Testing new AAV vector on fully differentiated retinal tissue in vitro made possible with human retinal organoids

A recent study by Riedmayr et al., in Nature communications (2023)

highlights the advantages of using human retinal organoids as a complex in vitro tissue to evaluate the transduction efficiency of new AAV vectors into retinal tissue.

The authors present a new dual AAV vector system based mRNA trans-splicing for the reconstitution of large coding sequences. This novel gene reconstruction approach aims to overcome packaging size limitations of current AAV vectors limited to 4.7 to 5.0 kilobases. The gene of interest is split into two parts and packaged into separate AAV vectors, typically referred to as dual AAVs. The split parts of the gene subsequently reconstitute the gene at the AAV mRNA level upon co-delivery of the dual AAVs into the target tissue.

The data presented shows the flexibility of the vector system by reconstitution of different split genes in a set of transduction experiments in HEK293 cells and human retinal organoids first and then completed by in vivo delivery in mouse models using a range of delivery routes (local and systemic).

The retinal organoids, obtained from Newcells biotech, are iPSC-derived, and recapitulate the complex structure of the human retina with laminar cell organisation mimicking embryonic development. They contain the outer photo receptor segment of the retina that responds to light. The organoids include retinal ganglion cells, horizontal cells, amacrine cells and photoreceptors (Including cone and rod photoreceptors). The temporal order of retinogenesis in vitro recapitulates the in vivo differentiation. The retinal organoids used in this publication were mature at D215 of differentiation, a time point when photoreceptor expression is at a peak. The temporal order of retinogenesis in vitro recapitulates the in vivo differentiation. The retinal organoids used in this publication were mature at D215 of differentiation, a time point when photoreceptor expression is at a peak. The experiments compared transduction of a full-length luciferase control gene and with a split luciferase with the reconstituted luciferase gene reaching 20% expression of the control, demonstrating that dual REVeRT AAVs can be used to transduce highly differentiated retinal tissue.

The study also showed that intra-vitreal delivery of full-length ABCA4 into a mouse model of Stargardt disease using dual REVeRT AAVs leads to reconstitution and correct localization of a therapeutic protein in vivo. Other delivery routes were also investigated. In addition to efficacy, further safety parameters such as tolerability, toxicity, or immunoreactivity need to be investigated in nonhuman primates before REVeRT can be used in clinical trials.