Uncovering the cellular mechanisms of morpholino antisense drug delivery to muscles.


A direct implications for multi-exon skipping strategies for Duchenne muscular dystrophy (DMD). A breakthrough has been achieved in the understanding why patients benefit so variably from molecular therapies for muscular dystrophies in clinical practice.

n a collaborative research setting the team of Sebahattin Cirak (CMMC and the Children’s Hospital Cologne) has, together with researchers of the Children’s National Medical Center in Washington DC/USA, deciphered the cellular mechanisms of morpholino antisense drug delivery to muscles. The outcome of this research has improved the understanding of how these therapeutic drugs target muscle tissue providing an avenue to develop improved treatments for Duchenne muscular dystrophy (DMD).

The research data has been published in the October issue of Nature Communication:
Myoblasts and macrophages are required for therapeutic morpholino antisense
oligonucleotide delivery to dystrophic muscle.

Novak JS, Hogarth MW, Boehler JF, Nearing M, Vila MC, Heredia R, Fiorillo AA,
Zhang A, Hathout Y, Hoffman EP, Jaiswal JK, Nagaraju K, Cirak S*, Partridge TA*.
Nat Commun. 2017 Oct 16;8(1):941.  *contributed equally

DMD is a lethal so far incurable neuromuscular disorder that is characterized by progressive muscular deterioration (e.g. muscular weakness). It is inherited in an X-linked recessive fashion affecting around one in 3,500–5,000 male births and is caused by frame-shifting mutations in the DMD gene coding for dystrophin, a cytoskeletal protein. The cytoskeletal protein Dystrophin forms together with other proteins the so-called “Dystrophin-associated protein complex”.

“In previous studies we were able to demonstrate, that the Dystrophin-associated protein complex (DAPC) is vital structure that maintains the plasma membrane integrity and function of muscle fibers” Dr. Sebahattin Cirak comments. He explains further: “Disruption of the DAPC leads to chronic myofiber degeneration, compensated in young DMD patients by activation of satellite cells. In later stages asynchronic regeneration and degeneration lead to complete failure of regeneration.”

Recently it has been shown that exon skipping could restore the reading frame and dystrophin expression. Antisense oligonucleotides (AONs) such as the drug EXONDYS51, a phosphorodiamidate morpholino oligomer (PMO), have been designed to modulate pre-mRNA splicing of the dystrophin transcript which lead to exon 51 skipping that changes the downstream reading frame of dystrophin. These findings were used to develop a personalized therapeutic approach to treat DMD patients carrying specific mutations in the DMD gene and subjected to preclinical and clinical trials. For Duchenne muscular dystrophy (DMD) dystrophin restoring therapies by exon-skipping are underway in late stages of clinical trials.

However, the analysis of the collected data of these trails showed a high variability of dystrophin protein restoration and expression. More striking was the observation that no evidence could be detected linking the association between the exon skipping efficacy and residual PMO, concentrations within muscles despite high doses, preference of systematically-delivered PMO to various muscle groups or fiber types.

Dr. Cirak comments: “These findings indicate the involvement of additional factors. An effective treatment of DMD with Dystrophin restoring therapies strongly depends on an accurate and efficient delivery of the therapeutic agent to their targets, the myonuclei of the skeletal muscle cells. These cells are located within the large muscle groups and distributed throughout the body. We hypothesized that the blood vascular system might play an important key factor by comprehensively transporting the therapeutic agent to the skeletal muscle cells.

To investigate this hypothesis, the Cirak and Partridge teams used an experimental mouse model of DMD, the so-called mdx model carrying an incorrect DMD gene version that, in analogy of its human counterparts, abolishes dystrophin expression. The researchers administered the phosphorodiamidate morpholino oligomer (PMO) labeled with a fluorescent tag and tracked the distribution of the therapeutic drug into the muscle fibers.

The researchers demonstrated that robust PMO uptake and efficient production of dystrophin following PMO administration coincide with areas of myofiber regeneration and inflammation. Moreover, in inflammatory foci PMO is maintained when entering macrophages, actively differentiating myoblasts and newly forming myotubes.

“From our observation we conclude that efficient PMO delivery into muscle requires two concomitant events: first, accumulation and retention of PMO within inflammatory foci associated with dystrophic lesions, and second, fusion of PMO-loaded myoblasts into repairing myofibers”, summarizes Sebahattin Cirak the research findings: The identification of these factors explains the variability in the clinical trials and is of importance for the improvement of this therapeutic approach for treating DMD. Our experimental set-up illustrates a successful example of translational research”.

The reseachers state that the understanding of these underlying cellular processes will further improve the therapeutic strategy as follows:
•    Treatment of young male patients affected with DMD immediately in early childhood, when spontaneous regenerative muscle activity is high. Possibly, immediately after birth once neonatal screening will be established.
•    Administration of higher doses of the therapeutic drug to facilitate an increased uptake
•    Improvement of the targeting strategies for antisense oligonucleotides into muscle cell to increase the clinical efficacy for DMD patients.  

Dr. Terence A. Partidge is a principal investigator in the Children´s Research Institute Center for Genetic Medicine (CGMR) and a Professor at the George Washington University School of Medicine and health Sciences (Wshington, DC, USA)

Dr. Sebahattin Cirak is a specialist in paediatrics and adolescent medicine. He is a principal investigator in the Medical Faculty and leading a DFG Emmy Noether Research Group in affilliation with the Center for Molecular Medicine Cologne and the Children Hospital of the University of Cologne.  

For further information, please contact:

Dr. Sebahattin Cirak