Spliceosome-Mediated Pre-mRNA trans-Splicing Can Repair CEP290 mRNA

Abstract

Ocular gene therapy with recombinant adeno-associated virus (AAV) has shown vector-mediated gene augmentation to be safe and efficacious in the retina in one set of diseases (retinitis pigmentosa and Leber congenital amaurosis (LCA) caused by RPE65 deficiency), with excellent safety profiles to date and potential for efficacy in several additional diseases. However, size constraints imposed by the packaging capacity of the AAV genome restrict application to diseases with coding sequence lengths that are less than 5,000 nt. The most prevalent retinal diseases with monogenic inheritance are caused by mutations in genes that exceed this capacity. Here, we designed a spliceosome mediated pre-mRNA trans-splicing strategy to rescue expression of CEP290, which is associated with Leber congenital amaurosis type 10 (LCA10) and several syndromic diseases including Joubert syndrome. We used this reagent to demonstrate editing of CEP290 in cell lines in vitro and in vivo in a mini-gene mouse model. This study is the first to show broad editing of CEP290 transcripts and in vivo proof of concept for editing of CEP290 transcripts in photoreceptors and paves the way for future studies evaluating therapeutic effects.

CRISPR Activation Enhances In Vitro Potency of AAV Vectors Driven by Tissue-Specific Promoters

“…Animals were housed and maintained at the University of Pennsylvania under Animal Care and Use Committee protocol 805890. Subretinal injections were performed as previously described 40 . Each retina received 1 × 10 9 vector genomes in a total volume of 1 μL.…”

“…Eyes were enucleated, fixed, and processed as frozen retinal sections, as described previously 40 . Sections were incubated in blocking buffer consisting of 10% normal goat serum (Cell Signaling Technology, Danvers, MA, USA) and 2% Triton X-100 (Sigma) and PBS (Corning, Corning, NY, USA) for 1 h at room temperature.…”

The molecular genetics of Joubert syndrome and related ciliopathies: The challenges of genetic and phenotypic heterogeneity

“…Gene therapy holds some promise in treating cystic renal disease in JS due to CEP290 mutations; an antisense oligonucleotide (ASO) has been used to promote alternative splicing and exon skipping to rescue an intronic mutation in this gene in patient-derived renal epithelial cells and in a mouse model of JS, with reduction of cystic kidney disease burden [81]. Similarly, a comparable approach to rescuing the retinal phenotype of LCA caused by the common intronic splicing defect in the CEP290 gene using gene therapy approaches has demonstrated some success in a cell model and in mice carrying the minigene [29]. Finally, as other articles in this special issue point out, there are novel strategies emerging to treat these and other manifestations of ciliopathy conditions ("Retinal disease in ciliopathies: recent advances with a focus on stem cell-based therapies review" by Chen, H et al, "Novel Treatments for Polycystic Kidney Disease" by Patil, A et al, and "Using human urine-derived renal epithelial cells to model kidney disease in inherited ciliopathies" by Sayer, J and Molinari, E.).…”

AKT3 Gene Transfer Promotes Anabolic Reprogramming and Photoreceptor Neuroprotection in a Pre-clinical Model of Retinitis Pigmentosa

“…Subretinal injections were performed as previously described. 47 Each retina received 1 mL of vector preparation. Eyes that received the AAV.eGFP vector alone were dosed with 2 Â 10 9 vector genomes.…”

“…Eyes were enucleated, harvested, and prepared as frozen sections as previously described. 47 Sections were incubated in blocking buffer containing PBS, 10% normal goat serum (Cell Signaling Technology, Danvers, MA, USA), and 2% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) for 1 h at room temperature. Afterward, sections were incubated in primary antibody solution overnight in a humidified chamber containing the previously described components and combinations of the following antibodies: rabbit anticone arrestin (1:400; ab15282, Millipore, Burlington, MA, USA), rabbit anti-phospho-S6-Ser240/244 (1:100; 5364, Cell Signaling Technology), rabbit anti-phospho-AKT-Ser273 (1:100; 4060, Cell Signaling Technology), mouse anti-rhodopsin (1:400; ab5417, Abcam, Cambridge, UK), rabbit anti-HA (1:100; 3724, Cell Signaling Technology), rabbit anti-Ki67 (1:400; ab15580, Abcam), mouse anti-PCNA (1:400; ab29, Abcam), chicken anti-GFAP (1:400; ab4674, Abcam), and rabbit anti-AKT (1:100; 4691, Cell Signaling Technology).…”