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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 342
AAV-CRISPR gene therapy is the innate and adaptive immune responses evoked by AAV vectors and Cas
proteins [31,33] .

Development of editing and delivery methods to reduce viral vector dosing to improve safety and reduce
immunogenicity, while maintaining/improving dystrophin restoration efficacy, off-target related safety
[33]
concerns and durability need to be addressed before clinical implementation .

Gene replacement therapy
For monogenic diseases that lead to absent protein expression, the goal of gene replacement therapy is to
deliver an intact copy of the mutated gene. In DMD, the transgene is delivered to muscle cells to allow the
[15]
expression of functional protein and then restore or attenuate the phenotype .
The DMD gene (2.3 Mb) and its full-length transcript (14 kb) exceed the carrying capacity of commonly
utilized AAV vectors, which is approximately 5 kb. To overcome this limitation, a truncated version of
dystrophin (mini/microdystrophin) was developed. The amount of functional microdystrophin required to
achieve clinical efficacy varies, and multiple studies in animal models (mice and canines) indicate that
[1]
approximately 20% of the normal dystrophin level may be sufficient to produce therapeutic effects .
Different degrees of disease improvement have been seen in the mouse model with a wide range of
dystrophin expression. Functional improvement was seen with as low as 4% dystrophin expression, while
20% levels prevented development of dystrophic symptoms. However, extrapolation of these data to human
patients remains uncertain and challenging due to interspecies differences in muscle biology and
[34]
correlations with clinical outcomes . Another factor to consider is that a truncated dystrophin is likely less
[35]
effective than the wild-type, and the durability of dystrophin expression is not fully known . Additionally,
the accuracy of dystrophin quantification techniques shows variability and sampling error should be
considered due to the substantial variation of dystrophin expression within myofibers, between myofibers
[30]
and even between muscles .

In general, gene replacement therapies for DMD have focused on the delivery of various micro-dystrophin
transgenes. Portions of the DMD coding sequence have been removed to preserve essential protein function
while reducing the size to address AAAV vector capacity limits. These constructs differ in terms of the
micro-dystrophin structure, tissue-specific promoter and the AAV serotype, such differences may influence
the transduction, efficacy and safety. [Table 1] summarize the distinctions between the FDA-approved gene
therapy and other investigational drugs currently in clinical trials [35,36] .

The therapeutic potential of AAV gene therapy depends not only on vector design optimization but also on
other strategies, including promoter selection, scaffold choice, and the use of adjunct peptides to enhance
transgene expression and minimize toxicity [37,38] .

In 2020, Mendell et al. demonstrated in a phase 1/2 study that treatment with SRP-9001 led to improved
microdystrophin expression, reduced creatine kinase levels, and increased NorthStar ambulatory assessment
(NSAA) scores . In the subsequent phase 3 randomized trial (NCT05096221) with the same molecule,
[39]
named delandistrogene moxeparvovec (Elevidys), part 1 - change from baseline NSAA score at 52 weeks-
did not reach statistical significance. Some of the secondary endpoints showed numeric differences favoring
treatment in time to rise (TTR), 10-meter walk/run (10MWR) and 95th percentile stride velocity [39,40] .

Based on this data, Elevidys was granted accelerated approval (AA) from the FDA in June 2023 to treat
patients with DMD aged four to five years. In June 2024, the label was expanded to a full approval for
ambulatory patients and AA for nonambulatory patients over four years of age.