Generation of humanized mouse models to support therapeutic development for SYNGAP1 and STXBP1 disorders

Abstract

Heterozygous variants in SYNGAP1 and STXBP1 lead to distinct neurodevelopmental disorders caused by haploinsufficient levels of post-synaptic SYNGAP1 and pre-synaptic STXBP1, which are critical for normal synaptic function. While several gene-targeted therapeutic approaches have proven efficacious in vitro, these often target regions of the human gene that are not conserved in rodents, hindering the pre-clinical development of these compounds and their transition to the clinic. To overcome this limitation, here we generate and characterize Syngap1 and Stxbp1 humanized mouse models in which we replaced the mouse Syngap1 and Stxbp1 gene, respectively, with the human counterpart, including regulatory and non-coding regions. Fully humanized Syngap1 mice present normal viability and can be successfully crossed with currently available Syngap1 haploinsufficiency mouse models to generate Syngap1 humanized haploinsufficient mice. Stxbp1 mice were successfully humanized, yet exhibit impaired viability (particularly males) and reduced STXBP1 protein abundance. Mouse viability could be improved by outcrossing this model to other mouse strains, while Stxbp1 humanized females and hybrid mice can be used to evaluate target engagement of human-specific therapeutics. Overall, these humanized mouse models represent a broadly available tool to further pre-clinical therapeutic development for SYNGAP1 and STXBP1 disorders.

Keywords: STXBP1; SYNGAP1; epileptic encephalopathy; gene-targeted therapies; humanized mouse model.

Figure 1.. Characterization of the Syngap1 humanized mouse model.

(A) Cartoon depicting the Syngap1 locus after humanization. FRT and F3 are two heterotypic recognition sequences used for FLP-mediated neomycin and hygromycin cassette removal. The human SYNGAP1 transgene is flanked with loxP sites. 5’ regulatory region indicates promoter and proximal enhancers. (B) qPCR from gDNA of wild-type (+/+), hybrid (Hu/+) and fully humanized (Hu/Hu) Syngap1 mice. Tert was used as endogenous control. (C) Body weights of Syngap1 model mice. (D) Genotypic ratios from the offspring of Syngap1^Hu/+ × Syngap1^Hu/+ breedings. Cartoon was created with BioRender. (E) RT-qPCR from cerebral cortex tissue of 9-week-old Syngap1 model mice. Atp5f1 mRNA was used as endogenous control. (F) SYNGAP1 western blot from samples in (E). ATP5F1 was used as endogenous control. (B, C, E and F) Data are represented as mean values ± SEM. Data points represent independent biological replicates. (E and F) White and gray data points indicate females and males, respectively. (B, E and F) One-way ANOVA with Dunnett’s multiple comparison test vs. wild-type (+/+). (C) 2-way ANOVA with Dunnett’s multiple comparison test vs. wild-type (+/+) (D) Chi-square test (df = 2, n = 45, p = 0.9048). SYNGAP1-AS, SYNGAP1 antisense transcript. FC, fold change. ns, non-statistically significant.

Figure 2.. Characterization of the Stxbp1 humanized mouse model.

(A) Cartoon depicting the Stxbp1 locus after humanization. FRT and F3 are two heterotypic recognition sequences used for FLP-mediated neomycin and hygromycin cassette removal. The human STXBP1 transgene is flanked with loxP sites. 5’ regulatory region indicates promoter and proximal enhancers. (B) qPCR from gDNA of wild-type (+/+), hybrid (Hu/+) and fully humanized (Hu/Hu) Stxbp1 model mice. Tert was used as endogenous control. (C) Kaplan-Meier survival curves of Stxbp1^Hu/+ and Stxbp1^Hu/Hu mice. (D) Body weights of Stxbp1 model mice. Red arrow indicates the last data available for male Stxbp1^Hu/Hu. (E) Genotypic ratios from the offspring of Stxbp1^Hu/+ × Stxbp1^Hu/+ breedings. Cartoon was created with BioRender. (F) RT-qPCR from cerebral cortex tissue of 8-week-old Stxbp1 model mice. Atp5f1 mRNA was used as endogenous control. (G) STXBP1 western blot from samples in (F). ATP5F1 was used as endogenous control. (B, D, F and G) Data are represented as mean values ± SEM. Data points represent independent biological replicates. (F and G) White and gray data points indicate females and males, respectively. (B, F and G) One-way ANOVA with Dunnett’s multiple comparison test vs. wild-type (+/+). (C) Mantel-Cox test. (D) 2-way ANOVA with Dunnett’s multiple comparison test vs. wild-type (+/+). (E) Chi-square test (df = 2, n = 94, p = 0.0503). FC, fold change. ns, non-statistically significant.