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Braun. J Transl Genet Genom. 2025;9:35-47 https://dx.doi.org/10.20517/jtgg.2024.79 Page 39

DNA-based therapies
Genome editing
Recently, genome editing has been proposed to restore the reading frame at the DNA level. CRISPR/Cas
gene editing may restore full-length dystrophin expression after a single treatment in dystrophic rodent and
dog models, although additional work is required to demonstrate the feasibility and safety of this approach
in humans. It has several drawbacks such as: (I) very low efficiency in deleting multiple exons, (II) lack of
efficiency in delivering the CRISPR-Cas system in vivo, (III) possible need for repeated treatments, and (IV)
[26]
off-target related safety concerns . In a first-in-man trial, a 27-year-old patient with DMD was treated with
a dose of 10 viral genomes (vg)/kilogram(kg) of body weight of an AAV of serotype 9 containing dSaCas9
14
(Staphylococcus aureus Cas9 in which the Cas9 nuclease activity has been inactivated). Eight days after
infusion, the patient sadly died after an initial mild cardiac dysfunction and pericardial effusion, followed by
acute respiratory distress syndrome and cardiac arrest. Given the timelines, genome editing is probably not
in question as it did not have enough time to take place. A postmortem examination showed severe diffuse
alveolar damage, probably elicited by an innate immune reaction to the high-dose AAV. Transgene early
expression was minimal, and there was no evidence of AAV serotype 9 antibodies or effector T-cell
reactivity in the organs .
[27]

Replacement gene therapy
Since DMD is a monogenic disease, gene replacement therapy is one of the most logical and promising
treatment options. The first gene therapy clinical trial was completed in 2004. It showed that local
intramuscular administration of a low dose (600 µg) full-length dystrophin plasmid in 9 Duchenne and
[28]
Becker patients allowed only very low and local (along the needle track) expression of dystrophin .
Intravascular delivery in isolated limbs (referred to as hydrodynamic limb vein (HLV)-HLV- injection) of
the plasmid showed a much higher muscle transfection efficiency (up to 40% of transfected limb muscles) in
primate, and in mouse (mdx) and dog (Golden Retriever Muscular Dystrophy) DMD models . Safety of the
[29]
locoregional HLV delivery of increasing volumes of saline buffer was asserted in a series [limb-girdle
muscular dystrophy 2A, Emery-Dreifuss muscular dystrophy, autosomal recessive autosomal dominant
Limb Girdle Muscular Dystrophy (LGMD), LGMD, and BMD] of volunteers in lower and upper limbs.
[30]
[31]
However, certain limits were imposed on the volume administered (not exceeding 20%-40% of the limb
volume to be administered) due to the internal pressure within the muscle tissues and the theoretical risk of
compartment syndrome in already weakened muscles. This limitation on the injected volume posed a
barrier to the use of plasmids in DMD.

Viral vectors may transduce skeletal muscle much more efficiently and more broadly. A few of them can
accommodate the full-length, 14 kb dystrophin coding sequence together with the associated promoter and
regulatory sequences, but they are either ineffective (lentiviruses) or too inflammatory (e.g., poxvirus) to be
considered [32,33] . As of today, AAVs outperform other vectors in delivering genes to skeletal muscles and the
heart. Moreover, they can infect both dividing and nondividing cells and enable relatively long-lasting and
stable gene expression. However, the dystrophin cDNA exceeds the capacity of AAVs (~5 kb).

Microdystrophin gene therapy
The initial discovery that truncated dystrophins retain functionality provided the rationale for using
[34]
minimized versions of dystrophin compatible with the capacity of AAVs. These highly modified, smaller
molecular weight versions of dystrophin, known as microdystrophins (~150 kDa compared to normal
427 kDa dystrophin), contain over half of the normal dystrophin amino acid sequence but are designed to
preserve essential functions of the protein. Microdystrophins could theoretically be applied to all DMD
[35]
patients irrespective of their mutation .