A Novel Recombinant Eklf-GATA1 Fusion Protein Reduces Sickling of Erythrocytes Cultured from CD34+ Cells with Sickle Cell Disease

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3506-3506
Author(s):  
Jianqiong Zhu ◽  
Kyung Chin ◽  
Wulin Aerbajinai ◽  
Chutima Kumkhaek ◽  
Hongzhen Li ◽  
...  
Keyword(s):  
Gene Expression ◽  
Sickle Cell Disease ◽  
Fusion Protein ◽  
Sickle Cell ◽  
Globin Gene ◽  
Erythroid Cell ◽  
Cell Disease ◽  
Cd34 Cells ◽  
Long Form ◽  
Globin Gene Expression

Abstract Current gene therapy approaches for treatment of hemoglobinopathies involve viral transduction of hematopoietic stem cells with antisickling globin genes. Hemoglobin A2 (HA2, α2δ2), expressed at a low level due to the lack of Eklf binding motif in its promoter region, is fully functional and could be a valid anti-sickling agent in sickle cell disease, as well as a substitute of hemoglobin A in β-thalassemia. We had previously demonstrated that two Eklf-GATA1 fusion proteins could significantly activate δ-globin expression in CD34+ cells from healthy and sickle trait donor's blood. Here we report the effects of Eklf-GATA1 on hemoglobin expression and phenotypic correction using erythrocytes cultured from CD34+ cells with sickle cell disease. We found that enforced expression of Eklf-GATA1 fusion protein enhanced globin gene expression in the erythrocytesas compared with vector control. The long-form Eklf-GATA1 up-regulated β-globin gene expression 2.0-fold, δ-globin gene expression 4.3-fold, and γ-globin gene expression 2.6-fold. The medium-form EKLF-GATA1 up-regulated δ-globin gene expression 2.3-fold and γ-globin 1.3-fold, but had no significant effect on β-globin gene expression. HPLC revealed a percentage of HA2+HbF was increased from 8.1 % in vector-transduced cells to 19.7% in medium-form Eklf-GATA-transduced-cells (p<0.01) and 14.4% in long-form Eklf-GATA-transduced-cells (p<0.01). Upon deoxygenation, the percentage of sickling erythrocyte was lower to 79.8% in medium-form Eklf-GATA-transduced cells as compared with 89.8% in vector-transduced-cells (p<0.05). Flow cytometry analyses of CD71/GPA and thiazole orange staining indicated that erythroid cell differentiation and enucleation were not affected by Eklf-GATA1. Our results shown that long form Eklf-GATA1 fusion protein has major effects on d- and g-globin induction than β-globin; the medium form Eklf-GATA1 elevated δ- and γ-globin expression without an effect on β-globin expression. Our results indicate that these fusion constructs could be a valuable genetic therapeutic tool for hemoglobinopathies, and warrant further preclinical study and evaluation. Disclosures No relevant conflicts of interest to declare.

scholarly journals A Novel Recombinant Eklf-GATA1 Fusion Protein Reduces Sickling of Cultured Mouse Erythrocytes

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3479-3479
Author(s):  
Jianqiong Zhu ◽  
Hongzhen Li ◽  
Wulin Aerbajinai ◽  
Chutima Kumkhaek ◽  
Kyung Chin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Sickle Cell Disease ◽  
Fusion Protein ◽  
Sickle Cell ◽  
Fusion Proteins ◽  
Globin Gene ◽  
Globin Chain ◽  
Cell Disease ◽  
Long Form ◽  
Globin Gene Expression

Abstract β-hemoglobinopathies are inherited disorders caused by mutations/deletions in the β-globin chain that lead to structurally defective β-globin chains or reduced (or absent) β-globin chain production. These diseases affect multiple organs and are associated with considerable morbidity and mortality, representing a major public health challenge. Current gene therapy approaches for the treatment of hemoglobinopathies involve viral transduction of hematopoietic stem cells with antisickling globin genes. Hemoglobin A2 (HA2, α2δ2), expressed at a low level due to the lack of Eklf binding motif in its promoter region, is fully functional and could be a valid anti-sickling agent in sickle cell disease, as well as a substitute of hemoglobin A in β-thalassemia. We had previously demonstrated that two Eklf-GATA1 fusion proteins could significantly activate δ-globin expression in human CD34+ cells. Here we report the effects of Eklf-GATA1 on hemoglobin expression and phenotypic correction using erythrocytes cultured from mouse hematopoietic progenitor cells with sickle cell disease. We found that enforced expression of Eklf-GATA1 fusion protein enhanced globin gene expression in the erythrocytesas compared with vector control. The long-form Eklf-GATA1 up-regulated β-globin gene expression 1.8-fold, δ-globin gene expression 3.3-fold, and γ-globin gene expression 1.7-fold. The medium-form EKLF-GATA1 up-regulated δ-globin gene expression 2.6-fold and γ-globin 1.3-fold, but had no significant effect on β-globin gene expression. HPLC revealed a percentage of HA2 was increased from 2.1 % in vector-transduced cells to 8.9% in medium-form Eklf-GATA-transduced-cells (p<0.01) and 6.3% in long-form Eklf-GATA-transduced-cells (p<0.01). Upon deoxygenation, the percentage of sickling erythrocyte was lower to 30.6% in medium-form Eklf-GATA-transduced cells as compared with 40.7% in vector-transduced-cells (p<0.05). Flow cytometry analyses of CD71/GPA and thiazole orange staining indicated that erythroid cell differentiation and enucleation were not affected by Eklf-GATA1. ChIP-sequencing analysis has demonstrated that Eklf-GATA1 fusion proteins and GATA1 having a similar protein-DNA binding pattern at a global level. Our results have found that the long form Eklf-GATA1 fusion protein has a major effect on δ-globin induction than β-globin; the medium form Eklf-GATA1 is able to elevate δ-globin expression without having an effect on β-globin expression. The above findings indicate that these fusion constructs could be a valuable genetic therapeutic tool for hemoglobinopathies, and warrant further preclinical study and evaluation. Disclosures No relevant conflicts of interest to declare.

scholarly journals Potentially therapeutic levels of anti-sickling globin gene expression following lentivirus-mediated gene transfer in sickle cell disease bone marrow CD34 + cells

2015 ◽  
Vol 43 (5) ◽  
pp. 346-351 ◽  
Author(s):  
Fabrizia Urbinati ◽  
Phillip W. Hargrove ◽  
Sabine Geiger ◽  
Zulema Romero ◽  
Jennifer Wherley ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Gene Transfer ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Globin Gene ◽  
Cell Disease ◽  
Cd34 Cells ◽  
Globin Gene Expression

Delta‐globin gene expression improves sickle cell disease in a humanised mouse model

2021 ◽  
Author(s):  
Susanna Porcu ◽  
Michela Simbula ◽  
Maria F. Marongiu ◽  
Andrea Perra ◽  
Daniela Poddie ◽  
...  
Keyword(s):  
Gene Expression ◽  
Sickle Cell Disease ◽  
Mouse Model ◽  
Sickle Cell ◽  
Globin Gene ◽  
Cell Disease ◽  
Globin Gene Expression

scholarly journals Gene Editing of KLF1 to Cure Sickle Cell Disease

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 30-31
Author(s):  
Kevin R. Gillinder ◽  
Casie Leigh Reed ◽  
Shezlie Malelang ◽  
Helen Lorraine Mitchell ◽  
Emma Hoskin ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Gene Editing ◽  
Globin Gene ◽  
Erythroid Differentiation ◽  
Erythroid Cell ◽  
Cell Disease ◽  
Hemoglobin Switching ◽  
Cd34 Cells ◽  
Klf1 Gene

Sickle cell disease (SCD) affects millions of people worldwide and represents the most common monogenic disease of mankind (1). It is due to a homozygous T to A transversion in the β-globin gene that results in an amino acid variant - G6V - and production of HbS, which polymerises in red blood cells (RBCs) under hypoxic conditions. This generates irreversibly sickled cells that fail to traverse the microcirculation, resulting in micro-infarcts, hypoxia and pain, or 'sickle cell crises'. During gestation RBCs utilise different sets of globin genes to produce embryonic and fetal hemoglobins (HbF), so it is not until after birth when adult hemoglobin (HbA) is first produced that the first signs of SCD become apparent. This process termed 'hemoglobin switching' has been the focus of research efforts for decades because it offers an opportunity to reactivate HbF in adult cells of patients with hemoglobinopathies. A number of transcription factors, including Krüppel-like factor 1 (KLF1), play critical roles in hemoglobin switching. KLF1 is an essential erythroid transcription factor that co-ordinates the expression of more than a thousand genes critical to the formation of adult RBCs. KLF1 directly binds the β-globin gene promoter to up regulate its expression, whilst regulating the expression of additional factors like BCL11A and LRF that directly repress γ-globin expression (HbF). Heterozygosity for loss of function mutations in KLF1 leads to a significant increase in HbF that is beneficial to patients with β-thalassemia. We propose this can be recreated by advanced gene editing techniques to provide an effective therapy for SCD. We have employed CRISPR-based gene editing to knockout the expression of KLF1 in human cells. We designed two separate sgRNAs with corresponding HDR templates to target the second exon of KLF1 and ablate its function. We optimised transfection protocols and tested the on-target specificity of our sgRNAs achieving &gt;90% efficacy in all cell types assayed. Using HUDEP-2 cells (2), a conditionally immortalised erythroid cell line which harbors three copies of KLF1 (3), we have demonstrated that these cells require at least one copy (&gt;1/3) for survival; heterozygous cells (+/-/- or +/+/-) proliferate at a reduced rate, but are able to differentiate normally. Using RNA-seq, we identified some genes, including ICAM-4 and BCAM, which are down-regulated accordingly in a KLF1 gene dosage-dependent manner. ICAM-4 and BCAM are cellular adhesion molecules implicated in triggering vaso-occlusive episodes (4; 5), so it is anticipated their reduced expression may provide additional benefit in treating SCD. Gamma-globin is upregulated 10-fold, BCL11A down-regulated 3-fold, and HbF+ RBCs generated at ~20% of total RBCs in KLF1 +/-/- HUDEP-2 cell lines. We also engineered the ablation of KLF1 in CD34+ cells harvested from the peripheral blood of SCD patients undergoing exchange transfusions. Following transfection of the two guides, we performed directed differentiation using an erythroid differentiation medium and analysed the levels of HbF. We observed HbF at levels of between 40-60% of total Hb by HPLC, and HbF+ cells of ~50% by FACS. There was no measurable block in erythroid differentiation by FACS. We documented the types of gene editing using a high throughout NGS assay (6). We compared efficiencies of CRISPR repair of the HbS mutation with CRIPSR damage of the KLF1 gene. Lastly, we transplanted gene-edited CD34 cells into NSGW41 mice (where human erythropoiesis is established) to determine the efficiency and safety of editing long term HSCs from SCD patients. We will report on the results of these xenotransplantation assays. Taken together these results reveal the potential utility in targeting KLF1 to cure SCD. References: Wastnedge, E. et al..J Glob Health 8, 021103 (2018). Kurita, R. et al.PLoS One 8, e59890 (2013). Vinjamur, D. S. & Bauer, D. E. Methods Mol Biol 1698, 275-284 (2018). Bartolucci, P. et al..Blood 116, 2152-9 (2010). Zhang, J., et al. PLoS One 14, e0216467 (2019). Bell, C. C., et al. BMC Genomics 15, 1002 (2014). Perkins, A. et al..Blood 127, 1856-62 (2016). Disclosures Kaplan: Celgene: Honoraria; Novartis: Honoraria. Perkins:Novartis Oncology: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Differences in Fetal Hemoglobin Induction in Sickle Cell Disease and beta-Thalassemia.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 555-555 ◽  
Author(s):  
Hassana Fathallah ◽  
Ali Taher ◽  
Ali Bazarbachi ◽  
George F. Atweh
Keyword(s):  
Gene Expression ◽  
Sickle Cell Disease ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Mrna Levels ◽  
Globin Genes ◽  
Thalassemia Intermedia ◽  
Cell Disease ◽  
Globin Mrna ◽  
Globin Gene Expression

Abstract A number of therapeutic agents including hydroxyurea, butyrate and decitabine have shown considerable promise in the treatment of sickle cell disease (SCD). However, the same agents have shown less clinical activity in β-thalassemia. As a first step towards understanding the molecular basis of the different clinical responses to these agents, we have studied the mechanisms of induction of fetal hemoglobin (HbF) by butyrate in BFU-E derived cells from 5 patients with SCD and 9 patients with β-thalassemia intermedia. Exposure to butyrate resulted in a dose-dependent augmentation of γ-globin mRNA levels in erythroid cells from patients with SCD. In contrast, induction of γ-globin expression in erythroid cells from patients with β-thalassemia intermedia was only seen at a high concentration of butyrate. The increase in γ-globin mRNA levels in patients with SCD and β-thalassemia intermedia was associated with opening of the DNA structure as manifested by decreased DNA methylation at the γ-globin promoters. Interestingly, butyrate exposure had markedly different effects on the expression of the β- and α-globin genes in the two categories of patients. Butyrate decreased the level of β-globin mRNA in 4 out of 5 patients with SCD (P = 0.04), while in β-thalassemia the levels of β-globin mRNA did not change in 7 patients and decreased in 2 patients after butyrate exposure (P = 0.12). Thus in patients with SCD, the effects of the induction of the γ-globin gene on the γ/(β+γ) mRNA ratios were further enhanced by the butyrate-mediated decreased expression of the β-globin gene. As a result, γ/(β+γ) mRNA ratios increased in all patients with SCD, with a mean increase of 31% (P = 0.002). In contrast, butyrate increased γ/(β+γ) mRNA ratios only in 4 out of 9 patients with β-thalassemia, with a more modest mean increase of 12% (P = 0.004). Interestingly, the decreased β-globin expression in patients with SCD was associated with closing of the DNA configuration as manifested by hypermethylation of DNA at the promoter of the β-globin gene while methylation of the same promoter did not change following butyrate exposure in patients with β-thalassemia intermedia. More surprisingly, the expression of the α-globin genes increased following butyrate exposure in 4 out of 9 patients with β-thalassemia, while the levels of α-globin mRNA decreased in 4 out of 5 patients with SCD. As a result, the favorable effects of the butyrate-induced increase in γ-globin gene expression on the α: non-α mRNA imbalance in patients with β-thalassemia intermedia were partly neutralized by the corresponding increase in α-globin gene expression. These differences may explain, at least in part, the more favorable effects of inducers of HbF in SCD than in β-thalassemia. Further studies are necessary to fully understand the molecular bases of the different responses to agents that induce HbF in patients with these disorders.

Preclinical Studies for Sickle Cell Disease Gene Therapy Using Bone Marrow CD34+ Cells Modified with a βAS3-Globin Lentiviral Vector

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3119-3119
Author(s):  
Fabrizia Urbinati ◽  
Zulema Romero Garcia ◽  
Sabine Geiger ◽  
Rafael Ruiz de Assin ◽  
Gabriela Kuftinec ◽  
...  
Keyword(s):  
Stem Cells ◽  
Gene Therapy ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Blood Cells ◽  
Globin Gene ◽  
Cell Disease ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Abstract 3119 BACKGROUND: Sickle cell disease (SCD) affects approximately 80, 000 Americans, and causes significant neurologic, pulmonary, and renal injury, as well as severe acute and chronic pain that adversely impacts quality of life. Because SCD results from abnormalities in red blood cells, which in turn are produced from adult hematopoietic stem cells, hematopoietic stem cell transplant (HSCT) from a healthy (allogeneic) donor can benefit patients with SCD, by providing a source for life-long production of normal red blood cells. However, allogeneic HSCT is limited by the availability of well-matched donors and by immunological complications of graft rejection and graft-versus-host disease. Thus, despite major improvements in clinical care, SCD continues to cause significant morbidity and early mortality. HYPOTHESIS: We hypothesize that autologous stem cell gene therapy for SCD has the potential to treat this illness without the need for immune suppression of current allogeneic HSCT approaches. Previous studies have demonstrated that addition of a β-globin gene, modified to have the anti-sickling properties of fetal (γ-) globin (βAS3), to bone marrow (BM) stem cells in murine models of SCD normalizes RBC physiology and prevents the manifestations of sickle cell disease (Levassuer Blood 102 :4312–9, 2003). The present work seeks to provide pre-clinical evidence of efficacy for SCD gene therapy using human BM CD34+ cells modified with the bAS3 lentiviral (LV) vector. RESULTS: The βAS3 globin expression cassette was inserted into the pCCL LV vector backbone to confer tat-independence for packaging. The FB (FII/BEAD-A) composite enhancer-blocking insulator was inserted into the 3' LTR (Ramezani, Stem Cells 26 :32–766, 2008). Assessments were performed transducing human BM CD34+ cells from healthy or SCD donors with βAS3 LV vectors. Efficient (1–3 vector copies/cell) and stable gene transmission were determined by qPCR and Southern Blot. CFU assays demonstrated that βAS3 gene modified SCD CD34+ cells are fully capable of maintaining their hematopoietic potential. To demonstrate the effectiveness of the erythroid-specific bAS3 gene in the context of human HSPC (Hematopoietic Stem and Progenitor Cells), we optimized an in vitro model of erythroid differentiation of huBM CD34+ cells. We successfully obtained an expansion up to 700 fold with >80% fully mature enucleated RBC derived from CD34+ cells obtained from healthy or SCD BM donors. We then assessed the expression of the βAS3 globin gene by isoelectric focusing: an average of 18% HbAS3 over the total globin present (HbS, HbA2) per Vector Copy Number (VCN) was detected in RBC derived from SCD BM CD34+. A qRT-PCR assay able to discriminate HbAS3 vs. HbA RNA, was also established, confirming the quantitative expression results obtained by isoelectric focusing. Finally, we show morphologic correction of in vitro differentiated RBC obtained from SCD BM CD34+ cells after βAS3 LV transduction; upon induction of deoxygenation, cells derived from SCD patients showed the typical sickle shape whereas significantly reduced numbers were detected in βAS3 gene modified cells. Studies to investigate risks of insertional oncogenesis from gene modification of CD34+ cells by βAS3 LV vectors are ongoing as are in vivo studies to demonstrate the efficacy of βAS3 LV vector in the NSG mouse model. CONCLUSIONS: This work provides initial evidence for the efficacy of the modification of human SCD BM CD34+ cells with βAS3 LV vector for gene therapy of sickle cell disease. This work was supported by the California Institute for Regenerative Medicine Disease Team Award (DR1-01452). Disclosures: No relevant conflicts of interest to declare.

scholarly journals Novel Lentiviral Vectors for Gene Therapy of Sickle Cell Disease Combining Gene Addition and Gene Silencing Strategies

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3973-3973
Author(s):  
Megane Brusson ◽  
Anne Chalumeau ◽  
Pierre Martinucci ◽  
Valentina Poletti ◽  
Fulvio Mavilio ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Gene Silencing ◽  
Sickle Cell ◽  
Globin Gene ◽  
Insertion Site ◽  
Lentiviral Vectors ◽  
Erythroid Cell ◽  
Erythroid Cells ◽  
Cell Disease ◽  
Hematopoietic Stem

Abstract Sickle cell disease (SCD) is due to a mutation in the β-globin (HBB) gene causing the production of the sickle β S-globin chain. The sickle Hb (HbS, a 2β S2) polymerizes, leading to the formation of sickle-shaped red blood cells that cause vaso-occlusions and organ damage. Transplantation of autologous hematopoietic stem/progenitor cells (HSPCs) transduced with lentiviral vectors (LV) expressing an anti-sickling β-globin transgene (βAS LV) is a promising curative treatment; however, it is partially effective in SCD patients, who still present elevated HbS levels. Here, we aim to improve LVs to boost therapeutic β-like globin levels without increasing the mutagenic vector load in HSPCs. We developed 2 novel LVs expressing βAS together with an artificial microRNA (amiR) targeting either the fetal Hb (HbF) repressor BCL11A (βAS/amiRBCL11A) or the β S-globin (βAS/amiRHBB). By downregulating BCL11A, amiRBCL11A re-activates the expression of the endogenous anti-sickling fetal γ-globin, which, together with βAS, should improve the clinical course of SCD; β S-globin downregulation should favor βAS incorporation in Hb tetramers, increase therapeutic Hb levels and ameliorate the SCD phenotype. First, we developed βAS/amiRBCL11A LV by inserting the amiR in multiple position of the βAS intron 2 under the control of HBB promoter/enhancers to limit BCL11A downregulation to the erythroid lineage and reduce potential amiR-related cellular toxicity and off-target effects. We showed that amiR insertion site did not affect LV titer nor βAS expression in a human erythroid cell line (HUDEP2). BCL11A downregulation in HUDEP2 led to γ-globin gene de-repression and a high proportion of HbF + cells (RTqPCR, HPLC, flow cytometry). Importantly, the total amount of therapeutic β-like globins was substantially higher in βAS/amiRBCL11A LV- than in βAS LV-transduced cells, with no impairment in cell viability or erythroid differentiation. In parallel, we designed 17 amiRs targeting HBB and generated the corresponding βAS/amiRHBB LVs. We tested these LVs in HUDEP2 and selected 2 amiRs efficiently downregulating β-globin at mRNA and protein levels (RT-qPCR and Western Blot). Of note, we modified the βAS transgene by inserting silent mutations that prevent its recognition by the amiR (βASm). Finally, we tested βAS/amiRBCL11A and βAS/amiRHBB LVs in HSPCs from SCD patients. HSPC-derived erythroid cells transduced with βAS/amiRBCL11A LV showed increased HbF levels, although HbS levels remained high. To further reduce β S-globin levels, we targeted the β S-globin mRNA using the βAS/amiRHBB LV. Efficient HSPC transduction by βASm/amiRHBB LV led to a substantial decrease of β S-globin transcripts in HSPC-derived erythroid cells compared to the βAS LV-transduced cells (RTqPCR) at a VCN/cell of 2. Notably, the amiR specifically down-regulated β S-globin, without affecting βAS expression. In βASm/amiRHBB- vs βAS LV-transduced cells, HPLC analysis showed that β S-globin downregulation led to a significant decrease of HbS, which represented 58% and 71% of the total Hb, respectively). This was associated with a significant increase of the therapeutic Hb in βASm/amiRHBB LV- vs βAS LV-transduced erythroid cells (38% and 27% of the total Hb, respectively). Importantly, we observed a substantial reduction of the proportion of HbS-positive cells in βASm/amiRHBB- vs βAS LV-transduced samples (from 96% to 70%; Figure 1A). The increased incorporation of βAS in Hb tetramers and the decrease in β S-globin led to a better correction of the sickling phenotype in mature RBCs derived from HSPCs transduced with βASm/amiRHBB LV- compared to βAS LV (55% and 84% of sickling cells, respectively; Figure 1B). A clonal assay of hematopoietic progenitors showed no impairment in HSPC viability and differentiation towards the erythroid and myeloid lineages upon transduction with bifunctional LVs. βASm/amiRHBB LV showed a standard lentiviral integration profile. Finally, we performed RNAseq to further evaluate the safety of our therapeutic strategy. In conclusion, we created a LV able to concomitantly silence the β S-globin and express βAS, achieving clinically relevant levels of therapeutic Hb and efficient correction of the sickling phenotype. Therefore, the combination of gene addition and gene silencing strategies can improve the efficacy of current therapeutic approaches, representing a novel treatment for SCD. Figure 1 Figure 1. Disclosures Cavazzana: Smart Immune: Other: co-founder.

Genetic Correction of Sickle Cell Disease by Co-Regulated γ-Globin Transgene Expression and ßS Interference.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1280-1280
Author(s):  
Selda Samakoglu ◽  
Yelena Usachenko ◽  
Tulin Budak-Alpdogan ◽  
Santina Acuto ◽  
Rosalba DiMarzo ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Branch Point ◽  
Transgene Expression ◽  
Globin Gene ◽  
Cell Disease ◽  
Globin Mrna ◽  
Tissue Specific ◽  
Human Cd34 ◽  
Cd34 Cells

Abstract RNA interference (RNAi) is a promising therapeutic strategy, but its application to stem cell-based gene therapy for the treatment of congenital or acquired disorders will require highly specific gene silencing. To ensure co-expression of a therapeutic transgene and a small interfering RNA (siRNA), we hypothesized that a promoter-less small hairpin RNA (shRNA) embedded within an intron could yield siRNA in tissue-specific fashion and thus achieve regulated RNAi. We demonstrate here that γ-globin expression and erythroid-specific siRNA generation can be achieved in mammalian cells, including human CD34+ cells. The shRNA was encoded under the transcriptional control of the human β-globin promoter, a prototypic tissue-specific Pol II promoter, and positioned at two different sites in the second intron or in the 5′-UTR of a recombinant human γ-globin gene. Three different genes were targeted in mouse erythroleukemia (MEL) cells, green fluorescent protein (EGFP), human sickle β-globin (β S) and endogenous mouse β-gobin. When cloned immediately upstream of the branch point, the siRNA was efficiently generated without altering γ-globin mRNA expression and processing, suggesting that hairpin positioning near the branch point is not detrimental to RNA splicing. When cloned near the 5′-end of the intron, the siRNA was structurally impaired, and the γ-globin mRNA levels greatly diminished. This strong effect of shRNA positioning is consistent with a quality control pathway of gene transcription, whereby introns harboring dsRNA stem loops are degraded if splicing is altered. The strong induction of interferon type I genes associated with the latter position but not the former correlated with the formation of small shRNA degradation products. Positioning of the shRNA in the 5′-UTR did not induce major interferon responses but severely compromised γ-globin expression. To further validate these findings in a clinically relevant model, we engineered an RNAi lentiviral vector in which the human sickle β-globin specific (β S) siRNA is embedded the second intron of a recombinant γ-globin gene. Following transduction of CD34+ cells from patients with sickle cell disease, γ-globin transgene expression was induced upon erythroid differentiation concomitant with a dramatic decrease of the β S transcripts. These findings fully support the principle of synergistic gene delivery and lariat-encoded RNAi in human CD34+ cells, demonstrating the feasibility of using lariat-embedded siRNA to potentiate globin gene transfer by reducing competition from endogenous β S globin chains. Importantly, a moderate decrease in β S expression may substantially improve SCD and abrogate the need for high level expression of the vector-encoded globin gene. This approach to regulate RNAi may find broad applicability in a wide range of disorders where the concomitant expression of a transgene and RNAi will enhance treatment safety and/or efficacy.

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