engleski

CRISPR/Cas9-Mediated Gene Editing Corrects Disease- Causing Mutation in Crigler-Najjar Syndrome Mice

AAV-mediated gene delivery for gene replacement therapy of monogenic liver disorders has demonstrated efficacy in several animal models and human clinical trials. However, since the therapeutic payload remains mostly episomal, the use of AAV vectors for liverdirected gene transfer during infancy or juvenile age is still limited in the long-term due to loss of vector DNA and efficacy, associated to hepatocyte duplication. Genome editing stands as a promising strategy for the treatment of genetic diseases, since it leads to a permanent and specific modification of the genome, that is transmitted to daughter cells upon proliferation.We previously demonstrated that promoterless genome targeting of the hUGT1A1 cDNA into the albumin locus (GeneRide) successfully rescued neonatal lethality in a mouse model of Crigler-Najjar syndrome. Therapeutic efficacy of GeneRide was subsequently increased by coupling the CRISPR/Cas9 platform. Here, we aimed at correcting the disease phenotype by editing a single base deletion present in the Ugt1a1 murine locus of Crigler-Najjar mice, responsible for the lethal phenotype. Neonatal mice were retro-orbital injected with two AAV8 vectors: one expressing the SaCas9 and the sgRNA, and the other one carrying the Ugt1a homology regions. Two sgRNAs were tested, Ex4-sgRNA, which targeted the point mutation, and NcoI-sgRNA, with a targeting site 120 bp upstream of the mutation. We observed 50% reduction in plasma bilirubin (TB) levels with the NcoI-sgRNA, which remained stable up to 5 months post-injection, while the correction with the Ex4-sgRNA was negligible. Next, we tested different Cas9:donor vector ratio combinations (1:1 ; 1:2 ; and 1:5), with the 1:5 ratio showing the higher efficacy in lowering TB levels. Finally, to further improve CRISPR/Cas9-mediated gene editing efficacy we used selected microRNAs regulating HR-mediated gene correction, identified by a robotic high-throughput approach in a library of 2, 042 human microRNAs. These miRNAs markedly enhanced CRISPR/ Cas9-AAV-based homologous recombination in the liver both in the albumin locus using eGFP reporter-system and in the mouse Ugt1a1 locus. In conclusion, we showed that CRISPR/Cas9 mediated gene editing in the Ugt1a1 locus was able to rescue the neonatal phenotype in Crigler-Najjar mice, and that HDR and therapeutic potential was further enhanced by selected miRNAs in the liver.

AAV, Gene Editing, hUGT1A1, CRISPR/Cas9

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