Mitchell J. Weiss - US grants

Recent discoveries in our laboratory offer new insights into normal erythroid biology and beta-thalassemia. The high- level production of hemoglobin that occurs during erythroid maturation is tightly coordinated so as to minimize toxicities caused by accumulation of individual alpha- and beta- globin subunits, which tend to precipitate in cells. Prior studies of normal and beta-thalassemic erythroid precursors predict that compensatory mechanisms exist to neutralize free alpha-globin. To learn more about the control of hemoglobin production, we isolated RNA transcripts that are induced by the essential transcription factor GATA-1, a global regulator of erythropoiesis. We identified Erythroid Differentiation Related Factor (EDRF), a small, abundant highly erythroid-specific protein that is strongly upregulated during terminal erythroid maturation and appears to be a direct GATA-1 target gene. We determined that alpha-globin is a specific EDRF binding partner in two independent protein interaction screens. EDRF interacts with free alpha-globin but not with beta-globin or hemoglobin A (alpha2beta2). Moreover, EDRF markedly inhibits precipitation of free alpha-globin in solution and in mammalian cells. Our findings raise the possibility that EDRF acts as a chaperone protein to prevent precipitation and subsequent toxicity of free alpha-globin in erythroid cells. Now that we have established a physical and functional connection between EDRF and alpha-globin in vitro and in heterologous cells, we will study the significance of this association in normal erythropoiesis. Structure-function analyses in Aim 1 will define the domains that are required for physical and functional interactions between EDRF and alpha-globin. In Aim 2, we will assess the biological role of EDRF and its association with alpha-globin in established cell lines and in primary erythroid cells derived from in vitro culture of EDRF gene-targeted embryonic stem (ES) cells. To this end, we have developed EDRF heterozygous and homozygous-null ES cells. In Aim 3, we will determine the hematopoietic consequences of altered EDRF expression in mice. By genetically manipulating EDRF and free alpha-globin levels, we will determine how their relative stoichiometry affects viability and differentiation of erythroid cells. Specifically, we will establish whether EDRF-null animals exhibit excessive alpha- globin precipitation in erythroid precursors, and whether altered EDRF gene expression affects the severity of beta-thalassemia, a disorder that is distinguished by alpha-globin precipitation. Our studies to characterize a highly expressed erythroid specific protein that prevents aggregation of free alpha-globin are important for understanding how hemoglobin chain balance is modulated by non-globin proteins during normal erythropoiesis and might provide a novel approach to alleviate the deleterious effects of excessive alpha-globin in beta-thalassemia.

[unreadable] DESCRIPTION (provided by applicant): GATA-1 is an essential hematopoietic transcription factor that coordinates proliferation arrest with cellular maturation, a function of relevance to normal tissue formation and cancer. Germline mutations in GATA-1 cause inherited dyserythropoietic anemia and thrombocytopenia, while somatic mutations are associated with acute megakaryocytic leukemia (AMKL). The extent of GATA-1 actions and its critical targets are not fully defined; here we propose to investigate its antiproliferative functions. Preliminary findings support the hypothesis that GATA-1 inhibits cell cycle progression by repressing mitogenic genes and activating antiproliferative ones. Remarkably, at least two oncogenes, Kit and Myc, are inhibited directly by GATA-1 in erythroid cells, highlighting a significant role for gene repression in the GATA-1 antiproliferative program. [unreadable] GATA-1 also triggers proliferation arrest and maturation of cultured GATA-1- megakaryocytes through pathways that are currently unexplored. Our current goals are to investigate the mechanisms by which GATA-1 regulates Myc, Kit (Aim 1), and four other potentially key cell cycle effector genes whose expression are controlled by GATA-1 (Aim 2), as identified in our preliminary studies. In Aim 2, we will also examine the functions of these genes by manipulating their expression in erythroid cells and determining the effects on the GATA-1 antiproliferative program. These experiments address the poorly understood role of GATA-1 in gene repression and characterize new molecular targets, several of which were not previously implicated in hematopoiesis. To complement our work on erythroid cells, we will investigate the effects of GATA-1 on megakaryocyte proliferation (Aim 3). Specifically, we will define the transcriptional program associated with proliferation arrest caused by normal GATA-1 and examine mutations previously associated with hyperproliferation and/or maturation defects. These findings will provide further insights into how altered GATA function causes AMKL. Moreover, comparative analysis between erythroblasts and megakaryocytes should reveal aspects of GATA-1-mediated cell cycle that are either common or unique to these related lineages. Our major long-term goals are to define the regulatory hierarchies through which GATA-1 orchestrates cell cycle arrest during normal hematopoietic differentiation and to apply this knowledge to the study of cytopenias and leukemia(s) associated with altered GATA-1 function [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Most leukemias are caused by somatic mutations that disrupt transcription factor function. One example is GATA-1, a nuclear protein required for erythroid and megakaryocytic maturation. Nearly all patients with Down's Syndrome-associated transient myeloproliferative disorder (TMD) or acute megakaryoblastic leukemia (AMKL) exhibit somatic GATA1 gene mutations that result in the exclusive production of an abnormal amino-truncated protein, termed GATA-1 short. We showed that in murine embryonic stem cells and embryos, loss of GATA-1 causes a previously unappreciated block at the bipotential megakaryocytic-erythroid progenitor (MEP) stage of hematopoiesis, where development also appears to be perturbed in AMKL. The leukemia-associated GATA-1 short protein fails to relieve this block and actually drives proliferation in a subset of arrested MEPs. Hence, we hypothesize that GATA-1 promotes normal MEP maturation and that derangements in this function contribute to Down's syndrome-associated TMD and AMKL. In particular, we believe that loss of specific GATA-1 functions activate an aberrant self-renewal program in MEPs, which contributes to development of the leukemic stem cell. Now, we will define the genetic program through which normal GATA-1 controls MEP development and investigate how this program becomes deranged by GATA-1 short in murine ES cells and in human fetal hematopoietic progenitors from both normal and trisomy 21 individuals. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Most leukemias are caused by somatic mutations that disrupt transcription factor function. One example is GATA-1, a nuclear protein required for erythroid and megakaryocytic maturation. Nearly all patients with Down's Syndrome-associated transient myeloproliferative disorder (TMD) or acute megakaryoblastic leukemia (AMKL) exhibit somatic GATA1 gene mutations that result in the exclusive production of an abnormal amino-truncated protein, termed GATA-1 short. We showed that in murine embryonic stem cells and embryos, loss of GATA-1 causes a previously unappreciated block at the bipotential megakaryocytic-erythroid progenitor (MEP) stage of hematopoiesis, where development also appears to be perturbed in AMKL. The leukemia-associated GATA-1 short protein fails to relieve this block and actually drives proliferation in a subset of arrested MEPs. Hence, we hypothesize that GATA-1 promotes normal MEP maturation and that derangements in this function contribute to Down's syndrome-associated TMD and AMKL. In particular, we believe that loss of specific GATA-1 functions activate an aberrant self-renewal program in MEPs, which contributes to development of the leukemic stem cell. Now, we will define the genetic program through which normal GATA-1 controls MEP development and investigate how this program becomes deranged by GATA-1 short in murine ES cells and in human fetal hematopoietic progenitors from both normal and trisomy 21 individuals. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): We are working toward a new perspective in understanding and manipulating the pathophysiology of ? thalassemia, a common and debilitating inherited anemia. A hallmark of this disorder is excessive free ? hemoglobin (Hb), an unstable protein that generates reactive oxygen species (ROS) and forms cytotoxic precipitates. We identified alpha hemoglobin stabilizing protein (AHSP), an abundant erythroid protein that enhances the solubility of free ?Hb and limits its biochemical reactivity. Ahsp-/- mice exhibit hemolytic anemia with Hb precipitates and excessive ROS. Moreover, loss of AHSP exacerbates ? thalassemia in mice, raising the possibility that altered AHSP function or expression could modulate ? thalassemia phenotypes in humans. Preliminary data support both mechanisms. First, we discovered a naturally occurring missense mutation, AHSP N75I, which impairs protein function and is associated with unexpectedly severe p thalassemia in two pedigrees. Second, AHSP appears to be a quantitative trait locus (QTL) whose expression varies considerably between different individuals. Moreover, reduced AHSP expression associates with more severe clinical disease in several independent studies of small p thalassemia cohorts and pedigrees. Together, these findings lead to the hypothesis that AHSP is a genetic modifier of ? thalassemia. We will test this by analyzing thalassemic populations for AHSP gene mutations, including N75I, and determining their effects on gene expression and/or protein function. In addition, we will study how variations in erythroid AHSP expression affect nascent ?Hb pools, oxidative stress and clinical severity in p thalassemic patients. Our findings should provide new insights into the mechanisms of normal erythropoiesis and the pathophysiology of ? thalassemia. Ultimately, this information could provide a basis for developing novel therapeutic approaches to mitigate the toxicities of free ?Hb in ? thalassemia. [unreadable] [unreadable] [unreadable]

Our studies seek to illustrate a new facet of hemoglobin (Hb) biology. We discovered alpha hemoglobin stabilizing protein (AHSP), an abundant erythroid protein that specifically binds free a globin, maintains its structure and limits its pro-oxidant activities. AHSP and (3 globin compete for binding to the same surface of a globin, although [3 globin binds much more tightly. Therefore, when (3 subunits are present, AHSP is displaced to allow for the formation of HbA (a2|32). Ahsp gene ablation causes hemolytic anemia with Hb precipitates, reflecting an essential role in erythropoiesis. Further preliminary data indicate several distinct molecular functions for AHSP to be explored in separate experimental aims. First, AHSP binds and detoxifies erythroid pools of free ccHb (a globin with heme), as evidenced by biochemical studies, X-ray crystallography of AHSP-aHb complexes and observations that loss of AHSP exacerbates P thalassemia in mice (Aim 1). Second, AHSP appears to serve as a chaperone for newly synthesized apo a globin (a globin without heme), thereby promoting its correct folding and subsequent incorporation into HbA (Aim 2). This function is supported by biochemical studies and our observation that even when the free aHb pool is depleted by a globin gene deletion, AHSP is still required for Hb stability in vivo. In addition, AHSP mRNA contains an iron response element (IRE) that extends transcript half-life when iron is limiting. This suggests potential roles for AHSP in the pathophysiology of iron deficiency or overload (Aim 3). Our overall view is that AHSP utilizes multiple mechanisms to protect against various genetic and environmentally induced imbalances in Hb homeostasis. Investigating these mechanisms will elucidate new basic principles of Hb biology and erythropoiesis. In addition, several practical long-term benefits are possible: First, understanding the functions of AHSP could illustrate novel therapeutic approaches to stabilize deleterious free a globin in human p thalassemias. Second, our studies may provide valuable details as to how AHSP can be exploited as a molecular chaperone to improve the manufacture of recombinant Hb-based artificial blood substitutes.

DESCRIPTION (provided by applicant): Our studies address a critical problem in hemoglobin (Hb) biology: how inherently unstable globin protein subunits are folded and maintained during normal and pathological erythropoiesis. We discovered alpha hemoglobin stabilizing protein (AHSP), an erythroid protein that specifically binds free ? globin subunit, stabilizes its structure and limits its pro-oxidant activities. Our preliminary studies suggest two distinct functions for AHSP. First, to detoxify excess ? globin that accumulates during normal erythropoiesis and in various anemias, particularly ? thalassemia. Second, to fold and stabilize newly formed ? globin subunits en route to HbA (?2??2) synthesis. Of potential importance to both functions, we discovered that degradation of AHSP mRNA is accelerated by iron, an essential component of HbA and determinant of nascent globin protein stability. Our overall view is that AHSP facilitates normal HbA synthesis and also buffers against imbalances that arise from genetic or environmental stresses, such as thalassemias and iron deficiency. Now we seek to better understand AHSP activities and their relevance to human health. Aim 1 uses mouse genetics to investigate AHSP functions in vivo. We will examine the consequences of manipulated AHSP expression in thalassemias and create Ahsp gene missense mutations in mice to probe mechanisms of AHSP protein function. Aim 2 studies the biochemical properties of AHSP. We will test in vitro if AHSP promotes reconstitution of HbA from its purified apo-globin and heme components and search for new erythroid proteins that interact with ? globin-AHSP complexes. Aim 3 examines the mechanisms by which iron regulates AHSP expression and the physiological implications of this pathway during altered iron homeostasis. If successful, our work will establish new basic principles of Hb biology and erythropoiesis. In addition, there are potential practical long-term benefits. For example, understanding how AHSP detoxifies excess ?Hb should illustrate novel therapeutic approaches for human ? thalassemias. Elucidating the role of AHSP in HbA synthesis may provide tools to optimize the manufacture of recombinant Hb-based blood substitutes. Finally, defining functional interactions between iron and AHSP could provide insights into the pathophysiology and management of iron overload and deficiency states. PUBLIC HEALTH RELEVANCE: Project Narrative: Our work examines how the blood oxygen carrier hemoglobin is stabilized and assembled during red blood cell formation. If successful, our experiments will enhance general knowledge about how blood is formed. In addition, we will provide new insights toward understanding and treating common and debilitating anemias such as thalassemia and iron deficiency.

DESCRIPTION (provided by applicant): Proper regulation of gene expression is essential to the normal development and health of organisms, whereas aberrant gene regulation is known to cause many genetic diseases, including some inherited anemias, and it is thought to be a major contributor to complex phenotypes such as susceptibility to common diseases. Understanding the molecular mechanisms of gene regulation may provide novel candidates for therapeutic interventions. Our studies aim for a deeper molecular understanding of global aspects of gene regulation in an important biological process, the maturation of erythroid precursor cells to become red blood cells. Building on our progress using patterns in sequence alignments to predict cis-regulatory modules for erythroid genes and deciphering functional correlations of their evolutionary history, we propose to acquire genome-wide information on biochemical features associated with regulation to reach a more complete understanding of gene regulation in erythroid cells. Specifically, we propose to use high throughput biochemical assays such as chromatin immunoprecipitation followed by hybridization to microarrays and deep re-sequencing to acquire data on genomic DNA sequences (Aim 1) occupied in vivo by critical tissue-specific transcription factors, (Aim 2) bound by histones with modifications associated with gene activation or repression, (Aim 3) in chromatin with an altered structure, and (Aim 4) transcribed in a mouse erythroid cell model that undergoes maturation upon restoration of the critical transcription factor GATA-1. Then we will (Aim 5) apply existing software and develop new data-processing algorithms to determine peaks of signals that are likely to represent the locations of the features targeted in aims 1-4. Aim 6 will mine the peak-calling results, along with raw data, multiple sequence alignments and other information to investigate their covariation structure and integrate them to predict cis-regulatory modules, classify the modules by function, identify motifs associated with specific protein occupancy, and deduce the phylogenetic depth of preservation of critical motifs in the regulatory modules. Aim 7 will experimentally test biological hypotheses that arise from the analyses in Aims 6 and 7, determining the extent to which we can validate the locations of protein occupancy and transcripts, the predictions of both positive and negative cis-regulatory modules by gain-of-function cell transfection assays, and the role of motifs implicated in occupancy by directed mutagenesis and in vivo binding assays. We will test whether the motif- constraint hypothesis for protein-occupied DNA segments involved in enhancement applies to transcription factors in addition to GATA-1, and we will conduct additional experiments probing deeper biological issues. This research will provide not only global insights into mechanisms and effects of gene regulation during erythroid maturation, but the techniques and analytical tools developed here can be applied to better understand the development and differentiation of any tissue. PUBLIC HEALTH RELEVANCE: Proper regulation of gene expression is essential to the normal development and health of organisms, whereas aberrant gene regulation can cause genetic diseases, and it appears to be a major contributor to susceptibility to common diseases. Understanding the molecular mechanisms of gene regulation may provide novel candidates for therapeutic interventions. Our studies collecting genome-wide data on many biochemical features associated with gene regulation, mining the data deeply to predict functional DNA sequences, and experimentally testing those bioinformatic predictions will provide global insights into mechanisms and effects of gene regulation during erythroid maturation and provide techniques and analytical tools to better understand the development and differentiation of any tissue.

DESCRIPTION (provided by applicant): This project addresses the NHLBI RC2 GO application entitled "Characterizing Differentiated Heart, Lung, and Blood Cells Derived by Reprogramming Human Embryonic and Induced Pluripotent Stem Cells." Emerging technologies to generate induced pluripotent stem cells (iPSCs) by reprogramming human somatic cells promises to revolutionize biomedical research and clinical medicine. Through in vitro culture methods, iPSCs can be differentiated into numerous cells types derived from all three germ layers. This raises the possibility that patient-derived iPSCs can be used to create relevant tissues for the study of many human disorders. In addition, iPSCs may provide starting material to manufacture transplantable cells for transfusion and regenerative therapies. However, the field is in its infancy and many core questions must be solved in order to realize these exciting long-term prospects. This proposal seeks to advance the use of iPSCs for the study of normal and pathological hematopoiesis. Multiple investigators with broad areas of expertise in hematopoiesis, embryonic stem cell/iPSC biology, chromatin biology, clinical hematology, bioinformatics, cell banking and bioethics/regulatory affairs will work together to pursue the following global issues 1) Mechanisms by which hematopoietic developmental potential might vary between different normal iPSC clones;2) The extent to which iPSC-derived hematopoietic precursors resemble normal ones with respect to cellular phenotypes, gene expression and epigenetic signatures;3) Whether hematopoietic disease phenotypes can be recapitulated by in vitro manipulation of patient-derived iPSCs. We will execute these studies using novel methods to create and culture iPSCs and state-of-the art tools to analyze and manipulate their resident genomes. Pursuit of these problems will serve as a framework in which to develop a facile infrastructure where investigators at our large pediatric institution can create, analyze, bank and distribute iPSCs from any patient of interest. If successful, this work will help to accelerate practical applications of iPSCs for the study and treatment of human diseases. This work will be based at Children's Hospital of Philadelphia with subcontracts to The Pennsylvania State University (State College, PA) and The Coriell Institute for Medical Research (Camden, NJ). The project will create 6 new jobs, thereby stimulating the economy in three different regions of the Northeastern United States. PUBLIC HEALTH RELEVANCE: Efforts to better understand blood production from patient-derived induced pluripotent stem cells (iPSCs) will enhance our understanding of blood disorders and generate new therapeutic approaches. Additionally, this work could create new general paradigms for studying the genesis of many normal tissues and their associated diseases.

Two pre-existing functional Core Facilities will be merged under this proposal and focused on supporting the UPENN/CHOP program in benign hematopoiesis. This combined service will be entitled the Human Stem Cell Core B and will be directed by Dr. Mitchell Weiss. The two SubCores are 1) SubCore B-1, the Hematopoietic Stem Cell (HSC) Facility at UPENN, directed by Martin Carroll, and 2) SubCore B-2, the Human Embryonic Stem (ES) and Induced Pluripotent Stem (iPS) Cell Facility, which is physically present at CHOP, and will be directed by Dr. Deborah French.

DESCRIPTION (provided by applicant): We seek to discover new, medically relevant mechanisms governing red blood cell formation (erythropoiesis). Recently identified small non-coding RNAs, termed microRNAs (miRs), profoundly influence normal development and stress responses in virtually all tissues. MiRs suppress protein synthesis by inhibiting the translation and stability of specific target mRNAs that are recognized via Watson-Crick base pairing. This proposal investigates the role of miRs 144 and 451, which are encoded on a single gene locus that is strongly induced during erythropoiesis. In zebrafish and mice, loss of miR-451 impairs erythropoiesis, sensitizes mature erythrocytes to destruction by oxidant stress and induces erythroid precursor apoptosis after acute anemia. Preliminary data indicate that miR-144/451 regulates iron uptake, survival, maturation, and mitochondrial energy metabolism during erythropoiesis through confirmed target mRNAs including Rab14, Myc, Ywhaz, and Cab39. We will investigate further the mechanisms through which miRs 144 and 451 control erythropoiesis at baseline, and during disease-related stresses including blood loss, iron deficiency, and unbalanced hemoglobin production (thalassemia). Our work should provide new insights into erythroid development and associated disorders including myeloproliferative syndromes and various anemias. Moreover, knowledge gained through our studies of erythropoiesis should be applicable to other biological processes where miR-451 is believed to function, including protection against myocardial stress, regulation of immune responses and as a tumor suppressor in numerous malignancies.

This new NIDDK P30 proposal seeks to establish a Center of Excellence in Hematology at the joint campuses of the University of Pennsylvania (UPENN) and the Children's Hospital of Philadelphia (CHOP) We believe that research and education in the field of benign hematology has achieved a critical mass a UPENN/CHOP. The 13 Investigators of this proposal are well funded by the NIH and published and have multiple co-authored papers and grants. Recent investigator driven enhancements in our research and clinical infrastructures include establishing: a) a Human Embryonic Stem Cell Center (2008); b) an overarching Blood Center focused on enhancing bench-to-bedside research in benign hematology (2009); and c) have recruited two new highly regarded investigators to establish a Comprehensive Adult and Pediatric Bone Marrow Failure Program to coordinate patient care as well as basic and translational research. Our educational efforts are supported by two major NIH training grants: An NIDDK benign hematopoiesis T32 and a NHLBI K12 award on benign hematology. As the number of UPENN/CHOP hematology investigators, research projects, clinical programs and trainees expand, it becomes increasingly important to focus our efforts into a cohesive, synergistic whole. To address this need, the current application seeks support to establish and maintain a multi-investigator, collaborative program on benign human hematopoiesis and associated diseases. Scientific efforts will involve three intertwined foci: 1) advancing our understanding of normal hematopoiesis and of BMF syndromes, 2) using such knowledge to develop novel therapeutic approaches for the hemoglobinopathies and BMF syndromes, as well as 3) using the knowledge gained under (1) to develop novel cellular-based therapeutics for the treatment of hematologic disorders. We believe that this propitious P30 RFA offers the opportunity to exploit and enhance our current strengths in benign hematology. Accordingly, we seek to develop an organized hematopoiesis program that will provide centralized state-of-the-art core facility technical support, offer pilot programs to attract young investigators and organize educational events. We anticipate that 5 years of such support will enhance our productivity as evidenced by important publications in the area of benign hematopoiesis, more synergistic research efforts, growth in the number of faculty and fellows focused on relevant areas and a further increase in our NIH-based funding, particularly multi-investigator grants.

DESCRIPTION (provided by applicant): We have recently discovered that hemoglobin ? is enriched in myoendothelial junctions, the anatomical location where endothelial cells and smooth muscle cells make contact in the resistance arteries. This was a significant finding because it demonstrated that hemoglobin ? had an important and active role outside of erythrocytes. This protein is one of only a few truly polarized proteins to be localized to endothelial-derived myoendothelial junctions, and the siRNA-induced decrease in the amount of the protein significantly altered arterial reactivity, including constriction to phenylephrine and dilation to acetylcholine. The mechanism we derived was based on evidence indicating that monomeric hemoglobin ? is a potent scavenger of nitric oxide, and that endothelial nitric oxide synthase (eNOS) and hemoglobin ? were found to be in a macromolecular complex. Based on this work, as well as a plethora of strong preliminary data, we hypothesize that hemoglobin ? at the myoendothelial junction is a novel regulator of nitric oxide signaling which can impact blood pressure regulation. We will test this hypothesis using two specific aims: 1.) investigate the effects of endothelial hemoglobin a gene ablation/over-expression on arterial function and 2.) elucidate how AHSP and eNOS regulate hemoglobin ? expression and dioxygenase activity at the MEJ. These aims will be elucidated using studies focused first on a floxed hemoglobin ? mouse as well as a hemoglobin a over-expressing mouse to determine the effects of deletion/over-expression of this protein in endothelium on arterial reactivity, whole tissue blood flow, peripheral resistance and blood pressure. In addition, a human model of the disease alpha thalassemia where 2 alleles of hemoglobin a are deleted will be used to study the effects of this genome-wide heterozygous deletion on the vasculature. Next we will investigate how the hemoglobin ? chaperone hemoglobin ? stabilizing protein (AHSP) may traffic hemoglobin ? to the myoendothelial junction or act directly as a regulator of the hemoglobin a redox state, altering the ability of nitric oxide to bind. The sum of this proposal unites and builds on the dat obtained from our previous R01 by allowing us for the first time to ask the direct question as to the function of the myoendothelial junction in intact animals. Indeed, we believe part of the answers could provide the basis for a completely new understanding of blood pressure control by the peripheral vasculature, as well as the derivation of unexplored pharmacological targets for control of hypertension.

DESCRIPTION (provided by applicant): We seek new, medically relevant insights into the biology of red blood cell (RBC) formation (erythropoiesis). In erythroid precursors, the ubiquitin proteasome system (UPS) identifies and eliminates endogenous proteins that become unnecessary or potentially deleterious during progressive maturation. The UPS also functions as a protective mechanism to eliminate toxic proteins that accumulate in RBC disorders, as we and others have demonstrated for ¿ thalassemia, a common anemia caused by imbalanced hemoglobin synthesis. While the UPS is believed to be critical for erythropoiesis, very little is known regarding the specific molecules involved. Large-scale genome wide association studies (GWAS) of human populations have identified numerous UPS components predicted to regulate erythropoiesis. We combined these GWAS with global transcriptome analyses to identify several potentially important UPS proteins expressed in RBC precursors. One interesting candidate that we have studied in depth is Trim58, a protein that marks other proteins for degradation and has also been implicated by GWAS to regulate the formation of platelets. We showed that Trim58 deficient RBC precursors exhibit faulty maturation, including impaired ability to expel the nucleus, a key step in mammalian erythropoiesis. Preliminary studies indicate that Trim58 facilitates enucleation by eliminating dynein, a molecular motor complex with multiple essential functions in virtually all other cell types. We will perform biochemical studies of purifed proteins and genetic manipulations of cultured RBCs to examine the mechanisms by which Trim58 degrades dynein and how this facilitates RBC precursor enucleation. To investigate potential dynein independent functions of Trim58, we will perform proteomic studies to identify its additional degradation targets (Aim 1). To examine Trim58 functions in vivo, we will ablate the gene in mice and determine the consequences on RBC and platelet formation at baseline and after exposure to various physiological stresses (Aim 2). Finally, we will use short hairpin RNAs to suppress the expression of additional GWAS-identified UPS candidates in cultured primary erythroid precursors and determine how this affects their maturation (Aim 3). Our studies aim to elucidate new pathways that promote erythropoiesis through regulated protein degradation. By altering these pathways through drugs or genetic manipulation, it should be possible to enhance ongoing efforts to generate RBCs in vitro for transfusion therapies and to treat various blood diseases caused by dysregulated erythropoiesis. More generally, our planned investigations synergize with GWAS to better understand how genetic variation influences medically relevant phenotypes.

Our multidisciplinary team of clinicians and researchers seeks novel patient-individualized approaches for understanding and managing pediatric acquired aplastic anemia (aAA), a rare but devastating condition characterized by bone marrow hematopoietic stem cell (HSC) hypoplasia with life threatening bleeding, anemia and infections. Pediatric aAA is believed to occur via immune cell attack of HSCs, but little more is known about the pathogenesis and current treatments are not mechanism-based. Some patients with aAA develop clonal hematopoiesis, which is typically viewed pessimistically as a sign of impending myelodysplasia or leukemia. However, this may not always be the case, as our preliminary studies have identified numerous aAA patients with clonal hematopoiesis who have been in healthy remission for years. Moreover, many of these patients harbor unique mutations within their dominant hematopoietic clones. Thus, we hypothesize that clonal hematopoeisis in aAA results from mutational events that impart a growth or survival advantage to HSCs or early progenitors, particularly in the face of disease-associated insults. We will use modern genomic approaches to define the scope of these mutations in a large cohort of aAA patients (Aim 1), follow the clinical course and genetic evolution of the patients longitudinally (Aim 2). Several unique aspects of our study enhance its likelihood of success: First, we are a team of investigators with broad, synergistic expertise in the clinical management of aAA, bioinformatics and genomics/genetics. The ability to follow all of the patients longitudinally in a comprehensive pediatric-adult bone marrow failure clinic at The Children's Hospital of Philadelphia and The Hospital of the University of Pennsylvania. Finally, our study will utilize a large clinically well-annotated tissue collection obtained serially from over 100 aAA patients over 13 years, consisting of DNA and cryopreserved skin, blood and bone marrow cells. We will continue to follow these patients clinically and procure additional samples throughout the study. If successful, our work will identify sets of genes and gene mutations that will sub-classify aAA molecularly to predict prognosis more accurately and to identify more effective, mechanism-based patient-specific therapies.

DESCRIPTION (provided by applicant): Defining the regulatory architecture of hematopoietic cells to elucidate lineage determination and differentiation can produce insights into developmental biology and can help identify targets with potential application to human diseases such as leukemias and anemias. Mouse hematopoiesis is a versatile system for studying gene regulation during differentiation because we can purify populations of progenitor and differentiated cells for genome-wide mapping of transcripts and regulatory sequences, and we can genetically manipulate critical proteins and cis-regulatory modules (CRMs) to study mechanisms of regulation. This application is for a renewal of a long-standing, productive collaboration among multiple investigators with complementary expertise in hematopoietic cell differentiation, gene regulation, genomics, bioinformatics and statistics. Our previous work laid a foundation of genome-wide data sets for transcriptomes, transcription factor occupancy and chromatin states in a cultured cell model for erythroid differentiation and in maturing primary cells in the erythroid and megakaryocytic lineages, which led to key new insights about regulation. We now propose to (Aim 1) generate genome-wide data on transcriptomes and informative epigenetic features in purified cells from each stage of differentiation from mouse hematopoietic stem cells to mature cells of the erythroid and myeloid lineages. For all cell types, including multilineage progenitor cells available only in small numbers, we propose to determine transcriptomes, DNA methylation, and chromatin accessibility (using a new method based on in vitro transposition). In more abundant cell types, we will use ChIP-seq to map transcription factors and histone modifications and also the chromosome conformation capture method Hi-C to build an interaction map of distal regulatory regions with target genes. We will then (Aim 2) conduct integrative, quantitative modeling to find genes differentially expressed and with different transcription factor binding patterns in the distinct lineages; within this set are candidates for genes involved in choice of cell lineage. A hypothesis-driven Bayesian network model will learn quantitative relationships between features, including expression level, and make predictions about how the system would behave after perturbation of both transcription factors and CRMs. We will then (Aim 3) conduct genetic manipulations to test hypotheses arising from integrative analysis in Aim 2. Specific hypotheses about genes involved in lineage choice will be tested by transduction of interfering or forced expression constructs into mouse fetal liver progenitor cells and bipotential cells in culture. Hypotheses from the quantitative modeling of determinants of levels of expression will be tested, targeting specific proteins (using transfections of cells with or withou GATA1) and CRMs (by Cas9-CRISPR-guided genome editing). The result of this proposed work will be deep, widely disseminated data on the regulatory landscape in multiple hematopoietic lineages and keener insights into how changes in regulatory proteins and chromatin lead to lineage choice and progressive differentiation.

Project Summary VISION: ValIdated Systematic IntegratiON of hematopoietic epigenomes Technological advances enabling the production of large numbers of rich, genome-wide, sequence-based datasets have transformed biology. However, the volume of data is overwhelming for most investigators. Also, we do not know the mechanisms by which the vast majority of epigenetic features regulate normal differentiation or lead to aberrant function in disease. We have formed an interdisciplinary, collaborative team of investigators to address the problem of how to effectively utilize the enormous amount of epigenetic data both for basic research and precision medicine. At this point, acquisition of data is no longer the major barrier to understanding mechanisms of gene regulation during normal and pathological tissue development. The chief challenges are how to: (i) integrate epigenetic data in terms that are accessible and understandable to a broad community of researchers, (ii) build validated quantitative models explaining how the dynamics of gene expression relates to epigenetic features, and (iii) translate information effectively from mouse models to potential applications in human health. These needs are addressed by the proposed ValIdated Systematic IntegratiON (VISION) of epigenetic data to analyze mouse and human hematopoiesis, a tractable system with clear clinical significance and importance to NIDDK. By pursuing the following Specific Aims, the interdisciplinary collaboration will deliver comprehensive catalogs of cis regulatory modules (CRMs), extensive chromatin interaction maps and deduced regulatory domains, validated quantitative models for gene regulation, and a guide for investigators to translate insights from mouse models to human clinical studies. These deliverables will be provided to the community in readily accessible, web-based platforms including customized genome browsers, databases with facile query interfaces, and data-driven on-line tools. Specifically, the proposed work in Aim 1 will build comprehensive, integrative catalogs of hematopoietic CRMs and transcriptomes by compiling and determining informative epigenetic features and transcript levels in hematopoietic stem and progenitor cells and in mature cells. CRMs will be predicted using the novel IDEAS (Integrative and Discriminative Epigenome Annotation System) method. Work proposed in Aim 2 will build and validate quantitative models for gene regulation informed by chromatin interaction maps and epigenetic data. Compiling and determining chromosome interaction frequencies will predict likely target genes for CRMs. Gene regulatory models will be built that predict the contributions of CRMs and specific proteins to regulated expression; these models will be validated by extensive testing using genome-editing in ten reference loci. Finally, work in Aim 3 will produce a guide for investigators to translate insights from mouse models to human clinical studies. This effort will include categorizing orthologous mouse and human genes by conservation versus divergence of expression patterns, assigning CRMs to informative categories of epigenomic evolution, and testing the interspecies functional maps experimentally by genome-editing.

Project Summary We seek funding for the highly successful and unique Red Cells Gordon Research Conference (GRC), which has been ongoing since 1979, and the accompanying Gordon Research Seminars (GRS) for trainees, which was initiated in 2013 and has received outstanding attendee scores for two previous meetings. The conferences provide a relaxed, collegial venue to unite diverse investigators working on all aspects of erythrocyte biology, from basic science to translational research on blood disorders. The upcoming Red Cells GRS/GRC meetings will occur July 15-16, 2017 and July 16-21, 2017, respectively, at the Salve Regina University in Newport, Rhode Island, located about 80 miles from the Boston International Airport. The Red Cells GRC will present the most cutting edge, unpublished research on diverse topics including the ontogeny of erythropoiesis, derivation of erythrocytes from pluripotent stem cells, membrane cytoskeletal proteins, genetic/epigenetic regulation of erythroid transcription, cytokine signaling pathways that stimulate erythropoiesis, iron/heme biology, specialized aspects of terminal erythroid maturation and anemias due to genetic and/or infectious causes. In addition, NIH program officers will discuss new funding and training initiatives, and a distinguished speaker will give a keynote address providing a historical overview of the field. The Red Cells GRS will occur over 1.5 days prior to the GRC and include short talks and poster sessions by postdoctoral fellows and graduate students, a keynote address, and workshops on mentoring and career development from successful scientists and physician-scientists from academia, industry and government. Selected trainee talks will be presented at the subsequent GRC. Overall, the Red Cells GRS/GRC serve two important missions of the NIH, particularly the Hematology Branch of the NIDDK and the Blood Diseases Branch of the NHLBI: 1) to promote health-related research that will improve our understanding of blood disorders and lead to new cures, and 2) to enhance the pipeline of new biomedical investigators through support, encouragement and exposure to outstanding scientific research.

PROJECT SUMMARY ? PROJECT 1 We aim to cure sickle cell disease (SCD) by merging new technologies for gene manipulation with recent insights into the perinatal ?-to-? globin gene switch. SCD is caused by mutations in HBB, which encodes the ?-globin subunit of adult hemoglobin (HbA, ?2?2). Elevated fetal Hb (HbF, ?2?2) caused by persistent postnatal??-globin (HBG1 and HBG2) gene expression alleviates pathologies of SCD. In hereditary persistence of fetal hemoglobin (HPFH), HbF exceeds 20% in all adult red blood cells (RBCs) and co-inherited SCD is clinically silent. Modern genetic studies reveal that the ?-to-? globin switch is mediated by BCL11A, a transcription factor that binds cis elements in the extended ?-globin locus, where contiguous HBG2, HBG1 and HBB genes compete for an upstream enhancer, termed locus control region (LCR). Hence, manipulation of human hematopoietic stem cells (HSCs) to reduce erythroid BCL11A expression or ablate its binding sites in the extended ?-globin locus favors HBG1/HBG2-LCR interactions and HbF expression in erythroid progeny. We will study both approaches as potential new gene therapies for SCD. Aim 1 is to develop novel lentiviral vectors (LVs) that express erythroid- specific BCL11A shRNA. Studies by others show that transduction of CD34+ cells with an LV encoding BCL11A shRNA driven by erythroid-specific regulatory elements raises HbF in RBCs generated by in vitro differentiation. However, this LV exhibits relatively poor hematopoietic stem cell (HSC) transduction, as measured by vector copy number (VCN) after long-term reconstitution of immunodeficient mice. We built two novel LVs that increase HSC transduction efficiency by 5- to 8-fold and raise RBC HbF to potentially therapeutic levels. We will study our novel LVs using in vitro culture assays and animal models to acquire additional preclinical efficacy and safety data and develop a production process (with GMP Core C) to support a clinical trial for adult SCD patients by year 3 of this study. Aim 2 utilizes genome editing-mediated non-homologous end joining (NHEJ) to raise adult RBC HbF, either by disrupting an erythroid-specific BCL11A gene enhancer, or by recapitulating a benign, naturally occurring form of HPFH caused by a 13-nucleotide HBG1 promoter deletion. In preliminary studies, both approaches raised HbF to potentially therapeutic levels in RBCs derived from normal or SCD patient CD34+ cells. We will optimize and compare these two gene editing approaches in cultured CD34+ cells, animal models and in vitro assays to identify the optimal method for altering HSCs to induce RBC HbF therapeutically with minimal off-target genotoxicity. Current gene therapy for SCD is promising, but expensive and not consistently effective. By comparing several new LV and gene editing approaches simultaneously, we hope to identify the safest, most effective and economical approach for curing this devastating disease that affects hundreds of thousands of Americans and millions of individuals worldwide.

PROJECT SUMMARY ? OVERALL This PPG is focused on developing safe and effective gene therapy approaches to treat sickle cell disease (SCD), Wiskott-Aldrich syndrome (WAS), and x-linked severe combined immunodeficiency (XSCID). Our overall approach is based on using self-inactivating lentiviral vectors as well as genome editing approaches to correct autologous CD34+ HSCs, which will be administered after busulfan-based conditioning and tested in a series of pre-clinical studies and human clinical trials. In Project 1, Dr. Weiss and his colleagues will validate a novel high-titer lentiviral vector for erythroid-specific expression of BCL11A shRNA and test this in a clinical trial for SCD gene therapy. In parallel, genome editing approaches will be developed to de-repress fetal hemoglobin (HbF) in adult red blood cells and tested in primary human CD34+ HSCs.  In Project 2, Dr. Rawlings and his colleagues will perform multi-center trials to evaluate the safety, feasibility, and efficacy of lentiviral gene transfer in WAS patients. The second aim will be to develop efficient means to use genome editing to correct WAS either by targeting safe harbor loci or by correcting the endogenous WAS allele. In Project 3, Dr. Sorrentino and his colleagues will prove the effectiveness of using the first lentiviral vector for XSCID, along with subablative busulfan conditioning, to treat both newly diagnosed infants and older children with XSCID. These studies will complete enrollment on two open XSCID gene therapy trials and are expected to lead to commercialization of this approach. Core A will provide essential administrative support and coordination between the various projects and centers. Core B will provide scientific expertise for development and production of viral vectors for all three projects. Core C will provide GMP manufacturing for all lentiviral vectors used all planned clinical trials, and provide GMP cell processing to generate transduced CD34+ HSCs for the St. Jude-sponsored gene therapy trials. Altogether, this P01 brings together the necessary expertise to fully develop gene therapy for these selected disorders in the context of a multi-center consortium providing diverse and multidisciplinary expertise in all required aspects of this work. Realization of the aims of this PPG will provide new approaches for treating these severe monogenetic disorders of the hematopoietic and immune system and provide novel and high impact scientific data that will broadly inform human lentiviral/HSC gene therapy.

PROJECT SUMMARY ? CORE A The overall function of Core A is to support Dr. Sorrentino in his role as overall Principal Investigator of the program by defining a chain or responsibility upon which the PPG is organized and establishing connectivity and communications between the various projects and participating centers. Core A will provide a closely interacting network of experienced administrators who work directly with the Project and Core leaders at St. Jude, the Children's GMP LLC in Memphis, Seattle Children's Hospital, intramural National Institutes of Health (NIH), Boston Children's Hospital, and University of California at San Francisco (UCSF). To do this, Core A will provide budgetary oversight and accounting for yearly grant expenditures, administrative support and organization for monthly teleconferences, assistance in preparation of progress reports and competitive renewal applications, and support for PPG-related travel and conferences. Core A will also support the External Advisory Board visit that will provide scientific review and advice for each of the various components and provide formal feedback following a site-visit review at the end of the third year of the upcoming cycle. Overall, Core A is an essential backbone for this multisite program project grant and will ensure seamless administrative and scientific interactions, particularly with the St. Jude Cores and external investigators.

PROJECT SUMMARY Despite advances in the medical care of sickle cell disease (SCD), most patients continue to experience severe pain, poor quality of life, progressive organ deterioration and premature death. Allogeneic hematopoietic stem cell transplantation (HSCT) can cure SCD but is associated with numerous toxicities and only 20% of patients have Human Leukocyte Antigen (HLA)-matched donors. Therefore, improved and more widely accessible curative therapies are needed. Genetic modification of autologous HSCs is a promising experimental approach for treating SCD that circumvents some of the problems associated with allogeneic HSCT, although the optimal technical strategies are not yet established. This proposal explores the use of adenosine base editors (ABEs) and prime editors (PEs) for genetic correction of SCD. In contrast to conventional genome editing, these novel approaches create precise nucleotide alterations independent of double-stranded DNA breaks (DSBs), which can cause structural DNA abnormalities, cell death or malignant transformation. Adenosine base editors convert targeted A·T base pairs to G·C pairs. Prime editors copy edited sequence information from a guide RNA template into a targeted DNA locus. We will test these potentially transformative tools in 3 different strategies for SCD therapy. Aim 1 employs ABEs to create HSC alterations that recapitulate hereditary persistence of fetal hemoglobin (HPFH), a benign genetic condition that alleviates the pathophysiology of co-inherited SCD by inducing the expression of red blood cell (RBC) fetal hemoglobin (HbF), a potent anti-sickling agent. We have used protein evolution strategies to create new high-efficiency ABEs that generate HPFH mutations at frequencies of up to 60% in CD34+ hematopoietic stem and progenitor cells (HSPCs), with HbF being induced to levels that inhibit hypoxic sickling of erythroid progeny. Aim 2 uses ABEs to convert the mutant SCD codon from valine to alanine, thereby generating ?Hemoglobin Makassar (HbG)?, a naturally occurring benign non- sickling variant. We have developed an altered PAM-specific ABE that converts HbS alleles to HbG in SCD donor HSPCs at frequencies of up to 80%, with inhibition of RBC sickling. Aim 3 employs prime editing to revert the mutant SCD codon to normal (Val?Glu), which we have shown to occur efficiently in the HEK293T cell line and now aim to optimize in HSPCs from affected individuals. Overall, our preliminary studies have shown proof of principle for three novel, independent editing approaches to treating SCD without the need to enrich for edited cells or to create DSBs. Through the proposed research, we seek to optimize the efficiency of these approaches in primary HSPCs and to further determine their safety and efficacy by using mouse models, in vitro culture methods and biochemical assays. Developing three approaches simultaneously will enable us to compare their outcomes directly and to determine the best therapeutic strategy to pursue in future clinical studies. More generally, our planned studies have the potential to generate new paradigms for using base editors and PEs to treat numerous genetic blood disorders via precise genetic manipulation of HSCs.