Fig. 4 |. RNA-seq shows robust immune response in AAV.miS1 dosed animals.
a–d, RNA sequencing of DCN-derived RNA in four vehicle-injected controls (n = 4) compared with dosed animals, including a pilot animal (n = 6) that received AAV.miS1 and was euthanized at day 31. a, PCA segregates vehicle and dosed groups on the basis of principal component 1 (PC1). b, Differential expression analysis between vehicle and AAV.miS1-treated animals. P values were determined by two-tailed Wald test. Adjusted P values (Padj) determined by Benjamini–Hochberg multiple testing correction. Genes reaching the threshold for significance (Padj < 0.1) are shown in blue; 2,086 (8.6%) of genes were significantly upregulated in samples from dosed NHPs and 313 (1.3%) genes were significantly upregulated in vehicle-treated animals. c, Top ten GO terms associated with differentially expressed genes when comparing dosed and vehicle-treated animals, half of which indicate an immune response (red). d, Heatmap of genes in GO:0002376 (immune system process) and GO:0006955 (immune response). e, Top, cartoon of the AAV.miS1 expression vector and alignment of Nanopore-sequenced vector DNA; bottom, RNA-seq reads align to the proviral vector used to generate AAV.miS1. Despite low detection of cross-packaged backbone by Nanopore sequencing (top), transcripts flanking the 3′ ITR map were detected in dosed samples, including in the pilot animal harvested at 31 days p.i. (white box). Transcripts mapping downstream of the 3′ ITR map to production plasmid DNA.