We developed a method of applying a small and safe electric current across the intact eye in vivo for a brief period following intravitreal vector administration. This significantly improved AAV-mediated transduction of retinal cells in wild-type mice following intravitreal delivery, with gene expression in retinal pigment epithelium and photoreceptor cells. The low-level current had no adverse effects on retinal structure and function. This method should be generally applicable for other AAV serotypes and may have broad application in both basic research and clinical studies.

Materials promoting viral gene delivery

“…Physical methods such as microinjection, 68,69 microfluidic, 70,71 sonication, 72,73 centrifugation, 74 cellular deformation, 75 laser irradiation 76,77 or electroporation 78,79 induce or facilitate cell entry of vectors by mechanical force, mainly through disrupting the plasma membrane. They are commonly applied in non-viral gene delivery because they transport the genetic information easily without the need for carriers and the otherwise low infectivity.…”

Trans-Ocular Electric Current In Vivo Enhances AAV-Mediated Retinal Transduction in Large Animal Eye After Intravitreal Vector Administration

“…These data provide evidence that transocular ECVM promotes the transduction efficiency of AAV8-CMV-GFP in normal rabbit and monkey retinas following intravitreal injection. In addition to the mouse retina shown previously, 9 this study demonstrates the utility of the ECVM technique to enhance penetration and expression of an AAV-vector construct into the retina of two other species, the rabbit and macaque, following administration into the vitreous. Although the ECVM effects were less in rabbit and NHP than we observed in wild type mouse, the present data were the first proof-of-concept that a similar process may be involved in the larger eyes of these species.…”

“…Specifically, GFP pixel area and intensity were measured along the entire medullated regions in fundus micrographs using an intensity threshold method with ImageJ software (http://imagej.nih.gov/ij/), as previously described. 9 In brief, the analysis used photographs with the entire medullated regions around the optic disc. An intensity threshold was applied by ImageJ software to select GFP-positive areas from background signals.…”

Recombinant Adeno-Associated Viral Vectors (rAAV)-Vector Elements in Ocular Gene Therapy Clinical Trials and Transgene Expression and Bioactivity Assays

“…Enzymatic digestion (proteasome inhibitors) of the ILM or ILM/outer limiting membrane (OLM) or disruptions of the ILM/OLM by the disease can alter rAAV infection and allow rAAV infection deeper into the retina [ 99 , 294 , 304 , 305 ]. Applying a low trans-ocular electric current also allowed efficient transduction of RPE and photoreceptors by rAAV8 upon intravitreal injection in adult mice [ 306 ]. Finally, the application of tyrosine kinase inhibitors might improve the passage of rAAV through the ILM or OLM [ 307 ].…”

“… A hypothetical model of the spread of rAAV capsids (serotypes 1, 2, 5 8, and 9) after intravitreal or subretinal injection in disease or non-disease mouse retinas in vivo based on the studies [ 99 , 294 , 304 , 306 , 308 , 309 ]. RPE, retinal pigment epithelium; OLM, outer limiting membrane, ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; GCL, ganglion cell layer; ILM, Inner Limiting Membrane.…”