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3673 Novel Sheep Model of Sickle Cell Disease Reproduces Human Clinical and Laboratory Parameters

Program: Oral and Poster Abstracts
Session: 113. Hemoglobinopathies, Excluding Thalassemia: Basic and Translational: Poster III
Hematology Disease Topics & Pathways:
Research, Sickle Cell Disease, Translational Research, Hemoglobinopathies, Diseases, Technology and Procedures, gene editing
Monday, December 12, 2022, 6:00 PM-8:00 PM

Caroline E. Kuczynski, BS1, Iuri V. Perisse, Ph.D.2*, Misha Regouski2*, Elmira Alipour, B.S.3*, Cecily LaPradd4*, Ying Liu, Ph.D.2*, Michael W. Beaty, M.D.4*, Daniel B. Kim-Shapiro, Ph.D.3*, Anthony Atala, M.D.1*, Irina Polejaeva, Ph.D.2*, Christopher D. Porada, Ph.D.1* and M. Graca Almeida-Porada, MD5

1Fetal Research and Therapy Program, Wake Forest Institute for Regenerative Medicine, Winston-Salem, NC
2Utah State University, Logan, UT
3Department of Physics, Wake Forest University, Winston-Salem, NC
4Wake Forest School of Medicine, Winston-Salem, NC
5Fetal Research and Therapy Program, Wake Forest Institute For Regenerative Medicine, Winston-Salem, NC

Sickle cell disease (SCD), the most common form of an inherited blood disorder, affects nearly 100,000 patients in the US and millions worldwide. SCD is caused by a point mutation in the β-globin gene that results in the replacement of the glutamic acid at position 6 of the protein with valine, causing polymerization of hemoglobin and red blood cell (RBC) sickling. The sheep β-globin gene shares approximately 87.5% identity with the human gene, and sheep hematopoietic development parallels that of humans, including a developmental γ- to β-globin switch, making sheep an ideal model in which to study SCD. After performing protein modeling to ascertain that insertion of the mutation present in human SCD patients into the sheep β-globin gene induced conformational changes consistent with human sickling, we used CRISPR/Cas9 editing technologies and subsequent somatic cell nuclear transfer (SCNT) to introduce the single nucleotide mutation present in human SCD into the β-globin locus of male sheep fibroblasts (both in a single allele and biallelically). Fibroblasts harboring the desired biallelic mutation were clonally expanded, and SCNT and subsequent embryo transfers were performed, that resulted in 3 SCD lambs. Here, we report on the hematological evaluation of these 3 animals.

Blood from three sickle cell edited lambs (SCEL) and age-matched controls (AMC) were evaluated by the same diagnostic methods used in humans: hematocrit, blood smears, reticulocyte counts, alkaline and capillary hemoglobin (Hb) electrophoresis, Hb solubility tests, and ektacytometry. Peripheral blood (PB) smears in SCEL exhibit sickled erythrocytes, acanthocytes, target cells, normoblasts, and polychromasia, while AMC displayed normal PB smears. Reticulocyte counts via flow cytometry were slightly elevated in SCEL when compared to AMC. All 3 SCEL displayed positive SickleDex tests at birth and at all time points analyzed thereafter – these tests were consistently negative in AMC. Alkaline and capillary Hb electrophoresis demonstrated the presence of an abnormal Hb with the same migration pattern as the human HbS, and the presence of HbF at birth. HbF waned progressively to disappear by 3 months, being replaced by HbS in SCEL and normal adult Hb in AMC. In addition, ektacytometry demonstrated a leftward shift of the elongation index–osmolality curves. These data indicate that introduction of the human 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. Our findings thus support the translational relevance of the SCD sheep model and highlight the potential for its future use in the development of novel therapies for SCD.

Disclosures: Kim-Shapiro: Beverage Operations, LLC: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: co-inventor on a patent related to the use of nitrite under cardiovascular conditions, and a co-author on patents related to treatment of hemolysis.

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