Figure 3. In vivo activity and concentration of antibody-PMOs in the CNS of adult transgenic mice bearing the human SMN2 gene.

Tail vein administration of 8D3₁₃₀-PMO and NIP228-PMO was given at 8 weeks of age and tissues were harvested 7 days postadministration. Splice switching activity of the compounds compared with saline treatment on the human SMN2 transgene was analyzed via quantitative PCR (qPCR) and Western blots (mean ± SEM). (A) qRT-PCR and (B) Western blot analysis of brain show distinct splicing activity between TfR-targeted 8D3₁₃₀-PMO and isotype control NIP228-PMO treatment groups. (C) PMO concentration as determined by ELISA. 8D3₁₃₀-scrambled PMO (8D3₁₃₀-scrPMO) is an 8D3₁₃₀-PMO conjugate used as negative control. (D) qRT-PCR from the thoracic region of the spinal cord shows elevated levels of activity. (E) Western blot analysis from the cervical region of spinal cord indicates little activity in this region. (F) PMO concentration of 8D3₁₃₀-PMO from the whole spinal cord. Statistical significance (representative P values) between 8D3₁₃₀-PMO versus saline (*) and 8D3₁₃₀-PMO versus NIP228-PMO (^#) was evaluated in GraphPad Prism. Data shown as the mean ± SD, n = 5–6 per group. qRT-PCR and Western blots were analyzed with 2-way ANOVA corrected for multiple comparisons using Dunnett’s test. ELISA for PMO concentration was analyzed with 1-way ANOVA corrected for multiple comparisons using Tukey test. P values adjusted to account for each comparison, confidence level 0.95%. *P, < 0.05; **P, < 0.005; ***P, < 0.0005; ****P, < 0.0001; ^#P, < 0.05; ^##P, < 0.005; ^###P, < 0.0005; ^####P, < 0.0001.