Recombinant erythroid Kruppel-like factor fused to GATA1 upregulates globin expr

National Heart, Lung, And Blood Institute

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Abstract

KLF1-GATA1 fusion protein upregulates -globin gene expression in erythroid cells cultured from human sickle CD34+ cells We have previously characterized a series of KLF1-GATA1 fusion proteins composed of different combinations of KLF1 transactivation domains and GATA1 DNA-binding domains, and have demonstrated that two of these fusion proteins could interact with the -globin promoter and increase -globin expression in primary CD34+ cells.32 To explore whether these fusion constructs could be a valuable genetic therapeutic tool for hemoglobinopathies, we overexpressed KLF1-GATA1 in CD34+ cells isolated from SCD patients using lentiviruses expressing KLF1-GATA1 fusion protein, cultured the transduced cells to the mature stage of erythroid, and evaluated the effects of KLF1-GATA1 on hemoglobin expression and sickling-phenotype correction. The long-form KLF1-GATA1 upregulated mean -globin gene expression 2.0-fold, mean -globin gene expression 4.3-fold, and mean -globin gene expression 2.6-fold; the medium-form KLF1-GATA1 upregulated mean -globin gene expression 2.3-fold and -globin gene expression 1.3-fold, but had no significant effect on mean -globin gene expression. Further, transduction of KLF1-GATA1 fusion proteins increased HbA2+HbF expression from 8.1 2.9% in vector-transduced cells to 19.7 3.7% in medium-form KLF1-GATA1transduced cells (p<0.01) and 14.4 3.2% in long-form KLF1-GATA1transduced cells (p<0.01), and HbS expression was reduced from 91.9 2.9% in vector-transduced cells to 80.3 3.7% in medium-form KLF1-GATA1transduced cells (p<0.01) and 85.7 3.2% in long-form KLF1-GATA1transduced cells (p<0.01), . Although the KLF1-GATA1 fusion proteins substantially increased the transcription of -like globin genes, the long-form KLF1-GATA1 upregulated -globin gene expression only very moderately (by a factor of 1.2-fold), and the medium-form KLF1-GATA1 had no impact on -globin gene expression. It has been reported that the effect of a single - or -thalassemia allele can be completely silent with no definable hematological abnormalities. Therefore, it is unlikely that the upregulation of -like globin gene expression (which does produce globin chain imbalance) by the fusion proteins would cause a thalassemia-like phenotype. KLF1-GATA1 fusion protein reduces hypoxia-related sickling without effect on differentiation and proliferation in erythroid cells cultured from human sickle CD34+ cells To evaluate the effect of KLF1-GATA1 fusion proteins on sickle phenotype correction, erythroid cells cultured from human sickle CD34+ cells and exposed to low-oxygen conditions were stained with thiazole orange, then subjected to cell sorting. Thiazole orange-negative cells were sorted directly into wells containing culture medium and incubated for additional 16 hours at 2% oxygen prior to imaging studies. Upon deoxygenation, the mean percentage of non-sickling erythroid cells significantly increased to 20.1 5.4% and 15.1 3.9% in medium- and long-form KLF1-GATA1transduced cells, respectively, as compared with 10.2 3.5% in vector-transduced cells. Transduction with KLF1 had no significant effect on erythroid cell-globin gene expression, HbA2+HbF expression, or sickling morphology. Our results suggest that, although the long-form and medium-form KLF1-GATA1 fusion proteins had different effects on inducing individual globin genes, both forms significantly reduced sickling morphology in erythroid cells cultured from human sickle CD34+ cells subjected to hypoxic conditions. Flow-cytometric analyses of transferrin receptor/glycophorin A expression and bromodeoxyuridine incorporation assays for erythroid cells cultured from human sickle CD34+ cells showed that both erythroid cell differentiation and proliferation were not affected by transduction with a KLF1-GATA1 fusion protein. KLF1-GATA1 fusion protein upregulates -globin gene and HbA2 expression and reduces hypoxia-related sickling in erythroid cells cultured from SCD mouse HSCs To investigate whether the effect of the KLF1-GATA1 fusion protein on hemoglobin expression and sickle phenotype in erythroid cells cultured from human sickle CD34+ cells could be extrapolated to mouse lineage-negative stem cells, Sca-1+c-Kit+Lin/low HSCs isolated from the bone marrow of SCD mice were transduced with lentiviral particles encoding the KLF1-GATA1 fusion protein, then cultured to differentiate into mature erythroid cells and evaluated. QT-PCR analysis showed that both long-form and medium-form KLF1-GATA1 fusion protein enhanced globin gene expression compared with vector control. Long-form KLF1-GATA1 upregulated mean gene expression of -globin, -globin, and -globin by 1.8-fold, 3.3-fold, and 1.7-fold, respectively. Medium-form KLF1-GATA1 upregulated mean -globin gene expression 2.6-fold, but had no significant effect on -globin expression. Transduction of KLF1-GATA1 fusion proteins increased HbA2 expression from 2.1 0.5% in vector-transduced cells to 8.9 1.9% in medium-form KLF1-GATA1transduced cells (p<0.01) and 6.3 1.4% in long-form KLF1-GATA1transduced cells (p<0.01) (Figure 4D). Transduction with KLF1 did not affect delta-globin gene or HbA2 expression. To evaluate the effect of KLF1-GATA1 fusion proteins on erythroid cell sickling under hypoxic conditions, KLF1- or KLF1-GATA1transduced SCD mouse HSCs were incubated in a 2% oxygen environment during the third week in culture, then subjected to SIFCA. Our data revealed that KLF1-GATA1 increased the mean percentage of hypoxia-related non-sickling erythroid cells from 38.1 8.0% in vector-transduced cells to 57.2 8.0% in medium-form KLF1-GATA1transduced cells and to 50.7 7.6% in long-form KLF1-GATA1transduced cells. Transduction with KLF1 had no impact on erythroid cell sickling induced by hypoxia. Flow-cytometric analyses of TER119/thiazole orange staining indicated that erythroid cell enucleation was not affected by KLF1-GATA1 transduction. Similarly, Giemsa staining showed a mostly uniform population of enucleated reticulocytes and erythrocytes in all the groups without a notable morphology difference. Therapeutic use of KLF1-GATA1 fusion protein improves the SCD phenotype in vivo We next determined the effect of the KLF1-GATA1 fusion protein on pathological changes in SCD-related organs. Mock- or KLF1-GATA1transduced SCD mouse HSCs were transplanted into SCD mice, then peripheral blood, spleen, kidney, and liver tissues were collected at 2024 weeks post-transplantation for evaluation. Compared with mock mice (SCD mice transplanted with mock-transduced SCD mouse HSCs), the SCD mice transplanted with KLF1-GATA1transduced SCD mouse HSCs (KG-M received medium from transduced SCD mouse HSCs and KG-L received long from transduced SCD mouse HSCs mice; collectively referred as to KG mice) exhibited a marked increase in red blood cell counts, hemoglobin levels, and hematocrit values, and significantly reduced reticulocytosis. The mean percentage of HbA2 in peripheral blood was increased, and the percentage of sickling cells was reduced in KG mice compared with mock mice. KG-M mice demonstrated a greater improvement in all these parameters compared with KG-L mice. Reduced SCD-related pathology was observed in the spleen, kidney, and liver tissues of KG mice compared with mock mice. The spleens of mock-transduced mice displayed excessive extramedullary hematopoiesis in the red pulp, with a dramatic loss of lymphoid follicular structure. Although these abnormalities were still present in the KG-L mice, the severity was markedly decreased. KG-M mice displayed a relatively normal splenic architecture (splenic red and white pulps observed), with mildly increased erythropoiesis. In addition, splenomegaly was substantially reduced in KG mice compared with mock mice. the SCD-related pathology of the kidneys in KG mice was substantially improved.