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.
Highlights
Leber congenital amaurosis (LCA) is the most severe subset of the set of diseases classified as retinitis pigmentosa, affecting 1 in 100,000 people with nearly 20 identified disease-associated genes
We initially designed a hypothetical pre-mRNA trans-splicing molecule consisting of flanking inverted terminal repeats, the cytomegalovirus immediate-early enhancer and promoter, the partial coding DNA sequence whose maximum size we were to determine, a canonical 50 splice site, 20 nt of intron spacer sequence, a putative binding domain, and a bovine growth hormone poly-adenylation signal sequence
For a 50 pre-mRNA trans-splicing molecule (PTM), the partial coding DNA sequence (PCDS) must end at the 30 end of an exon to allow for a 50 splice site
Summary
Leber congenital amaurosis (LCA) is the most severe subset of the set of diseases classified as retinitis pigmentosa, affecting 1 in 100,000 people with nearly 20 identified disease-associated genes. Children with LCA have trouble seeing in dim light (nyctalopia), have reduced visual resolution (visual acuity) and peripheral vision (visual fields), and often have abnormal rotatory eye movements (nystagmus) Whatever poor vision they have early in life gradually disappears due to the degenerative component of the disease.[1] Amelioration of one form of earlyonset retinal degeneration caused by the gene RPE65 has been demonstrated following sub-retinal administration of recombinant adeno-associated virus (AAV) encoding the complete RPE65 coding DNA sequence.[2,3,4] Other forms of inherited retinal degeneration may be amenable to rescue following this strategy; the genes associated with the most prevalent forms of retinal disease exceed the DNA coding capacity of AAV.[5] Mutations in CEP290 are designated as LCA type 10 (LCA10), and the complete coding DNA sequence is 7,440 nt (GeneBank: NM_025114.3). It is necessary to develop a gene therapy strategy that can correct a broad range of mutations in large genes such as CEP290 with AAV
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