Editing the Sickle Cell Disease Mutation in Human Hematopoietic Stem Cells: Comparison of Endonucleases and Homologous Donor Templates

Abstract Gene therapy and stem cell transplantation are attractive potential therapies for sickle cell disease (SCD). Previous studies have shown that the sickle environment is highly enriched for reactive oxygen species (ROS), but have not addressed whether or not the increased ROS may alter the bone marrow (BM) microenvironment or affect stem cell function. Using the Berkeley sickle mouse model, we examined the effects of sickle cell disease on hematopoietic stem cell function and the bone marrow microenvironment. We transplanted C57BL/6 (control) BM into C57BL/6 and homozygous sickle mice. Recipients received 2 × 106 BM cells and a conditioning regimen consisting of busulfan, anti-asialo GM1, and co-stimulation blockade (anti-CD40L and CTLA4-Ig). Following transplantation, sickle mice demonstrated increased donor cell engraftment in the peripheral blood compared to normal mice (58.3% vs. 33.1%, respectively). Similarly, BMT in a fully allogeneic system also resulted in enhanced engraftment in sickle recipients. Next we analyzed whether or not engraftment defects exist within the BM stem cell population of sickle mice. In vitro colony forming assays showed a significant decrease in progenitor colony formation in sickle compared to control BM. By flow cytometry, we determined that there was a significant decrease in the KSL (c-Kit+, Sca-1+, Lineage−) progenitor population within the BM of sickle mice. Cell cycle analysis of the KSL population demonstrated that significantly fewer sickle KSL cells were in G0 phase compared to control, suggesting that there are fewer quiescent stem cells in the BM of sickle mice. To assess the potential role of ROS and glutathione depletion in sickle mice, we tested the engraftment efficiency of KSL cells from untreated and n-acetyl-cysteine (NAC) treated control, hemizygous sickle (hemi), and sickle mice in a competitive repopulation experiment. Peripheral chimerism showed an engraftment defect from both hemizygous and homozygous sickle mice such that control KSL cells engrafted > hemi > sickle at a ratio of 1 : 0.4 : 0.25. Treatment with NAC for four months prior to transplantation partially restored KSL engraftment (control : hemi : sickle; 1 : 0.97 : 0.56 ). We have demonstrated that congenic and allogeneic BMT into sickle mice result in increased donor cell engraftment in the sickle recipients. Both the decreased number of KSL cells and the decreased percentage of quiescent KSL cells in the sickle mice indicate that more stem cells in the transgenic sickle mouse model are mobilized from the BM environment. The engraftment defect of sickle KSL cells that was partially ameliorated by NAC treatment suggests that an altered redox environment in sickle mice may contribute to the engraftment deficiencies that we observed.

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A Highly Sensitive Assessment Tool for the Gene Editing Efficiency of Sickle Cell Disease Hemoglobin Beta Gene

Blood ◽

10.1182/blood.v126.23.5557.5557 ◽

2015 ◽

Vol 126 (23) ◽

pp. 5557-5557

Author(s):

Mandula Borjigin ◽

Eric Brian Kmiec ◽

Rigumula Wu

Keyword(s):

Stem Cells ◽

Sickle Cell Disease ◽

Point Mutation ◽

Sickle Cell ◽

Gel Electrophoresis ◽

Gene Editing ◽

Cell Disease ◽

Transcription Activator ◽

Hematopoietic Stem ◽

Editing Efficiency

Abstract In sickle cell disease, a single point mutation in hemoglobin β gene (HBB) results in the substitution of valine for glutamic acid at position 6 of the β globin protein sequence, causing the deformation of red blood cells into a sickle (or crescent) shape. With the development of powerful gene editing tools, scientists are initiating the correction of the point mutation of HBB gene in CD34+ hematopoietic stem cells and induced pluripotent stem cells. Although the results are very exciting, the evaluation method of the gene editing is primitive. Currently, the modification at the mutation site is identified and quantified using Restriction Fragment Length Polymorphism (RFLP), which involves PCR amplification, restriction enzyme digestion and gel electrophoresis. The accuracy of the gene editing efficiency depends heavily on the quantification of the DNA bands in the gel images, which is inherently imprecise. We have developed a novel technique to quantify the correction efficiency of HBB gene editing using a fluorescence tagging of the edited DNA sequence. This method provides excellent sensitivity and accuracy, and saves time and labor, eliminating a process of gel electrophoresis. We demonstrate the assessment of gene editing in HBB of K562 cells, in which the wild type HBB (βA gene) is converted to mutant βs using the gene editing tools (i.e. Transcription Activator-Like Effector Nucleases (TALENs) and single-stranded oligo deoxynucleotides (ssODNs)). We present limited information here due to the sensitivity of the intellectual property, but will discuss in detail the experimental procedures and data at the American Society of Hematology meeting. Disclosures No relevant conflicts of interest to declare.

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

Blood ◽

10.1182/blood.v118.21.3119.3119 ◽

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.

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