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Braun. J Transl Genet Genom. 2025;9:35-47 https://dx.doi.org/10.20517/jtgg.2024.79 Page 41

AAV are thought to rarely integrate into the host genome and it is possible that, with successive cycles of
degeneration/regeneration (which may occur naturally throughout life), the therapeutic transgene will fade
with time due to some dilution effect. Whether this really happens and how long it takes remain unknown,
at least in humans. To avoid the risk of gradual loss of episomal transgenes and microdystrophin
expression, transgene expression must exceed the threshold (yet unclear) necessary. This has been reported
in dystrophic animal models [43,44] . Additionally, the “dilution” effect associated with muscle growth in very
young patients suggests that repeated administration of any DMD gene therapy will likely be needed.
However, the required interval between treatments and how to circumvent the presence of circulating AAV
neutralizing antibodies and the potential T cell response induced by the primary treatment remain to be
determined. Nonetheless, studies in Golden Retriever Muscular Dystrophy (GRMD) dog models have
shown that long-term (lifelong, > 10 years) clinical benefits [45,46] can still be expected.

Anti-AAV immunity

[47]
As seen in the general population , many DMD boys may have been exposed to AAVs, potentially
resulting in pre-existing immunity to the AAV serotype used for gene therapy [48,49] . Consequently, these
patients are currently excluded from receiving gene therapy. To address this limitation, immunosuppressive
strategies are being developed [50,51] . For instance, prior administration of IdeS (Imlifidase), a bacterial
[52]
protease that cleaves human antibodies (NCT06241950), is being investigated in DMD. Systemic AAV
13
14
gene delivery, with high doses of ~10 to 10 vector genomes/kg, has been shown to elicit the activation of
the innate immune response to the vector in large animal models and humans, including neuromuscular
patients . An adaptive T lymphocyte cell (CTL)-mediated immune response following AAV transduction
[41]
could lead to the clearance of transduced cells. However, this response is not expected to be detrimental, as
healthy skeletal muscle cells exhibit low MHC-I expression, which reduces the T-lymphocyte-mediated
reaction . In inflammatory contexts, such as in DMD patients, MHC-I antigens are overexpressed, and
[53]
muscles contain activated T cells, which would make DMD muscle tissue more susceptible to T-cell
immune responses . Additionally, given the high viral load used, a dose-dependent innate immune
[54]
response eventually initiates adaptive immune responses against both the viral capsid and the transgene.
[55]
Strategies like CpG depletion from rAAV vector constructs and enhancing vector production by
artificially increasing CpG methylation could partially alleviate this anti-AAV innate immune response.
Empty capsids may also be used as decoys for anti-AAV neutralizing antibodies, which means that pure
full-capsid batches may not necessarily be more effective than mixed preparations . On the other hand,
[56]
using different AAV strains may offer the possibility of selecting a suitable vector for patients who are
seropositive for a certain strain (and therefore, non-eligible for therapy as they would neutralize the injected
vector), provided there is no cross-reactivity.

Anti-transgene immune rejection

Recently, five cases of strikingly similar suspected unexpected serious adverse reactions (SUSARs),
including life-threatening myositis, were reported in DMD boys aged 7-9 years, who were recruited into
t h r e e s e p a r a t e m i c r o d y s t r o p h i n g e n e t h e r a p y t r i a l s ( N C T 0 4 2 8 1 4 8 5 , N C T 0 4 6 2 6 6 7 4 ,
Eudra-CT2020-002093-27). The three AAV products used in these trials contained different
microdystrophin transgenes, each under a distinct muscle-specific promoter, and were packaged in different
serotypes (AAV9, AAV8, AAVrh74). The kinetics of these SUSARs was consistent with transgene
expression, and laboratory findings suggested a cytotoxic T-cell immune response against dystrophin.
Notably, all the participants had similar DMD mutations, which pointed to epitopes located within exons 8-
11 containing hinge 1 and the beginning of the spectrin-like repeat domain, representing non-self epitopes