In utero rescue of cleft lip and palate in a humanized mouse model

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Abstract Craniofacial development is a complex process requiring coordinated proliferation, morphogenesis, fusion and differentiation of distinct facial prominences. The complexity of this process leaves it vulnerable to genetic and environmental perturbations, such that craniofacial malformations are a common human birth defect. Thus, about 75% of birth defects involve the head, face, and oral tissues - with orofacial clefting affecting ~1 in 700 live births. Orofacial defects such as CL/P (cleft lip with or without cleft palate) can impart a significant decrease in quality of life on those afflicted and present a major economic burden associated with treatment. The underlying genetic and developmental processes of embryonic facial development are strikingly similar in human and mouse, making the mouse one of the best available model systems to study human pathology. Despite this similarity, there are unfortunately very few cases in which the same gene mutation in mouse and human are known to cause CL/P. One notable exception is TFAP2A, the gene encoding transcription factor AP-2, which causes CL/P when mutated in mouse and is also linked to both human syndromic and non- syndromic CL/P. Notably, TFAP2A is mutated in human Branchio-Oculo-Facial Syndrome (BOFS) a monogenetic condition that presents with orofacial clefting, branchial skin anomalies, and eye defects. Previous clinical studies have indicated that missense mutations in TFAP2A generally cause more severe phenotypes than heterozygosity. Indeed, in vitro studies have shown that such missense mutations have a dominant negative mechanism of action - inhibiting DNA binding of a wild-type protein partner in the functional dimer. To study the etiology of human BOFS, we recently constructed a BOFS mouse model by conditionally placing a human missense mutation into the mouse Tfap2a locus. This humanized BOFS model recapitulates the craniofacial phenotypes observed in human patients including CL/P and branchial defects. This proposal will use this new model to test the hypothesis that CL/P caused by dominant negative BOFS mutations can be reversed by in utero treatment that alters the ratio between mutant and wild-type protein. In Aim 1 viral vectors will be used to add supplementary wild-type AP-2 early in development to titrate out the mutant protein. This approach will also be tested in related Tfap2a CL/P models in which there is insufficient AP-2 available. Aim 2 will employ anti-sense oligonucleotides to target and preferentially knockdown the mutant message in the BOFS mice. Finally, in Aim 3 a gene editing approach will be used to target the mutant BOFS allele. The expected outcome of the proposed research is a deeper understanding of delivery systems and therapeutic approaches for in utero treatments, and this should have a broad impact on how we approach human congenital dental and craniofacial disorders caused by dominant negative or loss of function mutations. Our Aims are aligned to the NIDCR RFA entitled “In utero Treatments of Congenital Dental and Craniofacial Disorders Using Precision Medicine Approaches” (RFA-DE-23-004) for which this application is targeted.