Animal models of Duchenne muscular dystrophy: from basic mechanisms to gene therapy

Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disorder. It is caused by loss-of-function mutations in the dystrophin gene. Currently, there is no cure. A highly promising therapeutic strategy is to replace or repair the defective dystrophin gene by gene therapy. Numerous animal models of DMD have been developed over the last 30 years, ranging from invertebrate to large mammalian models. mdx mice are the most commonly employed models in DMD research and have been used to lay the groundwork for DMD gene therapy. After ~30 years of development, the field has reached the stage at which the results in mdx mice can be validated and scaled-up in symptomatic large animals. The canine DMD (cDMD) model will be excellent for these studies. In this article, we review the animal models for DMD, the pros and cons of each model system, and the history and progress of preclinical DMD gene therapy research in the animal models. We also discuss the current and emerging challenges in this field and ways to address these challenges using animal models, in particular cDMD dogs.


CRKHR1
Unsequenced, dystrophin deficiency confirmed by immunofluorescence staining ENU chemically induced mutation on the C3H background, screened for and found to have an elevated CK, centrally nucleated myofibers and dystrophin deficiency. Aigner et al., 2009 Mdx52 Exon 52 deletion Targeted inactivation. On the C57BL/6 background. Hot-spot mutation. Araki et al., 1997 Mdx βgeo Insertion of the β-geo gene trap cassette in intron 63 LacZ replaced the CR and CT domain. All dystrophin isoforms are mutated. The full-length dystrophin-LacZ fusion protein is not detectable but Dp71-LacZ fusion protein can be detected. Wertz and Füchtbauer., 1998 DMD-null Entire DMD gene deletion Generated by Cre-loxP technology. Kudoh et al., 2005 Dp71-null Insertion of a β-geo cassette in intron 62. It disrupts Dp71 unique exon 1 Selective elimination of Dp71. Dp71 promoter driven LacZ expression. Similar LacZ expression pattern as mdx βgeo but muscle is not dystrophic. Dp71 deficiency is associated with early cataract formation in mice. Sarig et al., 1999;Fort et al., 2014 Dup2 Exon 2

Dp260 transgenic mdx
Transgenic over-expression of Dp260 in the mdx background Reduced but not completely prevented histopathology. Improved resistance to eccentric contraction injury but did not improve specific force. Warner et al., 2002 Dp260 in mdx/utrn -/-Transgenic over-expression of Dp260 in the utrophin/dystrophin dko background Severe lethal phenotype is converted to mild myopathy. Gaedigk et al., 2006 ∆17-48 transgenic mdx Transgenic over-expression of the naturally occurring ∆17-48 mini-dystrophin gene in the mdx background Two independent lines were generated. Both showed muscle protection. Phelps et al., 1995;Wells et al., 1992 and 1995

∆H2-R19 transgenic mdx
Transgenic over-expression of the synthetic ∆H2-R19 mini-dystrophin gene in the mdx background Completely reduced histopathology and normalized muscle force but did not restore sarcolemmal nNOS. Harper et al., 2002 Cardiac-specific ∆H2-R19 transgenic mdx Transgenic over-expression of the synthetic ∆H2-R19 mini-dystrophin gene in the heart of mdx mice Effectively protected but did not fully normalize the heart. Bostick et al., 2009

∆H2-R15 transgenic mdx
Transgenic over-expression of the synthetic ∆H2-R15 mini-dystrophin gene in the mdx background Complete correction of the dystrophic phenotype including nNOS and functional ischemia. Lai et al., 2009;Hakim and Duan 2013 Micro-dystrophin transgenic Transgenic over-expression of various synthetic micro-dystrophin genes in the mdx background Many lines are established for different microgenes. ∆R4-23 and ∆R4-23/C yield excellent protection but they don't restore nNOS. Hinge 2 in these two microgenes compromises function. ∆R2-15/R18-19/R20-23/C contains hinge 3 and is the only microgene capable of restoring nNOS. Harper et al., 2002;Li et al., 2011b;Sakamoto et al., 2002, Hakim et al., 2013Wang et al., 2008;Ferrer et al 2004 Fiona Transgenic over-expression of full-length utrophin in the mdx background Excellent protection but does not restore nNOS. Tinsley et al., 1998;Li et al., 2010 Laminin α1 transgenic mdx Transgenic over-expression of the laminin α1 chain in the mdx background Used to study laminin-111 protein therapy. Phenotype appeared to be very similar to mdx, without any benefit or harm. Gawlik Valentine et al., 1986;Cooper et al., 1988;Kornegay et al., 1988 GSHP MD Whole gene deletion Spontaneous mutation in the German short haired pointer (GSHP) breed. Schatzberg et al., 1999 Hybrid cDMD dogs with mixed genetic background and multiple mutations Various Generated by artificial insemination by crossing different cDMD breeds.
Resembles genetic diversity seen in human patients.