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Page 36 Braun. J Transl Genet Genom. 2025;9:35-47 https://dx.doi.org/10.20517/jtgg.2024.79

therapy. It piles up a number of barriers and difficulties that decades of research have tried to overcome
[1]
gradually. It is a rare disease. Its prevalence is currently around 1/5,000 boys worldwide . There are rare
cases of female patients or complications for female carriers of recessive mutations present on the X
chromosome (OMIM#300377, Xp21.2-p21.1). The relative rarity of the disease hampers large-scale clinical
trials.

DMD (MIM #310200), the largest human gene, spans 2.24 million base pairs. Mutations in this gene are the
primary cause of the disease. It contains multiple (seven) gene promoters, which drive the expression of
different mRNAs and protein isoforms (ranging from 40 kDa to the full-length 427 kDa) in a tissue-specific
and time-dependent manner . Clinical manifestations of DMD include progressive muscle degeneration
[2]
and waisting, typically becoming apparent between the ages of 2 and 5 years. Loss of ambulation often
occurs during the early teenage years, followed by the need for respiratory assistance in the early 20s and
premature death in the subsequent decade, due to respiratory and cardiac complications . Clinical
[3]
consequences and pace of disease course may vary depending on the mutation site and the genomic (such
as in-frame versus out-of-frame) alterations, which complicate the constitution of clinically homogeneous
trial cohorts. Other confounding factors in the assessment of the progression of muscle pathology, with or
without treatment, need to be taken into account, such as the initial motor improvement seen in young
patients due to age growth and development, which confound statistical interpretations. The first
gene-targeted therapeutic trials were based on the 6-min walk test, which proved to be poorly sensitive and
[4,5]
highly variable, especially in children . Today, the authorities mainly require clinical assessment scores
such as North Star Ambulatory Assessment (NSAA), which is not much more sensitive over short follow-up
periods.

The disease is multisystemic, affecting various tissues where the protein or its isoforms are expressed:
skeletal muscles, vascular and visceral smooth muscle, the heart, endocrine tissues, and the retina, as well as
central and peripheral neurons. Therefore, it is essential to ensure the distribution of vectors and their
expression throughout the body, even if the priority should be given to the life-threatening skeletal muscles
[6]
including respiratory muscles and the heart, which together represent 40% to 50% of the body’s mass .
Dystrophin is a structural, cytoskeletal membrane protein that plays a critical role as a mechanical link
between the extracellular matrix and contractile proteins, providing protection against force-related fiber
damage. To achieve this protective function, it is essential not only to target a large number of cells but also
to ensure robust and widespread expression of dystrophin. The expression must be sufficient to protect the
entire length of muscle fibers from membrane disruptions caused by the mechanical stress of contractions .
[7]

DMD is a chronic disease, which necessitates that molecular therapies remain effective throughout the
patient’s lifetime. This can be achieved either through a single administration or repeated administration of
the therapeutic vector, which, if viral, induces an immune response akin to a vaccine, preventing secondary
injections and even first-line treatment in seropositive patients. DMD is also a slowly progressive disease,
posing challenges for clinical trial design. Trails often require long follow-up periods and highly sensitive
clinical endpoints to achieve statistical significance. Consequently, such trials are typically limited to
patients with sufficient motor abilities. Additionally, DMD is a developmental disorder with potentially
deleterious consequences from the fetal period, which we do not know if post-natal treatment can reverse .
[8]

Skeletal muscles are regenerative tissues, and DMD is characterized by a succession of necrosis and
regeneration cycles, which raises questions about the persistence or the dilution of the transduced vectors
over time. The condition involves dystrophic changes, including histological rearrangements and the