Phenotypic analysis of human iPSC carrying addiction-associated gene variants

描述（由申请人提供）：识别与成瘾行为相关的遗传变异为使用细胞培养来模拟成瘾背后的分子和细胞机制提供了一个新的机会。观察到的遗传变异已被映射到细胞表面受体的氨基酸变化，可能影响涉及成瘾行为的神经回路。然而，细胞环境可能是决定这些基因功能的重要因素。利用携带已知遗传变异的药物滥用个体的成体细胞构建诱导多能干细胞（iPSC），为开发与成瘾相关的生理培养系统提供了一种手段。为了使培养细胞模型有用，它不仅必须表达受影响的基因，而且必须为研究药理学或细胞信号传导提供适当的细胞环境。目前还没有明确的期望，什么类型的ipsc衍生的培养将有助于研究基因变异改变的细胞生理。我们的假设是，与成瘾倾向相关的遗传变异的生理改变可以在来自供体淋巴细胞样本构建的iPSCs的培养神经元中建模。我们建议从供体淋巴细胞样本中构建多个iPSC细胞系，将这些iPSC分化为功能神经元，然后建立方法来测定变异来源细胞和野生型细胞之间可能的表型差异。这些细胞对于识别细胞和分子对滥用物质的反应，检查已知基因型对细胞表型的影响以及开发药物干预的新方法将是有价值的。

DESCRIPTION (provided by applicant): Identification of genetic variations associated with addictive behaviors provides a novel opportunity for using cell cultures to model molecular and cellular mechanisms that underlie addiction. The observed genetic variations have been mapped to amino acid changes in cell surface receptors, presumably affecting neuronal circuits involved in addiction behaviors. However, cellular context is likely to be important in determining the function of these genes. Construction of induced pluripotent stem cells (iPSC) from adult cells derived from drug abusing individuals carrying known genetic variants provides a means for developing physiologically relevant culture systems for understanding addiction. For the culture cell model to be useful it must not only express the affected gene but it must also provide an appropriate cellular context for studying pharmacology or cell signaling. There is no clear expectation about what type of iPSC-derived cultures would be useful in studying the cellular physiology altered by gene variants. Our hypothesis is that the altered physiology of genetic variants associated with addiction liability can be modeled in cultured neurons derived from iPSCs constructed from donor lymphocyte samples. We propose to construct several iPSC lines from donor lymphocyte samples, differentiate these iPSC into functional neurons, and then to develop methods to assay possible phenotypic differences between variant-derived cells and wild-type. These cells will be valuable for identifying cell and molecular responses to substances of abuse, to examine the effects of a known genotype on the cellular phenotype, as well as to develop novel approaches for pharmacologic intervention. PUBLIC HEALTH RELEVANCE: Drug abuse and addiction are a major burden to society, with the total cost of substance abuse in the U.S. exceeding $500 billion annually. Addiction is a chronic brain disease that leads to a compulsive desire for drugs despite significantly harmful consequences, including direct effects on health as well as detriments to society, including disintegration of families, child abuse, loss of employment, and a broad array of crimes. While addiction may be treatable, the discovery that some forms of addiction have an inherited component demonstrates that physiology contributes to addictive behaviors. To exploit new techniques of cellular reprogramming to prepare human neuronal cultures, we will establish induced pluripotent cell (iPSC) lines derived from donor blood samples carrying mutations in two genes known to be risk factors for addiction-an acetylcholine receptor and an opioid receptor. The blood lymphocyte samples already exist in the NIDA Center for Genomic Studies collection and will be used with permission from the collecting investigator. These iPSC will then be induced to differentiate into specific types of neurons for use in investigating the molecular bases of addiction and for screening new treatment therapies.