Abstract for laypersons

To model inherited vision loss, we have modified the genome of a pig to carry a sequence that cause rapid retinal degeneration in human patients. The mutation is dominant, i.e. its appearance in one of the two copies (alleles) of the GUCY2D gene deteriorates vision although the other allele is fully intact. Knowing from other human beings that one intact allele is sufficient for perceiving light in photo-sensitive cells, we consider two options to restoring function: Either reconstituting the original sequence or completely destroying the function of the mutated allele. We will tackle both strategies by therapeutic gene editing, an attempt that aims at direct manipulation of the causative defect at genomic level. The approaches will be tested and optimized in cell culture experiments and adapted for delivery into the eye of affected pigs and human beings. The preferred way to safely deposit gene editing tools into the retina and introduce them into photo-sensitive cells is their packaging in virus capsids. The limited capacity of such particles require the splitting of the gene editing complex into separate virus components and their self-assembly in the target cell. The optimization of virus-compatible self-assembly will allow the testing of tailored gene editing therapy for patients carrying the specific GUCY2D mutation and pave the way for novel therapeutic option in patients suffering mutations in diverse inherited retinal diseases.

Scientific abstract

A tailored pig model carries a segment identified in patients suffering a dominant negative variant of the GUCY2D gene. The humanization of the affected exon and its surrounding intronic environment facilitates the testing of human-specific gene editing tools. The dominant character of the mutation and the unsuspicious phenotype in human beings carrying only one intact GUCY2D allele suggest the pursuing of two distinct approaches: either the complete functional disruption of the mutated allele by gene disruption or the reconstitution of its original variant by gene reconstitution. We will follow and optimize both strategies in cell culture assays and adapt them for delivery into the eye of pigs and humans. Given the proven properties of AAV-based viral vectors for application of nucleic-acid-based therapies into the eye, we will work on separating gene editing constituents into distinct viruses and their self-assembly by intein-based protein splicing in photoreceptor cells. By this approach, we consider the constitution of capable prime editing systems from three precursors by double-intein-splitting. By testing patient-specific gene editing tools in the GUCY2D pig model, the project will immediately pave the way towards treating GUCY2D patients, but also reveal the general potential for sustained therapy of photoreceptor cells by gene editing in other inherited retinal diseases.