Item Details

title

Evaluation of a Novel Ovine Model of Sickle Cell Disease With Which to Develop Future Therapies

author

Kuczynski, Caroline Elise

abstract

Sickle cell disease (SCD) affects ~100,000 patients in the US and millions worldwide. A missense mutation (Glu6Val) in the β-globin gene (HBB) results in hemoglobin (Hb) polymerization and characteristic red blood cell (RBC) sickling. Sheep HBB shares ~87.5% sequence identity with humans, and their hematopoietic development parallels that of humans, including a developmental γ- to β-globin switch, making sheep an ideal model for SCD. After performing protein modeling to ascertain that introduction of the SCD mutation in sheep induced conformational changes consistent with human sickling, we used CRISPR/Cas9 and somatic cell nuclear transfer to generate lambs homozygous for the mutation, and three sickle cell edited lambs (SCEL) were born. One animal was bred to WT ewes, and numerous heterozygous offspring were born, analogous to humans with sickle cell trait. Here, we report on the hematological evaluation of the SCEL and heterozygous progeny. Blood from SCEL, age-matched controls (AMC), and heterozygotes was evaluated by human clinical diagnostic methods: hematocrit, blood smears, alkaline and capillary Hb electrophoresis, Hb solubility tests, and ektacytometry. SCEL exhibited abnormal RBC morphology (including sickled erythrocytes, acanthocytes, and polychromasia, while AMC were normal), an ektacytometric leftward shift of the elongation index–osmolality curves similar to human SCD patients, abnormal Hb identified by electrophoresis, and a positive test for insoluble Hb. The heterozygotes also exhibited degrees of similar RBC morphology, and the simultaneous presence of the abnormal and normal Hb upon alkaline electrophoresis. We also developed an Artificial Intelligence (AI) model to classify types of RBCs present within photomicrographs of peripheral blood smears from sheep and subsequently used it to analyze images from these animals. These data indicate that introduction of the SCD mutation in the sheep β-globin gene results in a similar phenotype at the blood level, thereby potentially producing a similar disease phenotype and progression in the sheep model. Findings from the heterozygous offspring indicate a similar phenotype at the blood level to humans with sickle cell trait. Our findings thus support the translational relevance of this model and highlight the potential for its future use in the development and testing of much-needed novel therapies for SCD.

subject

Animal Model

CRISPR/Cas9

Hematology

Hemoglobin S

Prenatal Gene Therapy

Sickle Cell Disease

contributor

Almeida-Porada, Graça (advisor)

Tisdale, John F (committee member)

Cho, Samuel S (committee member)

Lu, Baisong (committee member)

Polejaeva, Irina A (committee member)

date

2024-09-13T08:36:36Z (accessioned)

2024 (issued)

degree

Molecular Medicine and Translational Science (discipline)

embargo

2029-09-05 (terms)

2029-09-05 (liftdate)

identifier

http://hdl.handle.net/10339/109856 (uri)

language

en (iso)

publisher

Wake Forest University

type

Dissertation