Mitochondrial Gene Therapy

描述（申请人提供）：线粒体（mt）DNA突变与多种人类疾病相关，包括过早衰老、肌病、神经退行性疾病、糖尿病、癌症和不育症。鉴于这些疾病中的许多依赖于mtDNA的异质性状态和相关的阈值效应，很难基于植入前或产前遗传诊断提供准确的遗传咨询。目前，还没有治愈线粒体疾病的方法，可用的治疗方法只能改善症状和减缓疾病进展。该提案的主要目标是产生关于新的生殖选择的可行性、有效性和安全性的重要新见解，该生殖选择旨在最大限度地减少临床相关的非人灵长类动物模型中mtDNA缺陷的发生。我们的主要假设是mtDNA可以被一种新的方法有效地取代，即，纺锤体转移（ST）在成熟中期II（MII）卵母细胞，而不干扰随后的核-mtDNA相容性和发育能力。我们的初步研究证明了这种方法在恒河猴中的可行性和有效性。我们相信纺锤体移植后产生的重构卵母细胞将是几乎同质的，能够支持正常受精并有能力进行足月发育。为实现这一目标，我们提出以下具体目标：1.在恒河猴卵母细胞中开发有效的mtDNA替代方法。我们的工作假设是，未受精，成熟的MII停滞的卵母细胞是mtDNA干预的最佳阶段。我们将探讨几种ST程序，并评估其对纺锤体完整性，受精和体外胚胎发育的影响。我们还将评估ST与冻存卵母细胞的可行性和有效性。2.研究重构卵母细胞的发育潜力，并评估ST后代的mtDNA异质性和表观遗传特征。首先，我们提出了胚胎干细胞（ES细胞）和检查核型，多能性，印迹和mtDNA异质性在体外。接下来，我们将评估重建胚胎建立怀孕和产生正常婴儿的潜力，这是应用于人类之前的最终测试。我们还将调查ST后代的各种组织和器官中mtDNA变异体的分离。3.研究由ST产生的猴子的生长和发育，并检查ST雌性生殖系中的mtDNA传递。我们的假设是，mtDNA替代治疗不会影响ST后代的正常出生后生长发育。我们将研究从出生到5岁的实验创造的猴子，并与对照动物进行比较。由于雌性生殖系的遗传瓶颈，异质性雌性的一些后代可能从核供体遗传相当大一部分mtDNA。因此，我们将通过建立胚胎和ES细胞来研究从ST女性收集的卵母细胞中的mtDNA异质性。 公共卫生相关性：线粒体DNA突变与多种人类疾病有关，然而，线粒体疾病没有治愈方法，可用的治疗方法只能改善症状和减缓疾病进展。受精前卵子中的线粒体替换提供了一种潜在有效且伦理上可接受的策略，以避免受影响家庭中的大量线粒体疾病传播给儿童。在这项研究中，我们将探讨这种新的线粒体基因替代疗法在临床相关的非人灵长类动物模型的可行性，有效性和安全性。

DESCRIPTION (provided by applicant): Mutations in mitochondrial (mt)DNA are associated with a wide range of human diseases including premature aging, myopathies, neurodegenerative diseases, diabetes, cancer and infertility. In light of the fact that many of these disorders are dependent on the heteroplasmic state of the mtDNA and associated threshold effects, it is difficult to provide accurate genetic counseling based on preimplantation or prenatal genetic diagnoses. At present, there are no cures for mitochondrial disorders and available treatments only improve symptoms and slow disease progression. The main goal of this proposal is to generate important new insights concerning feasibility, efficacy and safety of novel reproductive options designed to minimize the occurrence of mtDNA- defects in a clinically relevant nonhuman primate model. Our main hypothesis is that mtDNA can be efficiently replaced by a novel approach, i.e., spindle transfer (ST) in mature metaphase II (MII) oocytes without interfering with subsequent nucleo-mtDNA compatibility and developmental competence. Our preliminary studies demonstrate the feasibility and efficacy of this approach in the rhesus monkey. We believe that reconstructed oocytes produced after spindle transfer will be nearly homoplasmic, capable of supporting normal fertilization and competent for full term development. To achieve this goal we propose the following specific aims: 1. Develop efficient mtDNA replacement approaches in rhesus monkey oocytes. Our working hypothesis is that unfertilized, mature MII-arrested oocytes are the most optimal stage for mtDNA interventions. We will explore several ST procedures and evaluate their impact on spindle integrity, fertilization and in vitro embryo development. We will also evaluate feasibility and efficacy of ST with cryopreserved oocytes. 2. Investigate developmental potential of reconstructed oocytes and assess mtDNA heteroplasmy and epigenetic profiles in ST offspring. Initially, we propose to derive embryonic stem (ES) cells and to examine karyotype, pluripotency, imprinting and mtDNA heteroplasmy in vitro. Next, we will evaluate the potential of reconstructed embryos to establish pregnancies and produce normal infants, the ultimate test before applications in humans. We will also investigate segregation of mtDNA variants in various tissues and organs of ST offspring. 3. Study growth and development of monkeys produced by ST and examine mtDNA transmission in the ST female germline. Our assumption is that mtDNA replacement therapy will not affect normal postnatal growth and development of ST offspring. We will study experimentally created monkeys from birth to age 5 in comparison to control animals. Due to the genetic bottleneck in the female germline, some offspring of heteroplasmic females may inherit a significant portion of mtDNA from the nuclear donor. Therefore, we will investigate mtDNA heteroplasmy in oocytes collected from ST females by creating embryos and ES cells. PUBLIC HEALTH RELEVANCE: Mutations in mitochondrial DNA are associated with a wide range of human diseases, however, there are no cures for mitochondrial disorders and available treatments only improve symptoms and slow disease progression. Mitochondrial replacement in eggs prior to fertilization offers a potentially efficient and ethically acceptable strategy to avoid transmission of the vast array of mitochondrial disorders in affected families to children. In this study, we will explore feasibility, efficiency and safety of this novel mitochondrial gene replacement therapy in a clinically relevant nonhuman primate model.