Fig 1: Evaluating the ability of clamp-G PNA to bind and inhibit TTR mRNA(A) Graphic representation of target site on TTR mRNA. The table contains PNA oligomer sequences to the TTR target site. PNA4-LBA, cGPNA5-L, and ScR-PNA6-L contain succinic acid (SA) at the 5′ end (N terminus) for conjugation with LBA ligand. Lysine (K) was added at the 3′ end (C terminus) of the PNA, and OOO represents the trioxo-miniPEG linker.(B) Dose-dependent gel shift assay of TTR target with PNA4, cGPNA5, and ScR-PNA6 at indicated concentration. Samples were incubated in physiological buffer (2 mmol/L MgCl2, 150 mmol/L KCl, and 10 mmol/L NaPi) for 16 h at 37°C, followed by PAGE separation.(C) Schematic representation of PNA inhibiting translation initiation of TTR mRNA. Plasmid containing TTR coding region, nucleotides, and T7 polymerase were used to make TTR mRNA via in vitro transcription. TTR mRNA was incubated with different concentrations of PNA, and in vitro translation was performed using reticulocyte lysate.(D) Representative western blot analysis (top) of TTR protein after PNA and TTR mRNA incubation in reticulocyte lysate. The graph (bottom) represents the quantification of TTR protein fold change relative to the control. ImageJ was used for quantification, the graph shows mean ± SEM (n = 3), and the p value is from two-way ANOVA.(E) Fold change in TTR mRNA levels in HepG2 cells treated with indicated doses of PNA4 and PNA4-L in comparison to ScR-PNA6.(F) Relative change in TTR mRNA levels in HepG2 cells treated with indicated doses of cGPNA5 and cGPNA5-L doses in comparison to ScR-PNA6. Graph shows mean ± SEM (n = 3) and p value is from two-way ANOVA.
Fig 2: Efficacy of TTR clamp-G PNAs activity in vivo(A) Workflow representing short-term evaluation of cGPNA5 in C56BL/6J mice post subcutaneous administration at 5 mg/kg.(B) Relative fold change in the levels of TTR in livers of ScR-cGPNA6-L, cGPNA5, and cGPNA5-L. Graph shows mean ± SEM (n = 3) and p value is from two-way ANOVA.(C) Quantification of relative fold change in plasma TTR protein levels of ScR-cGPNA6-L, cGPNA5, and cGPNA5-L by ELISA. Graph shows mean ± SEM (n = 3) and p value is from two-way ANOVA.
Fig 3: NANITE amplifies editing in vivo.(a) Experimental scheme for mouse experiments for editing the Ttr locus. Mice were hydrodynamically injected (HGD) with plasmids. Organs were collected 24 hours post-injection to quantify transfection efficiency (n = 3). Editing was monitored by TTR ELISA on blood collected on days 1, 7, and 14. Tissues were harvested at day 14 to assess editing and animal health (n = 7). (b) Representative immunofluorescence images of liver sections showing nuclei (magenta) and zsGreen (green). Scale bar as indicated. (c) Fraction of transfected cells in the liver. One field of view per mouse was quantified. No significant difference in the fraction of transfected cells was observed between Cas9 and NANITE conditions. (d) Serum TTR concentration 7 days post-administration. NANITE significantly reduced TTR levels. (e) Serum TTR concentration 14 days post-administration. NANITE significantly reduced TTR levels. (f) Next-generation sequencing of liver genomic DNA harvested 14 days post-injection. NANITE edited significantly more cells than saline or Cas9 groups. For (c–f), statistical significance was determined by one-way ANOVA with Tukey’s multiple comparison test. p-values are indicated. Data are presented as mean ± SEM (c: n = 3 mice; d–f: n = 7 mice).
Fig 4: AAVs induce dose-dependent CSF dyshomeostasis.(A) CSF CCL2 concentrations measured by ELISA at P7.5 following P0.5 ICV injection of escalating AAV doses.(B, C) Parenchymal microglial response. (B) Representative Iba1 immunostaining in P7.5 brain sections.(C) Quantification of Iba1+ cell density in parenchymal regions.(D, E) ChP microglial response. (D) Representative Iba1 immunostaining in the ChP. (E) Quantification of Iba1+ cell density in the ChP.(F) CSF transthyretin (TTR) concentrations measured by ELISA at P7.5 following P0.5 ICV injection of escalating AAV doses.(G–J) ChP protein analysis by immunoblot. (G) Representative blot for TTR and GFP in ChP lysates. (H) Relative GFP expression. (I) Relative TTR expression. (J) Correlation analysis between GFP and TTR levels in individual samples.(K, L) CSF ion disruption following inflammation. (K) Experimental timeline for LPS-induced inflammation and CSF collection in adult mice. (L) CSF potassium (K+) concentration measured by ICP-MS.
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