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 明显的面部隆起的分化。这一过程的复杂性使其容易受到遗传和 环境干扰，如颅面畸形是人类常见的先天缺陷。因此， 大约75%的出生缺陷涉及头部、面部和口腔组织--口腔面部裂伤的发病率约为1/700 活产。口腔面部缺陷，如CL/P(唇裂伴或不伴腭裂)可带来显著的 患者的生活质量下降，并与治疗相关的主要经济负担。 胚胎面部发育的潜在遗传和发育过程惊人地相似 人类和老鼠，使老鼠成为研究人类病理学的最好的可用模型系统之一。 尽管有这种相似性，但不幸的是，很少有小鼠和小鼠的相同基因突变 已知人类可引起CL/P。一个明显的例外是编码转录因子的基因TFAP2A AP-2，当突变时在小鼠中引起CL/P，也与人类综合征和非 综合征CL/P值得注意的是，TFAP2A在人类BofS综合征(BofS)中发生突变 单基因遗传性疾病，表现为口腔裂伤、鳃皮肤异常和眼部缺陷。 先前的临床研究表明，TFAP2A的错义突变通常会导致更严重的 表型而不是杂合性。事实上，体外研究已经表明，这种错义突变具有 抑制功能基因中野生型蛋白伴侣DNA结合的显性负作用机制 二聚体。为了研究人类BofS的病因学，我们最近用条件性方法建立了BofS小鼠模型。 将人类错义突变放入小鼠的TFAP2A基因座。这一人性化的BofS模式概括了 在人类患者中观察到的头面部表型包括CL/P和颧骨缺陷。这项建议 将使用这一新模型来检验由显性负BofS突变引起的CL/P可能是 通过改变突变蛋白和野生型蛋白之间的比例的宫内处理逆转了这种情况。在AIM 1病毒载体中 将用于在发育早期添加补充的野生型AP-2，以滴定出突变蛋白。这 该方法还将在相关的TFAP2ACL/P模型中进行测试，在这些模型中，可用的AP-2不足。目标 2将使用反义寡核苷酸来靶向并优先敲除 BofS小鼠。最后，在目标3中，将使用基因编辑方法来针对突变的BofS等位基因。这个 拟议研究的预期结果是对递送系统和治疗方法有更深入的了解 子宫内治疗的方法，这应该对我们如何对待人类产生广泛的影响 由显性阴性或功能丧失突变引起的先天性牙科和颅面疾患。我们的 AIMS与NIDCR RFA题为《先天牙齿和颅面畸形的宫内治疗》保持一致 使用精确医学方法的疾病“(RFA-DE-23-004)是本应用程序的目标对象。

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.