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: 9567422
• 负责人: FRANCIS S. COLLINS
• 金额: $ 109.7万
• 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/9567422
• 关键词: Adolescence; Affect; Age; Age-Months; Aging; Aging-Related Process; Alopecia; Amino Acid Sequence; Amino Acids; Animals; Antisense Oligonucleotides; Aorta; Atherosclerosis; Autophagocytosis; Bacterial Artificial Chromosomes; Biotin; Birth; Blood Vessels; Boston; CCI-779; Cardiovascular Diseases; Cardiovascular system; Cell Culture Techniques; Cells; Cessation of life; Child; Chromatin; Chromosome Segregation; Cleaved cell; Clinical Trials; Codon Nucleotides; Collaborations; Data; Defect; Developmental Bone Diseases; Disease; Disease model; Dominant-Negative Mutation; Ductus Arteriosus; Emery-Dreifuss Muscular Dystrophy; Engineering; Enzymes; Exhibits; Exons; Farnesyl Transferase Inhibitor; Fatty acid glycerol esters; Fibroblasts; Genes; Genetic Models; Hair; Heterozygote; Histologic; Histones; Homozygote; Human; In Vitro; Injection of therapeutic agent; Joints; Knock-in; Knock-in Mouse; Label; Laboratories; Lamin Type A; Lead; Longevity; Measurement; Mesenchymal; Messenger RNA; Methods; Mitosis; Modeling; Molecular; Mouse Strains; Mus; Muscle Cells; Mutation; Myocardial Infarction; Myocardium; Normal Cell; Nuclear; Nuclear Envelope; Nuclear Matrix; Nuclear Structure; Oligonucleotides; Organ; Organ Transplantation; Organism; Pathology; Patients; Pediatric Hospitals; Peptides; Peripheral; Pharmaceutical Preparations; Phase I Clinical Trials; Phenotype; Play; Point Mutation; Population; Premature aging syndrome; Process; Production; Progeria; Proteins; Proteomics; RNA; RNA Splicing; Reporter; Role; SDZ RAD; Safety; Saline; Sampling; Sirolimus; Site; Skeletal Muscle; Skin; Smooth Muscle Myocytes; Stroke; Subcutaneous Injections; Subcutaneous Tissue; Syndrome; System; Tail; Testing; Therapeutic; Therapeutic Intervention; Tissues; Transgenic Mice; Transgenic Organisms; Translating; Vascular Smooth Muscle; Vascular System; Vascular resistance; Veins; Werner Syndrome; Wild Type Mouse; base; bone; bone cell; comparative; experience; experimental study; human tissue; improved; interest; intravenous injection; mortality; mouse model; mutant; novel; open label; preclinical trial; prevent; progenitor; subcutaneous; therapeutic evaluation; tissue culture

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 90% of 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. Animals carrying the G608G mutation show progressive loss of smooth muscle cells in the media of large vessels. We tested the use of farnesyltransferase inhibitors (FTIs) in cell culture and the mouse model, to see if these drugs could provide benefit in HGPS by reducing the amount of progerin. The results were encouraging, and 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. This treatment is not a cure, however, and so the search for other therapeutic options continues. Homozygotes for the mouse G608G BAC transgenic have also now been bred, and show a considerably more severe phenotype, with death at 6-7 months of age. We have tested those animals to see if there might be therapeutic benefit from everolimus, a rapamycin analog, alone or in combination with FTIs. Rapamycin has been shown to extend 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. We conducted a drug trial in homozygous G608G mice to see if there might be therapeutic benefit from everolimus, and did see a modest improvement in lifespan. We have started a second trial on heterozygous G608G mice that are being treated with a combination of FTI and everolimus to determine if the drugs improve their vascular phenotype. We have also shown recently that everolimus has a beneficial effect on fibroblasts from patients with other LMNA mutations, including those that cause atypical Werners syndrome and Emery-Dreifuss muscular dystrophy. Based on the promising results from cell culture studies, and the encouraging safety profile of everolimus from long experience in organ transplantation, a Phase 1 trial of everolimus in children with progeria has just gotten underway at Boston Childrens Hospital. In yet another approach to the treatment of HGPS, we are investigating antisense oligonucleotide strategies to inhibit abnormal splicing at the cryptic G608G splice site. In collaboration with Sarepta Therapeutics, we tested several phosophorodiamidate morpholino oligonucleotides (PMOs) tiled across the cryptic G608G splice site in vitro in HGPS patient fibroblasts. Two of these candidate PMOs achieved reduction of progerin mRNA and protein, and were subsequently synthesized by Sarepta Therapeutics as proprietary peptide-conjugated phosophorodiamidate morpholino oligonucleotides (PPMOs). Using a GFP reporter system, we showed that intravenous and subcutaneous injection of PPMOs achieves excellent delivery to vascular smooth muscle cells of the mouse aorta. One of the PPMOs subsequently was shown to reduce progerin splicing in aorta, heart, and skeletal muscle in the G608G mouse model. Using this PPMO we have initiated a preclinical trial in our homozygous G608G transgenic mouse model. PPMO is being compared to saline control, administered by IV tail vein injection twice per week. Although mortality is due to myocardial infarction or stroke as a result of rapidly progressive atherosclerosis, HGPS patients also exhibit alopecia, bone and joint abnormalities, and subcutaneous fat loss. Thus cells and tissues derived from common mesenchymal progenitors are particularly susceptible to the pathology that results from the accumulation of progerin. Using a knock-in mouse model (LmnaG609G), we have initiated studies to characterize the molecular and cellular alterations in HGPS bone cell populations, which lead to defective tissue remodeling and progressive bone dysplasia. 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. Although progerin expression has been observed in human ductus arteriosus, and progerin is thought to play a role in the normal closure of this vessel, we did not observe any defects in ductus arteriosus closure in the homozygotes who make no progerin. Extensive histological analysis of the vessels and organs have thus far revealed no differences between the knock-in homozygotes and wild type mice. In a long term experiment, we are assessing whether these mice 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. Comprehensive understanding of how progerin drives HGPS phenotypes requires the identification of the protein interactors of lamin A/C and progerin. The high order insoluble structure of the nuclear envelope makes this challenging, even in cell culture, and has not been previously attempted in human tissues. We have developed a novel biotin proximity-based labeling approach and used it to identify lamin A/C and progerin interactors both in cell culture and directly from human tissue. We have identified instances where tissue culture did not faithfully replicate the interactome of primary tissues, and we have found multiple tissue-specific lamin A/C interactors. We have also identified proteins enriched or depleted in the presence of progerin. We have nowoptimized the method for quantitative comparative proteomics of primary human samples and are currently generating data and following up on initial findings.

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，在LMNA基因的密码子608中，有90％的HGP病例具有从头突变。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。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。我们测试了在细胞培养和小鼠模型中使用Farneylylansferase抑制剂（FTI）的使用，以查看这些药物是否可以通过减少孕激素量来提供HGP的好处。结果令人鼓舞，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。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G608G BAC转基因的纯合子，并且显示出更为严重的表型，死亡在6-7个月大。我们已经测试了这些动物，以查看单独的雷帕霉素类似物依维莫司（Everolimus）是否有治疗益处，或者与FTI结合使用。雷帕霉素已被证明可以延长野生型小鼠的寿命，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增加了增殖能力。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。 我们在纯合的G608G小鼠中进行了药物试验，以查看依维莫司是否可能有治疗益处，并且确实看到了寿命的适度改善。我们已经开始对杂合子G608G小鼠进行的第二次试验，该试验正在用FTI和依依他木斯的组合进行治疗，以确定这些药物是否改善了其血管表型。我们最近还表明，依维莫司对其他LMNA突变患者的成纤维细胞具有有益的作用，包括引起非典型Werners综合征和Emery-Dreifuss肌肉营养不良的患者。 基于细胞培养研究的有希望的结果，以及依然长期移植经验的令人鼓舞的安全性，依依他莫斯（Everolimus）对雌雄病儿童的1期试验刚刚在波士顿儿童医院开始进行。 在治疗HGP的另一种方法中，我们正在研究反义寡核苷酸策略，以抑制隐秘的G608G剪接部位的异常剪接。通过与Sarepta Therapeutics合作，我们测试了几个哲学缩影，在HGPS患者成纤维细胞中，在整个隐秘的G608G剪接位点上铺在隐秘的G608G剪接位点上。这些候选PMO中的两个实现了肌蛋白mRNA和蛋白质的降低，随后由Sarepta Therapeutics合成为专有的肽偶联的硫代偶然径向型摩pholino寡核苷酸（PPMO）。 使用GFP报告基因系统，我们表明PPMO的静脉注射和皮下注射可实现出色的小鼠主动脉血管平滑肌细胞的递送。 随后，其中一种PPMO被证明可减少G608G小鼠模型中主动脉，心脏和骨骼肌中的孕激素剪接。 使用此PPMO，我们在纯合G608G转基因小鼠模型中启动了一项临床前试验。 PPMO与盐水控制进行了比较，该盐水每周两次通过IV尾静脉注射给药。 尽管死亡率是由于快速进行性动脉粥样硬化而导致的心肌梗塞或中风引起的，但HGPS患者也表现出脱发，骨和关节异常以及皮下脂肪损失。因此，源自常见间充质祖细胞的细胞和组织特别容易受到孕产物积累的病理。使用敲击小鼠模型（LMNAG609G），我们已经开始研究以表征 HGPS骨细胞群体中的分子和细胞改变，导致组织重塑和进行性骨骼发育不良。 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。尽管已经在人类导管中观察到了孕激素的表达，并且人们认为雌蛋白在该血管的正常闭合中起作用，但我们没有观察到纯合子中没有产生progerin的纯合子的动脉动脉封闭中的任何缺陷。到目前为止，对血管和器官的大量组织学分析显示，纯合子敲击型和野生型小鼠之间没有差异。在长期实验中，我们正在评估这些小鼠是否具有特殊的寿命。 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。 对过程如何驱动HGP表型的全面理解需要鉴定层粘连蛋白A/C和孕激素的蛋白质相互作用。核包络的高阶不溶性结构甚至在细胞培养中都具有挑战性，并且以前尚未在人体组织中尝试过。我们已经开发了一种新型的基于生物素的基于生物素的标记方法，并将其用于鉴定细胞培养和直接从人体组织中的层粘连蛋白A/C和孕激素相互作用。我们已经确定了组织培养并未忠实地复制原代组织相互作用的实例，并且我们发现了多个组织特异性层粘连蛋白A/C相互作用。我们还鉴定出在孕激素存在下富集或耗尽的蛋白质。现在，我们已经将原代人类样品的定量比较蛋白质组学进行了优化，目前正在生成数据并跟进初始发现。