Ineffective behavioral rescue despite partial brain Dp427 restoration by AAV9-U7-mediated exon 51 skipping in mdx52 mice

The <i>mdx52</i> mouse model exhibits a common mutation profile associated with brain involvement in Duchenne muscular dystrophy (DMD), characterized by heightened anxiety, fearfulness, and impaired associative fear learning. Deletion of exon 52 disrupts the expression of two dystrophins found in the brain (Dp427 and Dp140) and is eligible for therapeutic exon-skipping strategies. We previously demonstrated that a single intracerebroventricular administration of an antisense oligonucleotide (ASO) targeting exon 51 of the <i>Dmd</i> gene could restore 5%-15% of Dp427 expression. This treatment reduced anxiety and unconditioned fear in <i>mdx52</i> mice, improved fear conditioning acquisition, and partially improved fear memory tested 24 h later. To improve the restoration of Dp427 and induce a long-lasting therapeutic effect, we employed a vectorized approach using an adeno-associated virus (AAV)-U7 small nuclear RNA vector to deliver antisense sequences to the brains of <i>mdx52</i> mice. We evaluated two AAV serotypes known for their brain transduction efficiency (AAV9 and RH10) and two delivery routes, intracisterna magna and intracerebroventricular (ICV) injections, to maximize brain targeting. Based on GFP expression data, we selected the AAV9 capsid and a bilateral ICV delivery route. Using this approach, we demonstrated that ICV administration of AAV9-U7-Ex51M induced exon 51 skipping and restored Dp427 expression in the brains of adult <i>mdx52</i> mice, though with significant variability among individuals. While a few mice showed high Dp427 expression levels, the average restoration was limited to approximately 6%-12%. In conclusion, inducing exon skipping in the brains of adult <i>mdx52</i> mice using the vectorized AAV9-U7 approach was less effective than synthetic ASO treatment and did not improve the emotional behavior of <i>mdx52</i> mice.