DMD is a progressive muscle-wasting disease caused by mutations in the DMD gene. GEN6050X (ss.AAV9.oTAM and ss.AAV9.hE50-sgRNA) is an intravenously cytosine base editing drug for DMD exon 50 amenable patients. A therapeutic gene ACTG1 was included in ss.AAV9.hE50-sgRNA vector, to provide a synergetic effect with restorated dystrophin protein. Understanding GEN6050X drug DMPK will facilitate the interpretation of toxicity and prediction of efficacy. In this study, the dynamics of vector DNA and transgene expression in wild-type B6 mice after one time tail vein injection with 2E14 vg/kg GEN6050X was investigated. After administration, the two vectors in blood fell rapidly with half-life as about 1.7 days for both components. The vector DNA predominantly distribution in liver, adrenal gland, heart and injection site on Day 8. The oTAM and hE50-sgRNA vectors DNA in top four tissues showed an gradual decrease from day 8 to day 92. The half-life of oTAM and hE50-sgRNA DNA concentration in these tissues ranged from 30.7 to 73.3 days within 3 months observation period. From Day 92 to Day177, the vectors DNA tend to be stable in heart, adrenal glands, and injection site except liver. On Day 177, the vector DNA is predominantly distributed in the liver, followed by heart, injection site, adrenal gland and other tissues.
hE50-sgRNA transcripts began to be detected in heart, liver and injection site on day 8. From day 22, hE50-sgRNA transcripts were predominantly distributed in heart, and remained stable thereafter until day 177. hE50-sgRNA transcripts peaked on day 8 in liver and injection site, and continuing decline within day 92 with half-life as 74.7 and 112.3 days, then tend to be stable from day 92 to day 177.
In the top 4 vectors DNA high biodistributed tissues, oTAM protein was detected only in cardiac tissue 22 days after drug administration, and remained below the detection limit in all other tissues.
The tissues with the highest concentrations of γ-actin protein were the spinal cord and brain. Slight to moderate but not meaningful increases of γ-actin protein were detected with 2E14 vg/kg treatment. In conclusion, the vector DNA will eliminate over time, the transcription of sgRNA tend to be major in target tissues over time. oTAM protein transiently to be detected in heart 22 days suggest non-consistently expression of base editing tool. The γ-actin protein change with GEN6050X treatment were within or around the normal physiological range.