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Gonzalez Castillo et al. J Transl Genet Genom. 2025;9:338-51 https://dx.doi.org/10.20517/jtgg.2025.57 Page 344

Transgene immunogenicity
Dystrophin-specific T cell activation is a major challenge in systemic gene therapy for DMD. Five patients
with DMD enrolled in three trials (NCT0428148, NCT04626674, Eudra-CT number, 2020-002093-27),
using different microdystrophin transgenes, promoters and AAV products (AAV9, AAV8 and AAVrh74),
experienced suspected unexpected serious adverse reactions. These patients presented between 3 and 6
weeks post infusion with symptoms of severe myositis that led to loss of ambulation and weakness of the
bulbar and respiratory muscles. The timing of these adverse reactions was consistent with transgene
expression and further studies suggested a T-cell-mediated response against specific microdystrophin
peptides contained within exons 8 through 11 [56,57] .

As a result, AAV clinical trials have been modified to exclude patients who have DMD deletions that
significantly overlap with those transgene sequences.

Pre-existing immunity
One of the major difficulties in AAV-mediated gene therapy is the presence of pre-existing Nabs against the
viral capsid; overall, up to 80% of the human population has antibodies against various serotypes .
[46]
Pre-existing immunity to AAV vectors is a barrier for treatment eligibility and is primarily due to prior
exposure to the wild-type AAV which leads to the formation of Nabs. Seroprevalence differs among
serotypes, with studies reporting 32%, 36% and 47% prevalence for AAVrh74, AAV9 and AAV8,
respectively. Given the degenerative nature of the disease, it is likely that redosing might be needed in the
future. Development of strategies such as pharmacological modulation, removal of circulating antibodies
(plasmapheresis), blocking innate immunity (complement antagonist), use of different AAV serotypes and
AAV capsid engineering are under investigation [45,58] .

RNA-mediated therapies
Exon skipping
Antisense oligonucleotides (ASOs) are short, single-stranded nucleotides that are capable of binding to a
specific region of RNA to promote exon-skipping. The main goal is to restore the disrupted reading frame
of the dystrophin gene, producing an internally truncated but functional dystrophin protein [1,59] .

These therapies are mutation-specific and benefit a subset of patients, approximately 27% of the DMD
population. Currently, there are four FDA-conditionally approved ASOs that target exons 45, 51 and 53
[Table 2]. Clinical trials have shown that ASOs restore dystrophin in patients with DMD, albeit at very low
levels. The FDA approval was granted based on the surrogate endpoint of dystrophin expression in muscle
biopsies of treated patients. However, none of these ASOs has received approval in Europe. Clinical data on
Eteplirsen, the first FDA-approved exon skipping therapy, has demonstrated slowing disease progression
[60]
compared with matched external controls . However, the data remains controversial and it is not yet fully
clear if the low levels of dystrophin restoration (even with high intravenous doses) are sufficient and further
clinical trials are ongoing to further assess the long-term functional efficacy and safety .
[61]

ASO treatments have several limitations related to delivery efficiency to the skeletal and cardiac tissues.
Approved ASOs are based on phosphorodiamidate morpholino oligomer (PMO) chemistry, which is
uncharged and does not bind to serum proteins, thereby limiting tissue distribution and bioavailability due
to renal clearance . Delivery to skeletal and cardiac muscle can be improved through pPMO peptide
[31]
conjugates, transferrin receptor-targeted antibody conjugation, and chemical modifications (see
Table 3) [62-64] .