Figure 3.

Mutant Abcd1 gene rescue in the ALD mouse model using HITI

(A) Scheme for HITI of the normal hABCD1 cDNA near the endogenous 5′ UTR of Abcd1 in the ALD model mouse. The start codon and most of exon 1 is replaced with a neomycin resistance gene in the mouse model. The blue arrows indicate the primers used to amplify the junction between endogenous Abcd1 and the HITI donor after integration. (B) Schematic representation of the AAV constructs used in this study. AAV9 #1 and #2 carry SaCas9-sgRNA and the HITI donor, respectively. (C) Timeline of the experiment. AAV9s were injected into 6-week-old mice, which were then euthanized at 9 weeks of age. (D) T7E1 assay to detect SaCas9 activity. Indel frequencies are indicated at the bottom of each lane. (E) Results from PCR analysis in which the integration junctions between endogenous Abcd1 and the HITI donor were amplified. Genomic DNA was extracted from tissues from two groups of three ALD model mice, transduced with either AAV9 #1+#2 or AAV9-EGFP; labels for the individual mice (1, 2, 3, 7, 8, and 9) are displayed at the top of each lane.