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Human Artificial Chromosomes for Cancer Research and Functional Genomics

用于癌症研究和功能基因组学的人类人工染色体

基本信息

批准号：
8937731
负责人：
VLADIMIR LARIONOV
金额：
$ 149.77万
依托单位：
DIVISION OF BASIC SCIENCES - NCI
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/8937731
关键词：
Affect Aneuploidy Antineoplastic Agents Autophagocytosis BRCA1 gene Binding Biochemical Biological Assay Biotechnology Boxing Cell Line Cell physiology Cells Centromere Chickens Chinese Hamster Ovary Cell Chromatin Chromatin Structure Chromosomal Instability Chromosome Segregation Chromosome Transfer Chromosomes Chromosomes, Artificial, Human Clinic Cloning DNA DNA Damage Development Disease Doxycycline Elements Epigenetic Process Evolution Flow Cytometry Frequencies Functional disorder Gene Delivery Gene Duplication Gene Expression Gene Silencing Gene Transfer Techniques Genes Genetic Genetic Recombination Genetic Transcription Genomics Goals Half-Life Hamsters Heterochromatin Histone Deacetylase Inhibitor Human Human Genome Insertional Mutagenesis Integrase Kinetochores Knowledge Lead Length Link Maintenance Malignant Neoplasms Measures Mediating Microtubules Mitochondria Mitosis Modification Molecular Mutation NBS1 gene PKD1 gene Pathway interactions Pharmaceutical Preparations Phenotype Plague Polycystic Kidney Diseases Polyploidy Population Preclinical Drug Evaluation Process Proliferating Regenerative Medicine Regulation Reporting Research Retroviral Vector Ribonucleotide Reductase Inhibitor Role Satellite DNA Site Speed Structure Susceptibility Gene System TOP1 gene TOP2A gene Tandem Repeat Sequences Tetanus Helper Peptide Therapeutic Time Transcriptional Activation Transfection Transgenes Tumor Suppressor Proteins Type I DNA Topoisomerases VP 16 Viral Vector Work anticancer research aurora kinase base cancer cell cancer genome cell type chromosome loss design and construction expression vector functional genomics gene correction gene delivery system gene function gene therapy genetic variant high throughput screening human disease inhibitor/antagonist interest kinase inhibitor metaplastic cell transformation mitochondrial dysfunction novel novel therapeutics polycystic kidney disease 1 protein prevent response screening segregation success transgene expression tumor growth tumor progression vector

项目摘要

Human Artificial Chromosomes (HACs) assembled from alphoid DNA arrays represent novel vectors that have a great potential for gene therapy, regenerative medicine, screening of anticancer drugs and biotechnology. HACs avoid the limited cloning capacity, lack of copy number control and insertional mutagenesis due to integration into host chromosomes that plague viral vectors. We previously constructed a synthetic HAC (tetO-HAC) that can be eliminated from cell populations by inactivation of its conditional kinetochore. The tetO-HAC was adapted for gene delivery and gene expression in human cells by insertion of a unique gene acceptor loxP site in chicken DT40 cells. Then the modified HAC was transferred to hamster CHO cells where a gene of interest may be inserted into the HAC and from which the gene-containing HAC can be moved to any desired recipient cell type via microcell-mediated chromosome transfer (MMCT) (Iida et al 2010). It was subsequently demonstrated the utility of the synthetic HAC for delivery of full size genes and correction of genetic deficiencies in human cells. Specifically genomic copies of several cancer-associated genes, including VHL and NBS1, were isolated by transformation-associated recombination (TAR) cloning, loaded into the HAC and successfully transferred into gene deficient cells. We have also shown that phenotypes arising from stable gene expression from the HAC can be reversed when cells are cured of the HAC by inactivating its kinetochore in proliferating cell populations. During the past year, we mainly focused on the analysis of three genes. The first gene, PKD1, encodes polycystin-1; mutations in this gene lead to polycystic kidney disease. The second is TOP1mt, which encodes a mitochondrial topoisomerase I. The TOP1mt gene has been previously discovered in our branch. Biochemical analyses indicate that TOP1mt deficiency in MEF cells results in mitochondrial dysfunctions, induction of the DNA damage response (DDR) pathways, and activation of autophagy. The third gene is BRCA1. Genomic copies of these genes were loaded into the tet-O HAC vectors and transferred into cell lines carrying inactivating mutations in the genes to clarify gene function, its regulation and link to disease. The most exciting results were obtained with the HAC module carrying the 90 kb genomic copy of the BRCA1 gene. As known, BRCA1 is involved in many disparate cellular functions, however, no unifying mechanistic framework that links the reported biochemical activity of BRCA1 to its tumor suppressor function has been identified yet. We demonstrated at first time that BRCA1 deficiency results in a specific activation of transcription of higher-order alpha-satellite repeats (HORs) assembled into heterochromatin domains in the functional kinetochore. At the same time no detectable elevation of transcription was observed within HORs assembled into centrochromatin domains. It is well known that of pericentromeric heterochromatin is essential for kinetochore function. Thus, we demonstrated a link between BRCA1 deficiency and kinetochore dysfunction. This supports the hypothesis that epigenetic alterations of the kinetochore initiated in the absence of BRCA1 may contribute to cellular transformation. In parallel with HAC-based gene expression studies we continue to work on optimization of the tet-O HAC vector itself. For situations in which several genes need to be inserted into the HAC, a multi-integrase HAC vector was designed and constructed. In the tetO-HAC, a gene-loading site was inserted into a centrochromatin domain critical for kinetochore assembly and maintenance. While this domain is permissive for transcription, there are no studies on a long-term transgene expression within centrochromatin. In our recent study, we compared the effects of different chromatin insulators on the expression of an EGFP transgene loaded into the tetO-HAC vector. Unexpectedly, insulator function was essential for stable expression of the transgene in centrochromatin that represents open chromatin structure. We infer that proximity to centrochromatin does not protect genes lacking chromatin insulators from epigenetic silencing. Barrier elements, such as gamma-satellite DNA and tDNA (both were previously discovered in our lab) that prevent gene silencing in centrochromatin would thus help to optimize transgenesis using HAC vectors. The tetO-HAC has an advantage over other HAC vectors because it can be easily eliminated from cells by inactivation of the HAC kinetochore via binding of chromatin modifiers, such as the tTS, to its centromeric tetO sequences. The opportunity to induce HAC loss provides a unique control for phenotypes induced by genes loaded into the alphoidtetO-HAC. However, inactivation of the HAC kinetochore requires transfection of cells by a retrovirus vector to achieve a high level of tTS expression, a step that potentially may lead to insertional mutagenesis. In our recent work, we describe a novel system that allows verification of phenotypic changes attributed to expression of genes from the HAC without a transfection step. We demonstrated that a single copy of tTA carrying 4 tandem repeats of the VP16 domain constitutively expressed from the HAC is capable to generate chromatin changes in the HAC kinetochore that are not compatible with its function. In new system inactivation of kinetohore followed the HAC loss is regulated by doxycycline. The newly modified tetO-HAC-based system has the potential for multiple applications in gene function studies. We have also applied our tetO-HAC for screening of drugs affecting chromosome instability (CIN). While CIN can act as a driver of cancer genome evolution and tumor progression, recent findings point to the existence of a threshold level beyond which CIN becomes a barrier to tumor growth. Our goal was to develop a new quantitative assay for identification of drugs that elevate CIN in cancer cells. For this purpose, the EGFP transgene was loaded into the tetO-HAC using Cre-loxP recombination. The presence of EGFP allows measuring of the HAC loss by flow cytometry. We have successfully used this assay to measure increased mis-segregation of chromosomes in response to different anticancer drugs (Lee et al., 2013b). Specifically, during the past year, a set of different inhibitors currently used in clinics, including HDAC inhibitors, PARP inhibitors, microtubule stabilizing and microtubule-destabilizing drugs, TOP1/TOP2 inhibitors, Chk1/Chk2 inhibitors, Aurora kinase inhibitors and ribonucleotide reductase inhibitors, were compared on their ability to induce chromosome loss. The new identified compounds that dramatically increase chromosome mis-segregation frequencies should expedite the development of new therapeutic strategies to target the CIN phenotype in cancer cells. During the past year, we worked to significantly increase the speed of our HAC-based assay using a modified EGFP with a reduced half-life (e.g., EGFP with a degron box sequence). Our goal is to convert an assay into high-throughput screening. To summarize, the tet-O HAC is the most advanced vector for expression of full-length genes and entire loci and for correction of genetic deficiencies in human cells. At the same time, the HAC provides a unique opportunity to measure chromosome instability and screen new anticancer drugs. Our new HAC-based flow cytometry assay may be also applied for identifying genes controlling chromosome segregation in human cells. In addition, the tetO-HAC was also used as a unique system to study a role of epigenetic modifications in the human kinetochore function (Bergmann et al., 2011, 2012). Recently we used a multi-integrase HAC vector to assemble a large block of lacO/gal4 alphoid DNA arrays in the existing HAC to clarify the role of heterochromatin in kinetochore assembly and maintenance.