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Page 42 Braun. J Transl Genet Genom. 2025;9:35-47 https://dx.doi.org/10.20517/jtgg.2024.79

[12]
for the patients . This observation resembles that in the boy reported by Mendell injected locally and who
[57]
had a deletion of exons 3-17 (of note, none of the 9 patients of the pioneer plasmid study where bearing
[28]
“at risk” deletions ). In response to these findings, the four AAV clinical trials have been amended so that
patients with genomic deletions that significantly overlap with transgene sequences are currently excluded
from dosing . Interestingly, various immunomodulatory treatments - such as pulsed-dose steroids,
[12]
intravenous immunoglobulins, plasmapheresis, and tacrolimus) - were used to manage the SUSARs across
the different programs, with a resolution of symptoms occurring within 3 months and tapering of the
immunosuppression. However, determining the optimal duration of immunosuppression to achieve
permanent tolerization of the transgene in “at-risk” patients will require further studies, given the particular
complexity of human immune responses. In this respect, the management of severe immunological events
reported by the three microdystrophin gene therapy sponsors may provide some guidance. Alternatively,
re-designed “de-immunized” epitopes without compromising their functionality are being explored in mdx
mice, but translating this approach to the more complex human immune context will require careful
attention .
[58]
Level of microdystrophin expression

It is clear that a major clinical goal is to achieve sufficient microdystrophin expression throughout the
skeletal and cardiac muscles. However, very little is known about the impact of microdystrophin
overexpression. Cardiac toxicity has been reported in transgenic mice with 100-fold overexpression of
microdystrophin . Recently, Hart et al. reported the accelerated onset of dilated cardiomyopathy, heart
[59]
[60]
failure, and death in dystrophic mice following AAV-induced overexpression of two of several
microdystrophin variants. Further studies are needed to better characterize the potential cardiotoxicity
elicited by overexpressed microdystrophin in the heart.

While microdystrophin gene therapy may prove to be a game-changer, it is not expected to fully restore
function in skeletal and cardiac muscles. The therapy is theoretically limited to shifting the phenotype from
DMD to a mild BMD phenotype, even with high levels of expression. This is supported by current trials,
where blood creatine kinase levels - an indicator of skeletal and cardiac muscle damage - seem to drop to
[57]
BMD levels, but not to those of healthy controls .

Alternative transgenes
Full-length or quasi-dystrophin

Researchers are developing dystrophin versions that are closer to the full-length protein, using
combinations of AAV vectors and leveraging the ability of AAV genomes to undergo intermolecular
concatemerization. A few studies have reported the use of dual AAV vectors for therapeutic correction in
DMD, and even a triple-vector system has been used to reconstitute the full dystrophin coding sequence
through a trans-splicing system, though with very low efficiency [61,62] . Full-length dystrophin can also be
generated using a triple AAV combination based on split inteins - small polypeptides that self-assemble and
undergo a protein trans-splicing reaction. This triple-vector combination improved muscle histopathology
and function in a mouse model of DMD . However, the translational potential of dual or triple AAV
[63]
vectors for human patients needs further investigation. For instance, homologous recombination efficiency
requires further optimization to reduce the total load of AAV vectors. Additionally, the formation of
aberrant products resulting from AAV Inverted Terminal Repeats (ITR) concatemerization requires in-
depth evaluation. Vectors that can efficiently transduce satellite cells would be particularly useful for
promoting muscle regeneration. Lastly, the quasi-dystrophin produced by this approach may bear a higher