Analysis of Fetal Hemoglobin Expression within Humanized Sickle Cell Disease Mice Overexpressing the TR2/4 Transgene.

Abstract Abstract 1619 Background: Sickle Cell Disease (SCD) is a chronic debilitating hematologic condition caused by a missense mutation within the adult beta globin gene leading to significant morbidity and mortality. Increased Fetal Hemoglobin production has been shown to significantly ameliorate SCD symptoms and improve survival. A novel specific DNA-binding factor DRED (direct repeat erythroid definitive) was recently identified that regulated epsilon and gamma globin gene expression (Tanimoto et al Genes Dev 2000). Purification of DRED revealed that it harbored the nuclear orphan hormone receptors, TR2/TR4, as its DNA binding core (Tanabe et al EMBO 2002). Overexpression of TR2/TR4 Transgene within Human Beta Globin Yeast Artificial Chromosome Transgenic Mice resulted in 4-fold induction of the gamma globin mRNA levels (Tanabe et al EMBO 2007). Therefore, we wanted to determine if the overexpression of TR2/TR4 within a humanized sickle cell disease model would result in fetal hemoglobin induction. Methods: Humanized Homozygous Knock-In UAB-Sickle Cell (UAB-Hbahα/hα Hbbhβs/hβs) Mice (Wu et al Blood 2006) was mated to TR2/TR4 Overexpressing Mice (TgTR2/TR4) to generate homozygous SS-TR2/TR4 compound heterozygotes (UAB-Hba hα/hα Hbb hβs/hβs TgTR2/TR4). We generated four 2–3 month old homozygous SS-TR2/TR4 transgenic mice and compared hemoglobin F levels, complete blood cell counts and % body weight (liver, spleen, kidney) to six 2–3 month old homozygous SS mice (Hbahα/hα Hbb hβs/hβs)without the overexpressing TgTR2/TR4. Tail PCR genotyping of all sickle cell mice (with and without TgTR2/TR4) and Hemoglobin F(Hgb F) and Sickle (HgbS) levels were confirmed by HPLC Hemoglobin electrophoresis. Results: The mean Hgb F: 7.8% (n=6, sd 1.63+/−) in the homozygous SS control mice vs. 16.5% (n=4, sd 2.64+/−)in the homozygous SS-TR2/4 Mice (2 Fold higher). Hematologic profile revealed a mean Hct: 25.2 (n=6, sd 5.50 +/−) mean MCV: 75.4 (n=6, sd 10+/−) and a mean WBC: 22.6 (n= 6, sd 13.9 +/−) in the homozygous SS control mice vs. a mean Hct: 31.25(n=4, sd 6.89+/−), mean MCV: 61(n=4, sd 3.5+/−) mean WBC: 16.3(n= 4, sd 5.99+/−) in the homozygous SS-TR2/TR4 mice. Lastly, initial organ (spleen, liver, kidney) pathology evaluation revealed decreased % body weight (bw) in homozygous SS TR2/TR4 Mice vs. homozygous SS controls: 1) Spleen %bw: 4.3% vs. 3.5% TgTR2/TR4), 2) Liver % bw: 8.8% vs. 7.7% TgTR2/TR4), and 3) Kidney %bw: 1.14% vs. 1.02% TgTR2/TR4). Conclusions: Our preliminary analysis revealed that TR2/TR4 overexpression within a humanized sickle cell disease mouse model resulted in a 2-fold induction of fetal hemoglobin based on HPLC hemoglobin electrophoresis. Further, increased TR2/TR4 overexpression improved anemia and organomegaly within sickle cell disease mice. TR2/TR4 may be an attractive target for fetal hemoglobin induction for the treatment of sickle cell disease. Ongoing studies will determine if TR2/TR4 decreases organ specific disease pathology. We will also determine the cellular distribution of fetal hemoglobin in future studies. Disclosures: No relevant conflicts of interest to declare.