Phenotypic analysis of human iPSC carrying addiction-associated gene variants

描述（由申请人提供）：与成瘾行为相关的遗传变异的鉴定为使用细胞培养物模拟成瘾基础的分子和细胞机制提供了新的机会。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，可能会影响成瘾行为所涉及的神经元回路。然而，细胞环境在决定这些基因的功能方面可能很重要。从携带已知遗传变异的药物滥用个体衍生的成体细胞构建诱导多能干细胞（iPSC）提供了开发用于理解成瘾的生理学相关培养系统的手段。为了使培养细胞模型有用，它不仅必须表达受影响的基因，而且还必须为研究药理学或细胞信号传导提供适当的细胞环境。对于哪种类型的iPSC衍生培养物在研究基因变异改变的细胞生理学方面有用，目前还没有明确的预期。我们的假设是，与成瘾倾向相关的遗传变异的生理学改变可以在来源于供体淋巴细胞样本构建的iPSC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几个iPSC系，将这些iPSC分化为功能性神经元，然后开发方法来测定变体衍生细胞和野生型细胞之间可能的表型差异。这些细胞将是有价值的识别细胞和分子的滥用物质的反应，检查一个已知的基因型对细胞表型的影响，以及开发新的药物干预方法。 公共卫生相关性：药物滥用和成瘾是社会的主要负担，美国每年药物滥用的总成本超过5000亿美元。成瘾是一种慢性脑部疾病，导致对毒品的强迫性渴望，尽管有严重的有害后果，包括对健康的直接影响以及对社会的危害，包括家庭解体，虐待儿童，失业和各种犯罪。虽然成瘾可能是可以治疗的，但某些形式的成瘾具有遗传成分的发现表明，生理学有助于成瘾行为。为了利用细胞重编程的新技术来制备人类神经元培养物，我们将建立诱导多能细胞（iPSC）系，该细胞系来自供体血液样本，该样本携带已知是成瘾风险因素的两个基因突变-乙酰胆碱受体和阿片受体。血液淋巴细胞样本已存在于NIDA基因组研究中心的收集中，并将在收集研究者的许可下使用。然后，这些iPSC将被诱导分化为特定类型的神经元，用于研究成瘾的分子基础和筛选新的治疗方法。

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.