G.P.96 : Dose escalation studies of rAAV9 U7snRNA targeting exon 2 show highly efficient skipping in the Dup2 mouse

Abstract

Duchenne Muscular Dystrophy (DMD) is a lethal X-linked disorder caused by truncating mutations in the <i>DMD</i> gene which result in an absence of the dystrophin protein. However, deletion mutations that preserve an open reading frame, lead to an internally truncated functional dystrophin which is associated with the milder Becker Muscular Dystrophy (BMD). This observation as led to exon-skipping therapies in DMD patients to restore the reading frame and generate a BMD phenotype. Our group has recently shown that mutations in the 5^′ exons of the <i>DMD</i> gene that disrupt the open reading frame (ORF) induce expression of a functional N-truncated dystrophin via IRES-driven translation initiation within exon 6. We have previously shown that the exon 5 IRES can be activated by disruption of the ORF by AAV9. U7snRNA-mediated skipping of exon 2 by intramuscular (IM) or intravascular (IV) injection. We have now extended those studies to address the minimal efficacious dose (MED), a prerequisite for clinical development. To assess dose ranging with IM delivery, TA (tibialis anterior) injections were performed at 5 different doses, ranging from 1.1×10¹⁰ to 1.1×10¹² total vector genomes (vg) in half-log steps (<i>N</i>=6 TAs per dose). A gradient of exon 2 exclusion was seen by RT-PCR at all doses, but high levels of properly localized dystrophin expression were seen at 3.5×10¹¹vg and above. Experiments to determine the correction of TA physiology at this dose are underway. Similarly, dose ranging experiments using IV tail vein injections with 5 doses (<i>N</i>=3 per dose) ranging from 1×10¹² to 1×10¹⁴vg/kg are underway, and results of both protein expression and physiology studies will be presented. Our results show that U7snRNA-mediated exon skipping in Dup2 is both titratable and highly efficient. The establishment of a IV MED for this vector will allow planning of IND-enabling GLP toxicology studies.