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

摘要 颅面发育是一个复杂的过程，需要协调的增殖，形态发生，融合和 不同面部特征的分化。这一过程的复杂性使其容易受到遗传和 环境扰动，例如颅面畸形是常见的人类出生缺陷。因此，在本发明中， 大约75%的出生缺陷涉及头部、面部和口腔组织，其中口面裂影响约1/700 活产口面缺陷，如CL/P（唇裂伴或不伴腭裂）， 这会降低患者的生活质量，并造成与治疗相关的重大经济负担。 胚胎面部发育的潜在遗传和发育过程在以下方面惊人相似： 人和小鼠，使小鼠成为研究人类病理学的最佳可用模型系统之一。 尽管有这种相似性，但不幸的是，在小鼠和小鼠中， 已知人类引起CL/P。一个值得注意的例外是TFAP 2A，编码转录因子的基因 AP-2 β，当在小鼠中突变时引起CL/P，并且也与人类综合征和非综合征相关联。 值得注意的是，TFAP 2A在人鳃-眼-面综合征（BOFS）中突变， 一种单基因疾病，表现为口面裂、鳃部皮肤异常和眼睛缺陷。 先前的临床研究表明，TFAP 2A中的错义突变通常会导致更严重的 而不是杂合性。事实上，体外研究已经表明，这种错义突变具有 显性负性作用机制-抑制功能性细胞中野生型蛋白伴侣的DNA结合 二聚体。为了研究人类BOFS的病因学，我们最近通过条件性地诱导人BOFS小鼠模型， 将人错义突变置于小鼠Tfap 2a基因座中。这个人性化的BOFS模型概括了 在人类患者中观察到的颅面表型，包括CL/P和鳃缺陷。这项建议 将使用这个新模型来检验由显性阴性BOFS突变引起的CL/P可能是 通过子宫内处理逆转突变体和野生型蛋白之间的比例。In Aim 1病毒载体 将用于在发育早期添加补充野生型AP-2以滴定出突变蛋白。这 方法也将在相关的Tfap 2a CL/P模型中进行测试，其中AP-2的可用性不足。目的 2将采用反义寡核苷酸靶向并优先敲低突变体中的突变信息。 BOFS小鼠。最后，在目标3中，将使用基因编辑方法来靶向突变的BOFS等位基因。的 拟议研究的预期成果是更深入地了解输送系统和治疗 子宫内治疗的方法，这应该对我们如何接近人类产生广泛的影响。 由显性阴性或功能缺失突变引起的先天性牙齿和颅面疾病。我们 目标与NIDCR RFA一致，标题为“先天性牙齿和颅面畸形的宫内治疗 使用精准医学方法治疗的疾病”（RFA-DE-23-004）。

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.