Functional Characterization of Trait Associated EnhancersDescription of Major Goals
The goals of this research are to synthesize genetics, biology, and technology to test the hypothesis that enhancer variation is a prevailing determinant of human traits. We will systematically disrupt blood cell trait-associated enhancers to investigate impact on blood cell development, the genetic architecture of blood cell traits, and define cis- and trans-acting determinants of hematopoiesis.
High-resolution and High Through-put genome editing to determine minimal repressive sequences within the beta-globin gene clusteDescription of Major Goals
This research is designed to test the hypothesis that NHEJ-mediated targeted indel production itself would be a favorable therapeutic genome editing strategy for sickle cell disease by disruption of endogenous β-globin gene cluster sequences that repress the expression of fetal hemoglobin.
Characterization of Enhancers Underlying Human Erythroid TraitsDescription of Major Goals
The goal of this project is to explore genetic determinants of human red blood cell traits that modulate erythroid enhancer elements. The aims are: to compare deletion of trait-associated enhancers to non-trait associated enhancers to investigate the genetic architecture of erythroid traits; and to disrupt trait-associated enhancers to identify variants and genes underlying erythroid traits.
Identification of sequences and factors required for HbF repressionDescription of Major Goals
The goals of this research are to utilize a high-resolution, high-throughput genome editing approach to identify genetically implicated noncoding sequences (in addition to the GWAS-marked BCL11A enhancer) associated with HbF level and to define trans-acting factors that physically and functionally interact with critical noncoding sequences at BCL11A and HBS1L-MYB.
Targeted Disruption of the BCL11A Erythroid Enhancer in Hematopoietic Stem Cells for Genetic Therapy of the Beta-HemoglobinopathiesDescription of Major Goals
This research aims to investigate the effects of disrupting the BCL 11 A erythroid enhancer in HSCs and ensure that the modified cells can produce all types of blood cells while also leading to elevated levels of HbF. In addition, we will investigate whether targeting a more specific, enriched HSC population will allow for increased rates of editing and a more effective therapy. The work proposed here will provide important advancements in creating a gene therapy treatment for the 13-hemoglobinopathies as well as improving the use of genome editing technologies in hematopoietic stem cells.
Research Supplement to Promote Diversity in Health-Related Research Associated with R03DK109232 Identification of sequences and factors required for HbF repressionDescription of Major Goals
The goal of this proposal is to identify mechanisms that regulate HbF level and could serve as rational targets for efforts to re-induce HbF for the β-hemoglobin disorders.
Biology studies of hematopoietic stem progenitor cells mobilized by plerixafor from patients with sickle cell diseaseDescription of Major Goals
The goal of this research is to study various features of the early blood cells that are collected during the plerixafor trial. We will test whether the plerixafor-mobilized cells are suitable for gene therapy and gene editing.
Genome Editing of Beta-Globin Gene Cluster Repressive Elements for Beta-ThalassemiaDescription of Major Goals
In this proposal, we will identify minimal sequences at the β-globin gene cluster necessary for BCL11A to repress γ-globin in β-thalassemia erythroid cells; and compare methodologies for safely and effectively performing genome editing of these repressive sequences in primary human β-thalassemia HSCs.
Epigenetic regulation of BCL11A in hemoglobin switchingDescription of Major Goals
The goals of this research are to discover molecular mechanisms underpinning hemoglobin switching. Specific aims include: to investigate mechanisms of BCL11A transcriptional regulation promoting the fetal-to-adult developmental transition; and to functionally evaluate regulatory elements at the BCL11A locus.
Identification of NuRD subunit domain(s) necessary for Y-globin silencing by functional CRISPR-Cas9 mutagenesis scanningDescription of Major Goals
The goal of this project is to explore whether a specific domain(s) of NuRD (Nucleosome Remodeling Deacetylase) subunits is necessary for γ-globin silencing and serves as a potential target for HbF reactivation therapy. The long-term goal of this project is to develop a compound(s) that induces HbF by targeting the epigenetic complex regulating globin switching.
Molecular identification of Psickle in reticulocytes generated from HUDEP-2 cells gene-edited to express Hemoglobin SS in the absence of HbFDescription of Major Goals
Molecular identification of Psickle in reticulocytes generated from HUDEP-2 cells gene-edited to express HbS. This project is designed to enable production of a human cellular model of HbS expression using gene editing and then initial characterization of Psickle activity in HbA and HbS expressing erythroid cells.