Friday, May 16, 2025
5/16/2025
Evaluating the therapeutic potential of gene replacement for SCYL1 deficiency syndrome - ABSTRACT SCYL1 deficiency syndrome is a rare, autosomal recessive, pediatric, multisystem disorder cause by inactivating mutations in the SCYL1 gene. The disease is characterized by recurrent episodes of liver failure, growth retardation, skeletal defects, as well as a range of neurological abnormalities including cognitive defects, ataxia, tremor, motor dysfunction, and neurodegeneration. SCYL1 encodes a protein whose function remains elusive Consequently, no treatments or cures have been developed or made available to treat the disorder. The genetic nature of the mutations, however, suggest that gene replacement therapy may represent an effective approach for its treatment. To test this hypothesis, we propose to engineer mouse models allowing for the inducible restoration of SCYL1 expression. Mice represent an excellent model to study SCYL1 deficiency syndrome as targeted disruption of the Scyl1 gene in mice recapitulates virtually all phenotypic changes seen in humans. In all, we propose to engineer two mouse models. The first model will make use of a loxP-flanked gene trap cassette containing a strong splice acceptor site followed by sequences encoding the enhanced green fluorescent protein followed by the bovine Growth Hormone polyadenylation signal. The cassette will be inserted within intron 1 of the mouse Scyl1 gene such that in the absence of the recombinase Cre, the cassette will act as a gene trap, preventing expression of the Scyl1 gene. In the presence of Cre, the cassette will be removed, allowing for expression of Scyl1 from its endogenous promoter. The second model will be engineered using a newly developed approach in our laboratory which makes use of short inverted artificial introns (iAI). In this system, a small DNA cassette – which contains sequences encoding a splice donor site, essential intronic sequences running antisense of the target gene and a strong splice acceptor site flanked by head-to-head lox71 and lox66 sites, followed by a weak splice acceptor site – is inserted within an exon of a target gene. In the absence of Cre, the DNA cassette is not recognized as an intron and remains within the transcript of the targeted gene. A string of stop codons in all three frames within the cassette, causes early translation termination, and prevents expression of the protein. In the presence of Cre, the iAI is flipped via recombination between the head-to-head lox71 and lox66 sites. The inversion of the cassette allows intronic sequences to be recognized by the splicing machinery which removes them, allowing for the normal expression of the Scyl1 gene. Both models will be characterized at the genomic level as well as functionally by crossing the mice with mice ubiquitously expressing CreERT2, a tamoxifen inducible Cre. The generation of these models and the successful application of gene replacement therapy in mice will provide the necessary evidence to move forward with more translational approaches for the treatment of the disorder.
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