Journal: bioRxiv

Article Title: Preventing acute neurotoxicity of CNS therapeutic oligonucleotides with the addition of Ca 2+ and Mg 2+ in the formulation

doi: 10.1101/2024.06.06.597639

Figure Lengend Snippet: The chemical structure of the di-siRNA HTT used throughout this figure contains 74mM of PO/PS backbones, alternating 2’-OMe and 2’-Fluoro modifications, 5’-(E)-VP modifications, and a glycerol-tetraethyleneglycol linker. (B) Timeline showing the surgical procedure and recovery period for wild-type mice injected bilateral ICV with oligonucleotides under isoflurane anesthesia. (Created with BioRender.com ) (C) Table outlining the acute tolerability scoring used to track adverse events that may not represent tonic-clonic seizures. The adverse events were scored based on an observational severity index (1 = least, 4 = most). A score of 20 represents the worst acute neurotoxicity observed during recovery, and a score of 0 represents no adverse events observed. (D) There was a dose-dependent increase in the severity of adverse events in wild-type mice injected with 0.125mM, 0.25mM, 0.5mM, or 1mM (∼28μg, ∼56μg, 112.5μg, and 225μg) di-siRNA HTT in 10μL 1XPBS buffer. Administering the 0.5mM and 1mM di-siRNA HTT resulted in significantly more severe abnormal behavioral phenotypes. Each data point represents one mouse (n= 4-6). *p=0.0163, ****p=<0.0001; data were analyzed using one-way ANOVA followed by Tukey’s post hoc test . (E) There was a significant difference in the time it took mice to be sternal following 1mM (225μg/10μL total dose) di-siRNA HTT injections compared to the 1XPBS controls. Each data point represents one mouse (n= 4-6). ***p=0.0010.; data were analyzed using one-way ANOVA followed by Bonferroni’s multiple comparisons test . (F) Normal huntingtin (HTT) protein levels in di-siRNA HTT -treated mice versus 1XPBS controls measured by ProteinSimple Wes one-month post-ICV injections in the frontal cortex (FC), striatum (S), medial cortex (MC), thalamus (T), and hippocampus (H). There was significant HTT protein lowering across all brain regions measured with all four di-siRNA HTT versus the 1XPBS vehicle control group (****p=<0.0001). Each data point represents one mouse and error bars represent mean values SEM (n=4). Data were analyzed using two-way ANOVA with Tukey’s multiple comparisons test.

Article Snippet: 5’-(E)-Vinyl tetraphosphonate (pivaloyloxymethyl) 2’-O-methyl-uridine 3’-CE phosphoramidite was used for the addition of 5’-Vinyl Phosphonate, 2ʹ-F, 2ʹ-OMe phosphoramidites with standard protecting groups were used to make the modified siRNAs (Hongene Biotech, Union City, CA).

Techniques: Injection