Single residue AAV capsid mutation improves transduction of photoreceptors in the Abca4−/− mouse and bipolar cells in the rd1 mouse and human retina ex vivo

Abstract

Gene therapy using adeno-associated viral vectors (AAV) for the treatment of retinal degenerations has shown safety and efficacy in clinical trials. However, very high levels of vector expression may be necessary for the treatment of conditions such as Stargardt disease where a dual vector approach is potentially needed, or in optogenetic strategies for end-stage degeneration in order to achieve maximal light sensitivity. In this study, we assessed two vectors with single capsid mutations, rAAV2/2(Y444F) and rAAV2/8(Y733F) in their ability to transduce retina in the Abca4-/- and rd1 mouse models of retinal degeneration. We noted significantly increased photoreceptor transduction using rAAV2/8(Y733F) in the Abca4-/- mouse, in contrast to previous work where vectors tested in this model have shown low levels of photoreceptor transduction. Bipolar cell transduction was achieved following subretinal delivery of both vectors in the rd1 mouse, and via intravitreal delivery of rAAV2/2(Y444F). The successful use of rAAV2/8(Y733F) to target bipolar cells was further validated on human tissue using an ex-vivo culture system of retinal explants. Capsid mutant AAV vectors transduce human retinal cells and may be particularly suited to treating retinal degenerations in which high levels of transgene expression are required.

Inclusion of the Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element Enhances AAV2-Driven Transduction of Mouse and Human Retina

“…We have previously shown that AAV2 has the capability of transducing human retinal explants cultured ex vivo 28, 39. Here, we report that the WPRE also improves GFP expression following transduction in retinectomized human retinal tissue, in spite of its inherent atrophic nature.…”

Assessment of AAV Vector Tropisms for Mouse and Human Pluripotent Stem Cell–Derived RPE and Photoreceptor Cells

“…The in vivo results consistently show greater photoreceptor transduction efficiency by serotypes AAV2/5, 2/8 and 2/9, compared with the archetypal AAV2/2 vector. Recently, screening for tropism and infectivity of human retinal cells has also been performed in ex vivo human retinal explants, although the results appear to be highly variable between individual explants [15][16][17] .…”

Improvement of Photoreceptor Targeting via Intravitreal Delivery in Mouse and Human Retina Using Combinatory rAAV2 Capsid Mutant Vectors

“…In view of these constraints, we instead turned to an ex vivo organotypic culture system that allows for the tropism of the hybrid vector rAAV2/2[MAX] to be determined on human retina directly. In contrast with previous studies where fresh peripheral retina was obtained during a retinectomy, 33 , 34 we cultured central retina acquired post mortem from donor eyes. While obtaining viable postmortem retinal samples proved to be challenging, the opportunity to evaluate the transduction profile of rAAV2/2[MAX] in samples containing the macula was deemed to be of substantial clinical relevance.…”

“…Indeed, tyrosine-to-phenylalanine point mutations have also been incorporated in rAAV2/8 and rAAV2/9 vectors, improving the transduction in a wide range of tissues, including the retina, kidney, and pancreas. 33 , 35 – 39 Although the 7m8 peptide was chosen due to its previously demonstrated impact on rAAV-mediated photoreceptor transduction, other cell-penetrating or cell-specific targeting peptides, such as the epidermal growth factor receptor tyrosine kinase inhibitor or human immunodeficiency virus Tat peptide, 40 could be incorporated in place of the 7m8 peptide in order to improve transduction of nonretinal cell types. The insertion of some peptides can have deleterious effects on the capsid stability 41 and therefore cannot be incorporated into the capsid.…”