Generation and delivery of AAV9-tMCK-DOK7

AAV9-DOK7 is a recombinant serotype 9 AAV encoding a wild-type human DOK7 transgene. DNA fragments containing a triple-tandem muscle creatine kinase (tMCK) promoter, a chimeric intron, a human DOK7 DNA coding sequence (NCBI GeneID: 285489), a shortened woodchuck hepatitis virus posttranscriptional regulatory element, and a simian vacuolating virus 40 polyadenylation signal were synthesized by GENEWIZ (South Plainfield, NJ, USA). Constructs were subsequently cloned into the baculovirus vector V445-ss-pFB by Virovek (Hayward, CA, USA). The resultant vector contained the DOK7 gene therapy construct flanked by AAV2 inverted terminal repeats and a Tn7L recognition sequence to make the vector compatible with baculovirus AAV production. AAV9-DOK7 was generated by Virovek using a baculovirus expression vector system-based process and Spodoptera frugiperda (Sf9) insect cells. The final product named AAV9-tMCK-DOK7 was formulated in phosphate-buffered saline (PBS) containing 0.001% Poloxamer 188.

Wild-type FVB/N mice were treated with either a high dose (5e11) or low dose (1.25e11) of AAV9-tMCK-DOK7 vector genomes (vg) via intravenous injection of the facial vein performed on the day of birth (postnatal day 1, P1).⁴³ The average body weight per mouse used in this study was 1.3 g. Untreated littermate mice were used as controls throughout. Body weight was recorded daily from P1 to P30 and then continually assessed a minimum of once weekly until the end of the experiment. Righting time was measured from P1 to P13.⁴³