Fig. 5: Reduced clinical disease manifestation in a mouse model of mucopolysaccharidosis type I treated with rAAV:HDR vectors.
a Schematic of rAAV:HDR:uncleaved, -cleaved, and control non-cognate spacer vectors to correct the Idua point mutation in MPS-I IduaW392X mice. b In vivo experimental regimen to correct the Idua mutation by injection of rAAV9 through the facial vein in neonate MPS-I mice. c IDUA specific activity in liver lysates of healthy mice compared to rAAV:IduaDonor:ncSpacer mice (negative control) and treated rAAV:HDR uncleaved/cleaved cohorts. The dashed lines indicate 0.2% (therapeutic threshold) and 3% of the IDUA activity detected in lysates from WT mice. d Level of glycosaminoglycan (GAG) accumulation in the livers of the same mice shown in (c). e β-d-glucuronidase specific activity in liver lysates of healthy mice compared to rAAV:IduaDonor:ncSpacer mice (negative control) and treated rAAV:HDR uncleaved/cleaved cohorts. f Bar graph showing the percentage of NHEJ, HDR, and imprecise NHEJ:HDR mix at Idua in the liver as measured by NGS sequencing of PCR amplicons from genomic DNA. g qRT-PCR data showing increase in the relative Idua mRNA in the liver normalized to Gapdh mRNA. h AAV copy numbers in MPS-I mouse liver tissues using the [sgRNA<>U6] [U6<>IduaDonor], [IduaDonor<>U1a], and [U1a<>Nme2Cas9] primer pairs. i, j Animals treated with rAAV:HDR:cleaved vectors exhibit reduced Nme2Cas9 mRNA (i) and sgRNA (j) expression levels as measured by qRT-PCR, though reductions do not reach statistical significance. Data in panels c–h are presented as mean values ± s.e.m. (n = 3 mice in the WT cohort, n = 4 mice in the rAAV:IduaDonor:ncSpacer and rAAV:HDR:uncleaved cohorts, and n = 5 mice in the rAAV:HDR:uncleaved cohort). p values are calculated using Student’s t-test (two-sided).
