Humanized synthetic chromosomes built in yeast for organ xenotransplantation

 描述(申请人提供)：人体器官供不应求。一个潜在的变革性解决方案是使猪器官的免疫系统人性化，以限制患者对异种移植器官的排斥反应。原则上，可以使用大的人造染色体将整个基因和途径放入猪细胞中，Boeke实验室将其称为合成猪人工染色体(SPAC)。这些哺乳动物人工染色体的当前版本存在局限性。该实验室将通过从“自下而上”彻底重新设计酿酒酵母中的SPAC来解决这些问题。这些基于酵母的SPAC可以很容易地用患者数据重新编程，并快速上传到猪细胞中。为了实现这种即插即用的能力，这一博士后建议的主要目标是使酿酒酵母染色质人性化，从而确保产生类似哺乳动物的染色体结构。该提案将集中于三个具体目标：1)使酵母的组蛋白人性化；2)建立H3K27甲基化；以及3)建立H3K9甲基化。此外，该提案将允许研究哺乳动物染色质在细胞中的从头建立。这将导致对甲基化的建立、维持和扰动机制的基本洞察，以及与人类基因表达相关的信息。这项提议将通过将人染色质构建到酵母中来实现两个创新和互补的目标：研究细胞中染色质建立的机制，以及生成能够制造猪人工染色体的细胞，旨在促进器官异种移植。长期目标是提供一种无限的方法来产生人性化的猪器官用于器官移植，从而促进人类疾病的治疗。

 DESCRIPTION (provided by applicant): The need for human organs far outstrips the supply. One potential transformative solution is to humanize the immune repertoire of pig organs in order to limit patient rejection of xenotransplanted organs. Whole genes and pathways can in principle be put into pig cells using a large artificial chromosome, which the Boeke lab refers to as synthetic Pig Artificial Chromosomes (sPACs). Current versions of these mammalian artificial chromosomes have limitations. The lab will address these issues by completely redesigning the sPACs from the `bottom up' in the yeast Saccharomyces cerevisiae. These yeast-based sPACs could be readily reprogrammed with patient data and quickly uploaded into pig cells. In order to achieve this `plug and play' capability, the primary goal of this postdoctoral proposal is to humanize the chromatin of S. cerevisiae, thus ensuring the generation of mammalian-like chromosome structure. The proposal will focus on three specific aims: 1) humanize the histones of yeast; 2) build in H3K27 methylation; and 3) build in H3K9 methylation. In addition, the proposal will permit the study of the de novo establishment of mammalian chromatin in a cell. This will lead to fundamental insight into methylation establishment, maintenance, and mechanisms of perturbation, information relevant to human gene expression. This proposal will achieve two innovative and complementary goals by building human chromatin into yeast: study the mechanisms of chromatin establishment in a cellular 'blank slate', and generate a cell capable of making pig artificial chromosomes designed to facilitate organ xenotransplantation. The long-term goal is to provide an unlimited method of generating humanized pig organs for organ transplantation, thus advancing the treatment of human disease.