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

人源化小鼠模型中唇裂和腭裂的子宫内抢救

基本信息

批准号：
10645829
负责人：
Hong Li
金额：
$ 45.78万
依托单位：
UNIVERSITY OF COLORADO DENVER
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-04-01 至 2028-01-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10645829
关键词：
Acceleration Adenine Affect Alleles Animal Model Antisense Oligonucleotides Binding Biological Models Branchio-Oculo-Facial Syndromes Cells Child Cleft lip with or without cleft palate Clinical Research Complex Congenital Abnormality Craniofacial Abnormalities DNA DNA Binding DNA Double Strand Break DNA Repair Defect Dental Development Developmental Process Disease Dominant-Negative Mutation Economic Burden Embryo Etiology Eye Face Fetal Development Frequencies Gene Mutation Genes Genetic Genetic Processes Genome Head Heterozygote Human Human Pathology In Vitro Individual Inherited Injections Link Live Birth Long-Term Effects Lung Mental Health Missense Mutation Modality Modeling Morphogenesis Mus Mutate Mutation National Institute of Dental and Craniofacial Research Newborn Infant Operative Surgical Procedures Outcome Parents Patients Phenotype Positioning Attribute Process Proliferating Proteins Quality of life RNA Splicing Research Research Support Site Skin Structural Congenital Anomalies System TFAP2A gene Technology Testing Therapeutic Titrations Tooth Diseases Transcript Viral Vector adeno-associated viral vector base editor cleft lip and palate craniofacial craniofacial development craniofacial disorder dimer family burden gene therapy hearing impairment humanized mouse in utero in vivo infancy knock-down loss of function mutation mRNA Stability mouse model mutant mutant mouse model off-target mutation operation oral tissue orofacial orofacial cleft physical conditioning postnatal precision medicine prevent psychologic repaired response transcription factor

项目摘要

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.

抽象的颅面发育是一个复杂的过程，需要协调的增殖，形态发生，融合和不同面部突出的区分。这个过程的复杂性使其容易受到遗传和环境扰动，使得颅面畸形是人类常见的先天缺陷。那，大约75％的先天缺陷涉及头部，脸部和口腔组织 - 口面裂，700中有〜1 活产。口面缺陷，例如Cl/p（带有或不带left裂的left唇裂）可以赋予显着的降低患者的生活质量，并呈现与治疗相关的重大经济燃烧。胚胎面部发育的潜在遗传和发育过程在人和小鼠，使小鼠成为研究人类病理学的最佳模型系统之一。尽管有这种相似性，但不幸的是，很少有小鼠中相同基因突变和已知人类引起Cl/p。一个值得注意的例外是TFAP2A，编码转录因子的基因 AP-2，在小鼠中突变时会导致Cl/P，并且也与人类综合征和非 - 综合征Cl/p。值得注意的是，TFAP2A在人类分支-Oculo-Facial综合征（BOF）A中突变单基因条件，带有口面裂，分支皮肤异常和眼部缺陷。先前的临床研究表明，TFAP2A中的错义突变通常会导致更严重表型比杂合性。实际上，体外研究表明，这种错义突变具有作用的主要负面机制 - 抑制功能中野生型蛋白伴侣的DNA结合二聚体。为了研究人类BOF的病因，我们最近通过有条件地构建了BOFS小鼠模型将人类的错义突变放入小鼠TFAP2A基因座。这种人源化的BOFS模型概括了在包括CL/P和分支缺陷的人类患者中观察到的颅面表型。这个建议将使用这个新模型来检验以下假设，即由负面BOF突变引起的Cl/P可以是在子宫治疗中反转，从而改变了突变体和野生型蛋白之间的比例。在AIM 1病毒向量中将在发育初期用来添加补充野生型AP-2，以滴定突变蛋白。这方法还将在相关的TFAP2A CL/P模型中进行测试，其中AP-2可用。目的 2将采用反义寡核苷酸来靶向并优先敲除 bofs小鼠。最后，在AIM 3中，基因编辑方法将用于靶向突变体BOF等位基因。这拟议研究的预期结果是对输送系统和治疗的更深入的了解在子宫治疗中的方法，这应该对我们如何接近人类产生广泛的影响先天性牙齿和颅面疾病是由功能突变占主导地位或功能突变丧失引起的。我们的目的与NIDCR RFA保持一致，标题为“在先天性牙齿和颅面的子宫治疗中使用精确医学方法的疾病”（RFA-DE-23-004）。