Advanced genetic engineering technology development

描述(由申请人提供) 摘要：美国国立卫生研究院设立了主任新创新者奖，以支持那些提出有可能产生异常高影响的高度创新项目的极具创造力的新调查者。十多年来，我一直在研究操纵脊椎动物基因组的新的创新方法。多年来，拥有在实验室模型系统中操纵特定基因的能力一直是遗传研究的黄金标准，但具体敲除(干扰)或敲入(添加)特定基因到基因组中的最有用的策略一直局限于小鼠。然而，实验室小鼠并不总是了解人类生理和疾病的最佳模型系统。如果我们能够将这些基因组工程方法推广到其他物种，这将对人类疾病研究产生巨大影响，因为我们最终将能够在最相关的动物模型中研究特定基因。最近，我和我的同事们是世界上第一个针对和敲除实验室大鼠基因的人，实验室大鼠是生理学家、行为学家、神经学家、营养学家以及对心血管和肾脏疾病感兴趣的研究人员研究最广泛的研究动物模型。这项技术成就被《科学家》评为2009年度十大创新之一。通过将锌指核酸酶(ZFN)技术应用于大鼠胚胎，生成特定基因的敲除动物模型变得非常迅速，因为它不需要改造干细胞，然后将这些干细胞转化为完整的动物。我们用这种方法非常成功，在过去的一年里，我们已经敲除了57个与人类高血压和肾功能衰竭有关的基因--这在两年前都是不可能的。然而，使用ZFN进行基因敲除只是这项技术所能做的事情的开始。具体地说，我们现在知道在大鼠胚胎中进行基因敲击的方法现在是可能的，但仍有许多有待探索的地方。我们建议了解敲门基因工程的ZFN的能力和局限性，以使快速和可重复的方法能够解决与任何脊椎动物模型系统中涉及的几乎任何疾病或发育过程的基因相关的假设。对我们来说，模型系统是大鼠，我们研究的疾病是高血压，但这项提议开发的创新方法将作为任何可以分离胚胎的模型系统的蓝图。具体地说，我们将使用ZFN敲击技术在大鼠中实现条件突变和全基因替换方法，以开发新的有价值的模型来解决与高血压相关的假说，并评估新的抗高血压疗法，并对大约28%的美国人和全球超过10亿受这种疾病影响的人产生影响。 公共卫生相关性：为人类疾病的治疗开发有效的靶向疗法的关键是了解基因、细胞和组织如何参与特定的疾病过程。多年来，对小鼠进行基因工程以了解这些角色一直是金标准，但我们已经开发出新的方法，可能适用于任何研究动物模型，包括大鼠。拟议的研究旨在建立高度创新和广泛适用的方法，在任何模型系统中研究细胞和组织中的特定基因，并开发更好的动物模型来测试高血压等疾病的治疗方法。

DESCRIPTION (Provided by the applicant) Abstract: The NIH created the Director's New Innovator Award to ""support exceptionally creative new investiga- tors who propose highly innovative projects that have the potential for unusually high impact."" I have been working for more than ten years on new and innovative approaches for manipulating vertebrate genomes. Having the ability to manipulate specific genes in laboratory model systems has been the gold standard of genetic research for many years, but the most useful strategies for specifically knocking out (disrupting) or knocking in (adding) specific genes precisely into the genome has been limited to mice. However, laboratory mice are not always the best model system for understanding human physiology and disease. If we can expand these genome engineering approaches to other species it will have a tremendous impact on human disease research because we will finally be able to study specific genes in the most relevant animal models. Recently, my colleagues and I were the first in the world to target and knock out genes in the laboratory rat, the most widely studied research animal model by physiologists, behavioralists, neuroscientists, nutritionalists, and by researchers in- terested in cardiovascular and kidney diseases. This technological achievement was named by The Scientist as one of the 'Top 10 Innovations' in 2009. By applying a Zinc-Finger Nuclease (ZFN) technology to the rat embryo, generating knockout animal models for specific genes becomes very rapid because it does not require the labor of engineering stem cells and then turning those stem cells into a whole animal. We have been very successful with this approach and have now knocked out 57 genes related to hypertension and renal failure in humans in the past year - something that was not possible even 2 years ago. Using ZFNs for gene knockout, however, is just the beginning of what can be done with this technology. Specifically, we now know that gene knockin approaches in the rat embryo are now possible , but much remains to be explored. We propose to understand the capabilities and limitations of ZFNs of knockin engineering to enable rapid and reproducible methods for addressing hypotheses related to genes involved in any vertebrate model system for virtually any disease or developmental process. For us, the model system is the rat and the disease we study is hypertension, but the innovative approaches developed by this proposal will serve as a blueprint for any model system where embryos can be isolated. Specifically, we will use ZFN knockin technology to enable conditional mutagenesis and whole gene replacement approaches in the rat to develop new and valuable models to address hypotheses related to hypertension and evaluate new anti- hypertensive therapies and make an impact on the estimated 28% of Americans and more than one billion people world-wide affected by this disease. Public Health Relevance: The key to developing effective targeted therapies for the treatment of human disease such is the understanding of how genes, cells and tissues are involved in specific disease processes. Genetic engineering in mice to understand these roles has been the gold standard for many years, but we have developed new approaches which are potentially applicable to any research animal model, including rats. The proposed studies aim to establish highly innovative and widely applicable approaches for studying specific genes in cells and tissues in any model system and to develop better animal models for testing therapies for diseases like hypertension.