On the use of D2.B10-Dmd mdx /J (D2. mdx ) Versus C57BL/10ScSn-Dmd mdx /J ( mdx ) Mouse Models for Preclinical Studies on Duchenne Muscular Dystrophy: A Cautionary Note from Members of the TREAT-NMD Advisory Committee on Therapeutics

Abstract

The C57BL/10ScSn-Dmd ^mdx /J (mdx) mouse model has been used by researchers for decades as a model to study pathology of and develop therapies for Duchenne muscular dystrophy. However, the model is relatively mildly affected compared to the human situation. Recently, the D2.B10-Dmd ^mdx /J (D2.mdx) mouse model was suggested as a more severely affected and therefore better alternative. While the pathology of this model is indeed more pronounced early in life, it is not progressive, and increasing evidence suggest that it actually partially resolves with age. As such, caution is needed when using this model. However, as preclinical experts of the TREAT-NMD advisory committee for therapeutics (TACT), we frequently encounter study designs that underestimate this caveat. We here provide context for how to best use the two models for preclinical studies at the current stage of knowledge.

TRANSLATABILITY OF DMD MOUSE MODELS

Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene that abolish production of dystrophin protein. The lack of dystrophin makes muscle fibers sensitive to damage during eccentric contraction, resulting in continuous myonecrosis, chronic inflammation and impaired muscle regeneration. Eventually, skeletal muscle tissue is replaced by fibrous and adipose tissues, resulting in loss of muscle function. In heart, fibrosis occurs as well, resulting in dilated cardiomyopathy. Even with good multidisciplinary care, most patients become wheelchair dependent before the age of 12, require assisted ventilation before the age of 20, and die in the 2^nd-4^th decade of life due to respiratory or heart failure [1].

Multiple strategic approaches aiming to restore dystrophin production or to reduce the downstream pathology are in development for Duchenne and a few are approved in the USA and Europe [2, 3]. Animal models in general are an imperfect reflection of human disease. Nevertheless, preclinical development of therapeutic approaches has benefited from animal models, especially the C57BL/10ScSn-Dmd ^mdx /J (mdx from here on) model, which carries a spontaneous nonsense point mutation in exon 23 of the mouse Dmd gene, resulting in lack of dystrophin [4]. Like in humans, lack of dystrophin results in myonecrosis for this mouse and cardiac problems develop later in life. However, the model compensates for skeletal muscle damage with hypertrophy and very active regeneration after an initial degeneration crisis between 3-6 weeks of age. With time fibrosis develops, but at much lower levels than in Duchenne patients. The only exception is the diaphragm, which has levels of > 20% fibrosis already at an early age. Deposition of adipose tissue, which occurs in Duchenne patients, is negligible for this model, even in the more severely affected diaphragm. Functionally, mdx mice show mild but detectable deficits over the first year of life, but then decline with pronounced dystropathology and slightly reduced lifespan [6–9]. However, it must be pointed out that this is in a laboratory setting where the mice have water and food ad libitum to provide nutrition for the extreme muscle regeneration and are safe from predators. Notably, if animals are exposed to exercise regimes using treadmill running, this exacerbates myonecrosis and pathology, resulting in increased fibrosis levels in skeletal muscles [5, 9]. Standardized operating procedures are available for these exercise protocols as well as for proper monitoring of other primary relevant readouts of potential clinical interest in relation to the known natural history of the mdx model [10].

Despite the milder pathology, the mdx mouse has been used to assess proof-of-concept for the currently approved therapies (exon skipping and stop codon read through) [11, 12]. In addition, compounds targeting fibrosis or inflammation, or improving muscle regeneration or mass, have been tested preclinically in this model [13]. However, the limited pathology in skeletal muscle clearly differs from the human situation and has posed challenges on translating results from mouse to human. In Duchenne patients, myonecrosis and pathology is more severe than in the mdx model. Therefore, extrapolating and anticipating clinical effects of treatments that showed beneficial effects in the mouse is difficult. Larger animal models, such as the golden retriever muscular dystrophy model and the pig model, are more severely affected [14, 15]. However, these models are more difficult and expensive to work with routinely.

Recently, the D2.B10-Dmd ^mdx /J mouse model (D2.mdx from here on) was developed as an alternative to use in preclinical studies. This model carries the same dystrophin mutation as the mdx mouse, but has been crossed into the DBA/2J genetic background, which is more prone to fibrosis formation due to more active transforming growth factor (TGF)-beta signaling resulting from a variation in the latent TGF-beta binding protein 4 (LTBP4) [17, 18]. Notably, LTPB4 also is a genetic modifier of disease progression in Duchenne patients [19]. In D2.mdx mice, skeletal muscles contain significant amounts of necrosis and fibrosis (10% for the tibialis anterior, 20% for the gastrocnemius and the triceps, 25% for the quadriceps, and 30% for the diaphragm at 10 weeks of age [20–22]). This is accompanied by inflammation. In addition, muscle regeneration is impaired due to an annexin 6 mutation and consequently the D2.mdx model is atrophic [16]. The model is also characterized by calcium deposits in skeletal muscle and heart that start to appear even before mice are 1 months of age [21, 22]. These deposits are occasionally seen also in mdx mice and Duchenne patients, but to a much lesser extent than in D2.mdx mice. Finally, D2.mdx mice show a clear functional deficit that remains relatively stable over time.

Based on these findings, the D2.mdx mouse was heralded as a better model for therapy development for DMD. However, it is now clear from the first long term natural history studies published for this model that, unexpectedly, the levels of pathology, such as calcification, reduce rather than increase with time [18, 21]. Furthermore, heart calcification is observed in both D2.mdx and DBA/2J wild type mice, thus impairing cardiac function in healthy controls as well [21, 23]. These findings anticipate potential complications for preclinical studies. First, it will be difficult to show reduced pathology in longer term therapeutic studies when these levels will reduce also without intervention. Secondly, for studies aiming to study heart pathology, it will be difficult to extricate which of the pathological aspects are due to the lack of dystrophin and which are due to the genetic background.

RECOMMENDATIONS

The TREAT-NMD advisory committee for therapeutics (TACT) is a multidisciplinary committee that aims to de-risk clinical development of therapies for neuromuscular diseases [24]. Both academic and industry drug developers can submit their preclinical and clinical data and future plans for a confidential review (see more on www.treat-nmd.com/tact). The authors of this commentary are preclinical experts on this committee and have noticed recently that preclinical study designs from applicants often fail in rigor and robustness, with additional potential bias for the use of short term proof-of-concept data for justifying trials in patients [25]. When using the D2.mdx mouse model, a further bias might be added, if applicants do not take the challenges and limitations of this model into account.

For approaches aiming to restore dystrophin either model can be used, but the mdx model might be preferred due to the wealth of reference data that is available for this model. This facilitates study design and power calculations. For approaches aiming to target the pathology (or to study the effects of dystrophin restoration on pathology), the D2.mdx mouse offers the possibility to study the impact on muscle pathology in short-term studies for both skeletal muscle and diaphragm. For the mdx model, early-stage studies are more challenging, due to the ongoing de- and regeneration between 3 and 6 weeks of age, which thereafter stabilizes. However, for longer term studies, the mdx mouse would be a better model especially when the pathology is exacerbated with treadmill running, since disease pathology ameliorates with age in the D2.mdx model. Also without treadmill running, the mdx model can be used for extensive long-term studies, as beyond 12 months of age pathology starts to progress further. Moreover, the inclusion of wild type mice of matching genetic backgrounds is vital for studies assessing effects on muscle functionality, as performance differs between inbred strains. Caution should be used in any case to clearly define the aim of the study and to apply the best protocol accordingly, based on available guidelines on husbandry, mouse sex, use of controls, blinded design and analysis, etc., as this is believed to reduce the risk of failure in the translational exercise [5].

FUTURE PERSPECTIVES

Due to the limited availability of natural history data for the D2.mdx model, study design and power calculations are more challenging. Detailed natural history of the functional and histological aspects of this model over time are sorely needed to add further evidence to available results [18, 21]. The authors of this paper are currently conducting a cross sectional natural history study in D2.mdx and mdx mice, with their respective wild types, where functional data is collected for up to 52 weeks and histological analyses will be performed at specific time points, based on a previous consensus meeting of the ‘of mice and measures’ project coordinated by Charlie’s Fund and TREAT-NMD [26]. The current study is conducted at the University of Bari and the Leiden University Medical Center with funding from Duchenne UK and Charlie’s Fund. Publications of these studies are forthcoming when data analysis is complete. These studies will shed more light on the specific differences between mdx and D2.mdx mice, e.g. the fact that myonecrosis appears to continue throughout life for mdx mice, while for D2.mdx mice it appears to stop at an as yet unidentified timepoint. The study will also provide the community with a collection of tissues and blood isolated from both models and their respective wild types at different timepoints. We anticipate that the study results will facilitate the implementation of SOPs and guidelines, and the achievement of a large consensus of the scientific community, which will help future study design for both mdx and D2.mdx models.