Date of Award

6-2021

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Biomedical Sciences

Track

Microbiology, Immunology, and Biochemistry

Research Advisor

Mitchell J. Weiss, MD, PhD

Committee

Steven R. Goodman, PhD; Kui Li, PhD; Tony N. Marion, PhD; Shannon McKinney-Freeman, PhD; Jian Xu, PhD

Keywords

gene editing, hematology, sickle cell disease

Abstract

Sickle cell disease (SCD) is caused by a mutation of the β-globin gene (HBB), resulting in abnormal hemoglobin molecules that polymerize when deoxygenated, forming “sickle” shaped red blood cells (RBCs). Sickle RBCs lead to anemia, multi-organ damage and pain crises, beginning the first year of life. The onset of symptoms coincides with the developmental switch of β-like globin gene expression from fetal stage γ-globin to adult stage β-globin, resulting in a shift from fetal hemoglobin (HbF, α2γ2) to adult hemoglobin (HbA, α2β2). Some individuals harbor rare genetic variants in the extended β-globin gene cluster that cause constitutively elevated postnatal HbF, a benign condition known as hereditary persistence of fetal hemoglobin (HPFH) which alleviates symptoms of co-inherited SCD. Previously, we showed that CRISPR-Cas9-mediated genome editing can recreate a naturally occurring HPFH variant in the γ-globin (HBG1 and HBG2) promoters. Disruption of a TGACC nucleotide motif within this region by Cas9-mediated non-homologous end joining in human erythroid cells or their progenitors caused induction of HbF by interfering with recruitment of the transcriptional repressor, BCL11A. This strategy results in potent HbF induction in human cells and is a promising therapeutic strategy. However, the efficiency of genome editing and the level of HbF induction required to arrest or reverse the pathologies of SCD are unknown.

In this work, we investigated the utility of humanized mouse models for SCD to answer this question. We further characterized the genomic configurations of two models: Berkeley mice, which harbor multiple tandem copies of three separate transgenes encoding human α-globin, sickle β-globin (βS) and a segment of the locus control region (LCR) a powerful enhancer that drives high-level erythroid-specific expression of linked genes; and Townes mice, in which the endogenous α-globin gene is replaced by the homologous human gene and the endogenous β-globin gene is replaced by human γ-globin (γA) and βS-globin genes. Genome editing of human γ-globin promoter in the Berkeley mouse induced a massive DNA damage response and cell death caused by the accumulation of multiple double-stranded DNA breaks (DSB) within the highly repetitive human transgene. In contrast, it was possible to achieve high-level editing of the single copy human γ-globin gene in the Townes model. However, induction of HbF was approximately 10-fold less that what occurred after generating the same edits in human cells, possibly because the mouse model lacks essential non-coding DNA regulatory sequences. Together, these limitations rule out the Berkeley mouse for DSB-inducing gene-editing purposes and the Townes mouse for HbF induction by regulatory element targeting.

Despite these limitations, we determined the Townes model to be a good candidate for a base editing strategy to directly alter the SCD mutation. This work sought to edit the sickle T to a G, resulting in the Hb G-Makassar variant suspected to be benign and non-sickling. Recipient mice transplanted with successfully edited (55-60%) Townes HbSS Lin- cells show marked improvement in blood count values and splenomegaly. This Hb G-Makassar strategy allows for a better understanding of the levels of hematopoietic stem cell editing required to correct the SCD phenotype.

Declaration of Authorship

Declaration of Authorship is included in the supplemental files.

ORCID

https://orcid.org/0000-0002-5622-8069

DOI

10.21007/etd.cghs.2021.0540

2021-013-Woodard-DOA.pdf (319 kB)
Declaration of Authorship

Available for download on Thursday, December 30, 2021

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