Many children with DS (trisomy 21) develop hematological abnormalities, ranging from mild to severe. Virtually all individuals with DS show signs of disordered hematopoiesis, including leukopenia, lymphopenia, and macrocytosis. Approximately 10% of DS infants develop a transient myeloproliferative disorder (TMD);10- 30% later develop acute leukemia, most often acute megakaryoblastic leukemia (AMKL). Truncation mutations of the GATA1 transcription factor are consistently present in DS TMD and AMKL and its effects appear dependent upon constitutional trisomy 21 which itself causes a hyperproliferative state in multiple lineages. The pathways that underlie hematopoietic defects in DS patients are poorly understood. This proposal seeks to investigate hematopoiesis in DS, using a highly novel method of "chromosome therapy" by site-specific targeting of a human XIST (X-inactive specific transcript) transgene to silence the entire trisomic chromosome 21 (Chr21) in DS induced pluripotent stem (iPS) cells. This system, already in hand, provides rapid and robust silencing of Chr 21 genes, thus allowing direct comparison of parallel cultures of otherwise identical DS stem cells, with and without over-expression of Chr21 genes.
Specific Aim : How does whole chromosome silencing of the trisomic Chr21 in DS iPS cells correct the hematopoietic defects of DS? We will address this question in 3 subaims:
Sub aim A. We will determine whether silencing of trisomic Chr21 corrects the abnormal in vitro hematopoietic phenotype of DS. We will measure production of hematopoietic progenitor and mature cells from human DS iPS cells, with and without doxycycline induction of a Chr21-targeted XIST transgene, using flow cytometry and hematopoietic colony assays.
Sub aim B. We will determine whether silencing of trisomic Chr21 affects expression of Chr21 and non- Chr21 genes. We will examine the transcriptome of corrected and uncorrected DS hematopoietic cells by high-throughput RNA sequencing and analyze gene expression pathways associated with the hematopoietic phenotype identified in subaim A.
Sub aim C. We will determine whether GATA1s expression induces Chr21-dependent myeloproliferation and gene expression in DS iPS cells. Wild type GATA1 will be replaced by a knock-in GATA1s allele in DS iPS cells with inducible Chr21 silencing. The phenotype and gene expression profile will be analyzed in trisomic versus disomic GATA1s hemizygous hematopoietic progenitors to determine the effect of combined trisomy 21 and GATA1 truncation on the networks identified in subaim B. This focused, high-impact study should provide a proof of principle for a novel form of "chromosomal therapy" potentially applicable to hematopoietic and other complications of DS. The proposed experiments should also identify potential therapeutic targets for treatment or prevention of DS hematopoietic defects.
This project will capitalize upon and extend a highly innovative means to study and advance therapeutic treatment of Down syndrome (DS), based on the ability to silence (i.e. neutralize) the extra Chr 21 that causes this disorder. DS is a highly common disorder in which children have a greatly increased risk of blood abnormalities, including myeloproliferative disorder and leukemia. Understanding the abnormal blood formation in Down syndrome is important for the development of treatments, and is relevant to understanding the basis for blood disorders in people more generally. We will use a novel method to directly compare human DS cells with and without the extra chromosome silenced, to reveal the genes and pathways most consistently perturbed by trisomy 21, and identify a subset of genes markedly deregulated on other chromosomes. In addition, this project will lay the foundation for the potential development of a high risk/high payoff new concept of chromosomal therapy, applicable to hematopoietic and other complications of Down syndrome.