The "humanized" hDMD mouse

Exon skipping strategy is a sequence-specific therapeutic approach. Since the human and mouse target sequences can be markedly different, the ideal pre-clinical validation would be to a target human DMD gene, in a mouse experimental background. We have previously engineered transgenic, “humanised” DMD (hDMD) mice, carrying an integrated and functional copy of the full-length human DMD gene [JT den Dunnen, Human Genetics, LUMC].    

"Embryonic stem (ES) cells were genetically modified through fusions with yeast spheroplasts carrying a YAC of 2.7 Mb that contained the full-length (2.4 Mb) human DMD gene. This YAC was previously reconstructed by homologous recombination of smaller overlapping YACs in yeast. ES-cells showing integration of one copy of the full-size YAC, as assessed by PFGE mapping, exon-PCR analysis across the entire gene, and metaphase FISH analysis, were then used to generate homozygous hDMD mice. Transgenic hDMD mice do not appear to be physically affected by the genetic modification. The engineering of these mice was authorised by the Dutch Ministry of Agriculture (LNV); project nr. VVA/BD01.284 (E21)."                                                                  [from 1]

On RNA level, RT-PCR analyses using either mouse- or human-specific primers demonstrated correct transcription of the human DMD gene in muscle tissue.

                       

On protein level, expression of human dystrophin in hDMD mouse muscle was specifically detected by immunohistochemical analysis using a human-specific antibody (MANDYS106). The antibody DYS2 cross-reacts with both human and mouse dystrophin.

                       

The hDMD mouse model allows the direct testing of human-specific AONs and target sequences in a mouse experimental background. As a first proof of principle for human sequence-specific DMD exon skipping in vivo, we we have shown the induction of specific skipping of the hDMD exons 44, 46, and 49, whilst the endogenous mouse transcripts were not affected [1]. This underlines that AONs, based upon specific design, can be highly sequence-specific small molecule drugs.

References

1       Bremmer-Bout M, Aartsma-Rus A, de Meijer EJ et al: Targeted exon skipping in transgenic hDMD mice:
        A model for direct preclinical screening of human-specific antisense oligonucleotides. Mol Ther 2004; 10:
        232-240.  
        Abstract

2        't Hoen PAC, de Meijer EJ, Boer JM et al: Generation and characterization of transgenic mice with the
         full-length human DMD gene. JBC; 2007 epub
        Abstract