Reduced retinal transduction and enhanced transgene-directed immunogenicity with intravitreal delivery of rAAV following posterior vitrectomy in dogs

Abstract

Adeno-associated virus (AAV) vector-based gene therapy is a promising treatment strategy for delivery of neurotrophic transgenes to retinal ganglion cells (RGCs) in glaucoma patients. Retinal distribution of transgene expression following intravitreal injection (IVT) of AAV is variable in animal models and the vitreous humor may represent a barrier to initial vector penetration. The primary goal of our study was to investigate the effect of prior core vitrectomy with posterior hyaloid membrane peeling on pattern and efficiency of transduction of a capsid amino acid substituted AAV2 vector, carrying the green fluorescent protein (GFP) reporter transgene following IVT in dogs. When progressive intraocular inflammation developed starting 4 weeks post IVT, the study plan was modified to allow detailed characterization of the etiology as a secondary goal. Unexpectedly, surgical vitrectomy was found to significantly limit transduction, whereas in non-vitrectomized eyes transduction efficiency reached upwards to 37.3% of RGC layer cells. The developing retinitis was characterized by mononuclear cell infiltrates resulting from a delayed-type hypersensitivity reaction, which we suspect was directed at the GFP transgene. Our results, in a canine large animal model, support caution when considering surgical vitrectomy before IVT for retinal gene therapy in patients, as prior vitrectomy appears to significantly reduce transduction efficiency and may predispose the patient to development of vector-induced immune reactions.

A Novel Method Combining Vitreous Aspiration and Intravitreal AAV2/8 Injection Results in Retina-Wide Transduction in Adult Mice

“…24 In contrast, posterior vitrectomy and posterior hyaloid membrane peeling, in combination with transductions using capsid mutant AAV2 particles, recently were applied to a canine model but failed to increase GFP signals in the retina. 54 Boyd et al 54 observed an inflammatory response possibly against the GFP transgene and a reduced level of GFP-positive cells following vitrectomy. As our vitreous aspiration is different from vitrectomy in larger animal models and humans, it is difficult to compare these studies directly.…”

Assessing retinal ganglion cell damage

“…The greatest challenge when utilizing AAV vectors for in vivo labelling of RGCs is to overcome the inner limiting membrane, which acts as a physical barrier to achieving a high transduction rate. [54][55][56] Administering AAV vectors via intravitreal injection has been shown to be a useful method for longitudinal labelling and imaging ( Figure 5). 57 This technique provides the ability to quantify an estimate of cell density in a living animal and monitor changes in cell labelling.…”

Looking into the future: Gene and cell therapies for glaucoma

“…The use of the self‐complementary AAV vectors and the recent development of novel capsid mutated AAV particles resulted in an expanded toolkit for targeting of the trabecular meshwork and other tissues within the anterior segment of the eye (Figure 3). 62,77,78,80,92‐95 The self‐complementary AAV genome facilitates the generation of double‐stranded DNA for more efficient and long‐term transduction of trabecular meshwork cells in several species, with long‐lasting effects for at least 2 years in monkeys 62,77,91 . Capsid mutant AAV2 (AAV of serotype 2) showed the highest transduction of the trabecular meshwork, followed by AAV5, AAV6, and AAV8 serotypes as shown in the mouse, rat, and cultured human trabecular meshwork cells and perfused anterior chambers 78,80,93 .…”

“…The ciliary body epithelium can be reached by gene therapy vectors via intravitreal injection (Figures 2 and 3). 94,117 Recently, a team of investigators achieved a reduction of aqueous humor production in experimental mouse models of corticosteroid and microbead‐induced ocular hypertension following an intravitreal AAV administration that resulted in disruption of the aquaporin 1 ( AQP1 ) gene within the ciliary epithelium by use of CRISPR‐Cas9 gene editing 117 …”

“…The latter has been demonstrated mostly with AAV in several animal species, including mice, rats, dogs, and monkeys with differences in transduction efficiency partially due to species‐specific variations in retinal inner limiting membrane thickness (Figure 4). 94,186‐192 Techniques for sub‐inner limiting membrane injection of AAV have been described to improve transgene delivery to the retina 193,194 . While other intraocular injection methods could also be considered, for example subretinal and suprachoroidal techniques, the intravitreal administration of viral vector appears to be the most effective to target RGCs 191,195 …”