Fig. 6 |. Systemic treatment of HSA_LR DM1 mice with RNA-targeting Cas9 leads to sustained expression in various tissues, eliminates toxic RNA foci and reverses DM1-related mis-splicing.

a, mRNA levels of Cas9 (blue) and HSA_LR (CUG repeat levels, orange) in the quadriceps muscle of mice that were treated with either control or RCas9-CTG 16 weeks after treatment with 2 × 10¹¹ vg of AAV9-RCas9-CTG in P0 (neonatal) HSA_LR mice. n = 3 for each condition. Statistical significance was determined using a one-tailed Student’s t-test; **P = 0.0071. The centre lines show the median values, the box limits indicate the 25th and 75th percentiles as determined using R and the whiskers extend to 1.5× the interquartile range from the 25th and 75th percentiles; outliers are represented by dots. b, The distribution of mRNA levels of GFP gRNA (green) and Cas9 (grey) levels in various indicated tissues measured using RT–qPCR at 4 and 8 weeks after lateral tail vein injection of 1 × 10¹² vg of AAV9-RCas9-CTG vectors in adult (aged 8 weeks) HSA_LR mice (n = 3). The levels were normalized to the levels in TA muscle at 4 weeks. 4 w veh., vehicle treatment at 4 weeks. The centre lines show the median values, the box limits indicate the 25th and 75th percentiles as determined using R and the whiskers extend to 1.5× the interquartile range from the 25th and 75th percentiles; outliers are represented by dots. c, RNA-FISH analysis of CUG-repeat RNA in the quadriceps muscle 4 weeks after lateral tail vein injection of either vehicle control or 10¹² vg of AAV9-RCas9-CTG (100 nuclei were counted in 3 sections from n = 3 mice) in adult HSA_LR mice (aged 8 weeks). Scale bar, 50 μm. d, Quantification of RNA-FISH analysis of CUG-repeat RNA foci in adult HSA_LR mice (aged 8 weeks) in c. Statistical significance was determined using a one-tailed Student’s t-test; **P = 0.0015. n = 3 each. The centre lines show the median values, the box limits indicate the 25th and 75th percentiles as determined using R and the whiskers extend to 1.5× the interquartile range from the 25th and 75th percentiles; outliers are represented by dots. e, HSA RNA levels in the quadriceps muscle 4 weeks after lateral tail vein injection of either vehicle control or 10¹² vg of AAV9-RCas9-CTG in adult HSA_LR mice (aged 8 weeks). n = 3. Statistical significance was determined using a one-sided Student’s t-test; *P < 0.024. f, Splicing of alternative exon 22 in Atp2a1 assessed using RT–PCR in quadriceps 16 weeks after temporal vein injection of P0 (neonatal) HSA_LR mice with either saline or 10¹¹ vg of AAV9-RCas9-CTG (top). Splicing of alternative exon 22 in Atp2a1 was assessed using RT–PCR in the quadriceps 4 weeks after lateral tail vein injection of adult HSA_LR mice (aged 8 weeks) with either vehicle or 10¹² vg of AAV9-RCas9-CTG (bottom). Total n = 3 mice for each condition, each lane represents measurements from a single mouse.