Hutchinson-Gilford Progeria syndrome--a model for the genetics of aging.

哈钦森-吉尔福德早衰综合症——衰老遗传学模型。

• 批准号: 9152721
• 负责人: FRANCIS S. COLLINS
• 金额: $ 106万
• 依托单位: NATIONAL HUMAN GENOME RESEARCH INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9152721
• 关键词: Adolescence; Adult; Affect; Age; Aging; Aging-Related Process; Alternative Splicing; Amino Acid Sequence; Amino Acids; Anatomy; Animals; Autophagocytosis; Bacterial Artificial Chromosomes; Birth; Breeding; Bulla; CCI-779; Cardiovascular Diseases; Cardiovascular system; Cell Culture Techniques; Cells; Child; Chromatin; Chromosome Segregation; Cleaved cell; Clinical Trials; Codon Nucleotides; Complex; Data; Disease; Dominant-Negative Mutation; Emery-Dreifuss Muscular Dystrophy; Engineered Gene; Enzymes; Exons; Farnesyl Transferase Inhibitor; Feedback; Fibroblasts; Genes; Genetic Models; Hair; Heterozygote; Histones; Homozygote; Human; Individual; Knock-in Mouse; Laboratories; Lamin Type A; Length; Life; Longevity; Measurement; Messenger RNA; Mitosis; Modeling; Molecular; Mouse Strains; Mus; Mutation; Myocardial Infarction; Normal Cell; Nuclear; Nuclear Matrix; Organism; Patients; Peripheral; Pharmaceutical Preparations; Phase I Clinical Trials; Phenotype; Play; Point Mutation; Premature aging syndrome; Process; Production; Progeria; Proteins; RNA; RNA Splicing; Role; SDZ RAD; Siblings; Sirolimus; Site; Skin; Smooth Muscle Myocytes; Stroke; Structure; Subcutaneous Tissue; Syndrome; Telomere Shortening; Testing; Therapeutic; Therapeutic Intervention; Transgenes; Transgenic Organisms; Translating; Vascular System; Vascular resistance; Weight; Werner Syndrome; Wild Type Mouse; Work; base; beta-Galactosidase; interest; mouse model; mutant; open label; prevent; programs; research study; senescence

Hutchinson-Gilford progeria syndrome (HGPS) is the most dramatic human syndrome of premature aging. Children with this rare condition appear normal at birth, but by age 2 they have stopped growing, lost their hair, and shown skin changes and loss of subcutaneous tissue that resemble the ravages of old age. Untreated, they rarely live past adolescence, dying almost always of advanced cardiovascular disease (heart attack and stroke). Our laboratory discovered that nearly all cases of HGPS harbor a de novo point mutation in codon 608 of the LMNA gene. This mutation, denoted G608G, causes disease by creating an abnormal splice donor, generating an mRNA with an internal deletion of 150 nt. This is translated into a mutant form of the lamin A protein (referred to as progerin) that lacks 50 amino acids near the C-terminus. Normally lamin A is post-translationally processed to add a farnesyl group at the C-terminus, and then the last 18 amino acids are cleaved off by the enzyme ZMPSTE24 to produce mature lamin A. Progerin lacks the recognition site for this final cleavage, and so remains permanently farnesylated. We have shown that this abnormal protein acts as a dominant negative to disrupt the structure of the nuclear scaffold. Data from our group has also demonstrated that progerin interferes with proper chromosome segregation during mitosis, and alters the distribution of various histone chromatin marks. Our lab has developed a mouse model for HGPS, by inserting into the germline a 164 kb bacterial artificial chromosome (BAC) containing the human LMNA gene, engineered to carry the G608G mutation. Recent work has demonstrated the complex anatomy of the transgene insert, but an intact copy of LMNA is included. Animals carrying the G608G mutation show progressive loss of smooth muscle cells in the media of large vessels. Thus, the mouse model nicely replicates the cardiovascular phenotype of HGPS. We have tested the use of farnesyltransferase inhibitors (FTIs), to see if these drugs could provide benefit in HGPS by reducing the amount of the toxic progerin protein. Treatment of HGPS fibroblasts growing in cell culture demonstrates that FTIs are capable of reversing the dramatic nuclear blebbing that is the hallmark of the disease. A trial of FTIs in the HGPS mouse model has demonstrated that this drug treatment is capable of preventing and even reversing the cardiovascular phenotype. A four year open label clinical trial of FTIs in 30 children with the disease demonstrated benefit to the vascular system, with reduction in peripheral vascular resistance in most of the 28 children treated. Survival was also modestly extended compared to historical controls. This treatment is not a cure, however, and so the search for other therapeutic options continues. Homozygotes for the mouse BAC transgenic have also now been bred, and show a considerably more severe phenotype. We are now testing those animals, as well as an independent line from Carlos Lopez-Otin (a knock-in of the progeria mutation into the mouse germline) to see if there might be therapeutic benefit from everolimus, a rapamycin analog, alone or in combination with FTIs. Rapamycin has been shown to expand lifespan in wild type mice, and its use on HGPS fibroblasts causes an improvement in phenotype with reduced nuclear blebbing and increased proliferative ability. We have shown that in cell culture, rapamycin acts by increasing turnover of progerin aggregates by activating autophagy. An application for Phase 1 trial testing of everolimus in children with progeria is currently under consideration by the FDA. We are currently studying whether everolimus has an effect on fibroblasts from patients with other LMNA mutations, including those that cause atypical Werners syndrome and Emery-Dreifuss muscular dystrophy. We have found that everolimus decreases nuclear blebbing, extends survival in culture, and eliminates senescence-associated beta-galactosidase expression in most of the atypical LMNA mutation lines we have tested. Of considerable relevance to the study of normal human aging, we have also shown that progerin is made in small amounts in normal individuals, and appears to increase in quantity as cells approach senescence. Our data points to an interesting connection between shortening of telomeres and activation of alternative splicing of dozens of genes, including production of progerin from a normal LMNA gene. In this way, senescence apparently proceeds by a positive feedback loop, once a cell has reached its maximum life span. We are interested in knowing what would happen to normal cells or organisms if progerin production was completely prevented. To pursue this, we have created a mouse knock-in model that alters the sequence of mouse exon 11 to block the use of the cryptic splice site, without changing the encoded amino acid sequence. The knock-in heterozygotes and homozygotes are viable, and quantitative RNA measurements have confirmed that homozygotes make no progerin at all. Interestingly, the homozygotes appear to keep growing beyond adulthood and to achieve greater weight and length than their wild type siblings. We are interested to see if they have exceptional longevity. Cell culture and whole animal studies of this progerin-free mouse strain are underway, and might reveal what role progerin plays in natural aging.

Hutchinson-Gilford Progeria综合征（HGP）是早熟的最戏剧性人类综合征。出生时这种罕见状况的儿童看起来很正常，但是到2岁时，他们已经停止生长，失去头发，表现出皮肤变化和皮下组织的丧失，类似于老年的破坏。他们很少经过治疗，很少生活过青春期，几乎总是死于心血管疾病（心脏病发作和中风）。我们的实验室发现，HGP的几乎所有病例都具有LMNA基因密码子608中的从头突变。该突变表示G608G，通过产生异常的剪接供体引起疾病，产生一个内部缺失150 nt的mRNA。这将转化为层粘连蛋白A蛋白（称为雌激素）的突变形式，该蛋白在C末端附近缺乏50个氨基酸。通常，层lamin a进行后处理，以在C-末端添加Farnesyl基团，然后通过酶ZMPSTE24裂解最后18个氨基酸，以产生成熟的lamin a。因此仍然是永久性的。我们已经表明，这种异常蛋白质是破坏核支架结构的主要阴性。来自我们组的数据还表明，孕激素会干扰有丝分裂过程中适当的染色体分离，并改变各种组蛋白染色质标记的分布。 我们的实验室通过将含有人工LMNA基因的164 kb细菌人造染色体（BAC）插入生殖线中开发了用于HGP的小鼠模型，该模型设计为携带G608G突变。最近的工作证明了转基因插入物的复杂解剖结构，但包括完整的LMNA副本。携带G608G突变的动物显示大血管介质中平滑肌细胞的逐渐丧失。因此，小鼠模型很好地复制了HGP的心血管表型。我们已经测试了Farnesylsylansferse酶抑制剂（FTI）的使用，以查看这些药物是否可以通过减少毒性孕激素蛋白的量来提供HGP的好处。在细胞培养中生长的HGP的成纤维细胞的治疗表明，FTI能够逆转该疾病标志的戏剧性核溢出物。 HGPS小鼠模型中FTI的试验表明，这种药物治疗能够预防甚至逆转心血管表型。在30名患有该疾病的儿童中，FTI的四年开放式标签临床试验对血管系统有好处，在接受治疗的28名儿童中，大多数人的周围血管耐药性降低。 与历史对照相比，生存也适度扩大。但是，这种治疗方法无法治愈，因此继续寻找其他治疗选择。 现在也已经繁殖了小鼠BAC转基因的纯合子，并显示出更为严重的表型。我们现在正在测试这些动物，以及来自Carlos Lopez-otin的独立线（对小鼠种系的后代突变的敲击），以查看是否可以从依维莫司（Everolimus），雷帕霉素类似物（单独）或中获得治疗益处。结合FTI。雷帕霉素已被证明可以扩大野生型小鼠的寿命，并且其在HGPS成纤维细胞上的使用会导致表型的改善，而核爆失能力降低并提高增殖能力。我们已经表明，在细胞培养中，雷帕霉素通过激活自噬来增加孕激素骨料的周转来起作用。 FDA目前正在考虑对Everolimus进行1期试验测试的申请。我们目前正在研究Everolimus是否对其他LMNA突变患者的成纤维细胞有影响，包括引起非典型Werners综合征和Emery-Dreifuss肌肉营养不良的患者。我们发现，在我们测试过的大多数非典型LMNA突变线中，依依他木降低了核流失，扩展培养物中的存活并消除了与衰老相关的β-半乳糖苷酶表达。 与正常人衰老的研究相关，我们还表明，正常个体中的肌蛋白量少量，并且随着细胞接近衰老的数量，摄入量似乎增加了。我们的数据表明，端粒缩短和激活数十个基因的替代剪接之间的有趣联系，包括从正常LMNA基因产生孕激素。这样，一旦细胞达到其最大寿命，衰老显然会以正反馈循环进行。 我们有兴趣知道，如果完全阻止了孕激素的产生，正常细胞或生物会发生什么。 为了追求这一点，我们创建了一个小鼠敲入模型，该模型改变了小鼠外显子11的序列，以阻止隐秘剪接位点的使用，而无需更改编码的氨基酸序列。 敲入杂合子和纯合子是可行的，定量RNA测量已证实纯合子根本没有过程。 有趣的是，与野生型兄弟姐妹相比，纯合子似乎保持超越成年，并达到更大的体重和长度。 我们有兴趣看看他们是否具有杰出的寿命。 细胞培养和对这种无雌激素小鼠菌株的整个动物研究正在进行中，并且可能揭示了孕激素在自然衰老中的作用。