Envisioning the development of a CRISPR-Cas mediated base editing strategy for a patient with a novel pathogenic CRB1 single nucleotide variant.

BACKGROUND: Inherited retinal degeneration (IRD) associated with mutations in the Crumbs homolog 1 (CRB1) gene is associated with a severe, early-onset retinal degeneration for which no therapy currently exists. Base editing, with its capability to precisely catalyse permanent nucleobase conversion in a programmable manner, represents a novel therapeutic approach to targeting this autosomal recessive IRD, for which a gene supplementation is challenging due to the need to target three different retinal CRB1 isoforms. PURPOSE: To report and classify a novel CRB1 variant and envision a possible therapeutic approach in form of base editing. METHODS: Case report. RESULTS: A 16-year-old male patient with a clinical diagnosis of early-onset retinitis pigmentosa (RP) and characteristic clinical findings of retinal thickening and coarse lamination was seen at the Oxford Eye Hospital. He was found to be compound heterozygous for two CRB1 variants: a novel pathogenic nonsense variant in exon 9, c.2885T>A (p.Leu962Ter), and a likely pathogenic missense change in exon 6, c.2056C>T (p.Arg686Cys). While a base editing strategy for c.2885T>A would encompass a CRISPR-pass mediated "read-through" of the premature stop codon, the resulting missense changes were predicted to be "possibly damaging" in in-silico analysis. On the other hand, the transversion missense change, c.2056C>T, is amenable to transition editing with an adenine base editor (ABE) fused to a SaCas9-KKH with a negligible chance of bystander edits due to an absence of additional Adenines (As) in the editing window. CONCLUSIONS: This case report records a novel pathogenic nonsense variant in CRB1 and gives an example of thinking about a base editing strategy for a patient compound heterozygous for CRB1 variants.

Tropism of engineered and evolved recombinant AAV serotypes in the rd1 mouse and ex vivo primate retina.

There is much debate on the adeno-associated virus (AAV) serotype that best targets specific retinal cell types and the route of surgical delivery-intravitreal or subretinal. This study compared three of the most efficacious AAV vectors known to date in a mouse model of retinal degeneration (rd1 mouse) and macaque and human retinal explants. Green fluorescent protein (GFP) driven by a ubiquitous promoter was packaged into three AAV capsids: AAV2/8(Y733F), AAV2/2(quad Y-F) and AAV2/2(7m8). Overall, AAV2/2(7m8) transduced the largest area of retina and resulted in the highest level of GFP expression, followed by AAV2/2(quad Y-F) and AAV2/8(Y733F). AAV2/2(7m8) and AAV2/2(quad Y-F) both resulted in similar patterns of transduction whether they were injected intravitreally or subretinally. AAV2/8(Y733F) transduced a significantly smaller area of retina when injected intravitreally compared with subretinally. Retinal ganglion cells, horizontal cells and retinal pigment epithelium expressed relatively high levels of GFP in the mouse retina, whereas amacrine cells expressed low levels of GFP and bipolar cells were infrequently transduced. Cone cells were the most frequently transduced cell type in macaque retina explants, whereas Müller cells were the predominant transduced cell type in human retinal explants. Of the AAV serotypes tested, AAV2/2(7m8) was the most effective at transducing a range of cell types in degenerate mouse retina and macaque and human retinal explants.