Megabase-Scale Transgene De-Duplication to Generate a Functional Single-Copy Full-Length Human DMD Transgenic Mouse Model

Abstract

AbstractThe development of sequence-specific precision treatments like CRISPR gene-editing therapies for Duchenne Muscular Dystrophy (DMD) requires sequence humanised animal models to enable the direct clinical translation of tested strategies. The current available integrated transgenic mouse model containing the full-length humanDMDgene, Tg(DMD)72Thoen/J (hDMDTg), has been found to have two copies of the transgene per locus in a tail-to-tail orientation, which does not accurately simulate the true copy number of theDMDgene. This duplication also complicates the analysis when testing CRISPR therapy editing outcomes, as large genetic alterations and rearrangements can occur between the cut sites on the two transgenes. To address this, we performed long read nanopore sequencing on hDMDTg mice to better understand the structure of the duplicated transgenes. Following that, we performed a megabase-scale deletion of one of the transgenes by CRISPR zygotic microinjection to generate a single-copy, full-length, humanised DMD transgenic mouse model (hDMDTgSc). Functional, molecular, and histological characterisation show that the single remaining human transgene retains its function and rescues the dystrophic phenotype caused by endogenous murineDmdknockout. Our unique hDMDTgSc mouse model can potentially be used to further generation of DMD disease models, suited for the pre-clinical assessment of sequence-specific therapies.