In the context of SETBP1 haploinsufficiency disorder (SETBP1-HD) pathobiology, critical uncertainties persist. Firstly, it remains unclear whether SETBP1 haploinsufficiency exerts detrimental effects early during brain development. Secondly, the question of whether the correction of SETBP1 levels after the clinical onset is sufficient to reverse the symptoms associated with SETBP1-HD remains unanswered. These uncertainties currently impede the exploration of novel molecular therapies based on gene correction, particularly in symptomatic patients.

To address these challenges, we proposed the creation of a reversible knock-out mouse model for SETBP1-HD. Our approach involves generating a knock-in mouse model wherein one Setbp1 allele is inactivated, yet its expression can be conditionally restored. The disruption of the Setbp1 gene involves the insertion of a STOP cassette flanked by loxP sites in the intron between exons 3 and 4. Validation of correct integration in the Setbp1 gene locus was confirmed through Southern blot analysis. The STOP cassette can be selectively removed upon Cre recombinase expression, thereby restoring the functional allele.

Thus, this murine model harbors one silenced allele of the Setbp1 gene (Setbp1Stop/+), closely resembling the human SETBP1-HD condition. Importantly, it can be converted into a functional allele (Setbp1REC/+) on demand, mimicking the full correction of the pathological mutation. Leveraging this unique experimental system, we are positioned to conclusively address a pivotal question concerning SETBP1-HD: Can the symptoms be reversed after their onset?

To validate the efficacy of the inserted STOP cassette in silencing gene expression and its correct removal through Cre-mediated loxP sequence recombination, our new mouse line has been crossed with a constitutive Cre-expressing mouse line (CMV::Cre mouse strain). Genomic PCR, mRNA, and protein expression analyses have confirmed the correct functionality of the engineered locus. This novel experimental model allows us to compare a full SETBP1-HD condition (un-reverted animals, Setbp1Stop/+, no Cre) with healthy controls (WT-like, fully reverted animals: Setbp1Stop/+; CMV::Cre a.k.a. Setbp1REC/+), as well as intermediate conditions through different Cre activation scenarios in time and space.

Given that Setbp1 haploinsufficiency mice exhibit behavioral abnormalities, ongoing detailed analyses are being conducted on aspects such as hyperactivity, anxiety, and social deficits in a model in which the Cre is active in post-natal period. These analyses aim to assess the potential rescue of defects following post-natal restoration of Setbp1 expression to normal levels.