Keywords : Gene therapy, Sickle Cell Anemia, Stem cells, lentivirus, β-globin
Project type: GBE-492 Genetic Engineering Project
I. INTRODUCTION, BACKGROUND and AIM
Sickle cell anemia is a devastating genetic disease which causes premature death. Sickle cell disease (SCD) is caused due to single amino acid change (glutamine to valine at position 6) in β chain of hemoglobin molecule, which causes the hemoglobin molecules to aggregate. These aggregated hemoglobin molecules make the red blood cells (RBCs) stiff and deformed (sickle shaped). These deformed cells block the normal blood flow. Although this disease is well-characterized, but no long-term treatment 1 is still available for this disease.
The discovery of the sickle cell-anemia in 1949 by Linus Pauling and his team made them call it as “a molecular disease”. There is a disappointing lag in the various forms of therapy and applied knowledge to find treatment for the disease1. Scientific community is trying to find a cure for genetic diseases, which could be possible by genetic manipulation or gene therapy. 2,3 This involves either editing a faulty gene, or inserting a functional gene. Hence gene therapy could be a potential curative therapy for diseases (such as SCD) which are caused by single mutation.
Hanna and colleagues have used gene therapy for SCD treatment in mice.4Another important contribution in this field was the research by Romero and colleagues who have used transfer and consistent expression of anti-sickling β-globin gene in human SCD bone marrow CD34+ progenitor cells.5 Previously an attempt was made to cure the sickle cell disease and effectiveness of a lentivirus based transduction method was demonstrated in a mouse model. An important feature of this method is that it avoids GMCSF induced granulocytosis which results in severe vascular crisis and death.6
The aim of this project is to use a lentiviral based antisickling β-globin gene transduction of purified human hematopoietic stem cells (hHSC) 7 to treat human sickle cell anemia.
II. REVIEW OF RELATED LITERATURE
Sickle cell anemia is characterized by the sickle shaped hemoglobin blood cells. These cells greatly differ from the normal cells such that they are more rigid and cause blocking in the path of the blood. Sickle cells also have shorter life span wit 10 to 20 days which is far from the normal cycle of 120 days. The disorder affects the circulation of oxygen of blood from the lungs to the entire part of the body. When the sickle shaped cells block the blood vessels, it causes episodes of pain in the body. The symptoms for the disease include delayed growth, jaundice, and strokes. Sickle Cell Anemia is a genetic disease that has no known cure. It affects around 300,000 individuals worldwide. It is an autosomal recessive disease with molecular basis of displacing the glutamic acid and replaced with valine in the amino acid beta-globin structure. In effect, the deoxygenated hemoglobin changes its conformation.
In the molecular level, humans receive two types of genes from their parents, that is the normal beta genes “A” and abnormal genes of “S”. Genes inherited from parents can be AA, AS, SC, and SS. AS are healthy (phenotype) people but carriers of the disease. Sickle cell disease develops at the combinations of SC and SS. Children born with parents with the genotype AS will have a probability of 25% risk of getting the disease. 11
The sickle cell anemia is one of the first identified and understood disease in the molecular level. Despite this fact, the long term treatment and cure for this disease is still unknown. There are measures for the fetal hemoglobin induction for the inhibition of the polymer formation. Other measures seek for the infection prevention and treatment, measures for pain control, and the complications of the surgeries. In the current stream, there are several groups that utilize the lentiviral vectors for gene transfer of globin to the mononuclear cells of the bone marrow. This practice resulted to the beta-thalassemia and sickle cell anemia correction among the mouse models for human disease. 6
III. RESEARCH QUESTION
Can lentivirus based antisickling β-globin gene transduction of unmobilized6, purified human hematopoietic stem cells lead to cure human sickle cell disease?
III. MATERIALS and METHODS
Antisickling lentiviral vector (lenti-βAS3) will be designed based on an earlier work of Levasseur and coworkers.6 The human bone marrow cells will be obtained from the femurs of volunteer patients using standard biopsy procedure. They will be stained with a biotinylated specific antibody against specific antigen of hHSC followed by an antibiotin magnetic bead and fluorescein isothiocyanate (FITC)-conjugated streptavidin. The cells will then be isolated on a magnetic-activated cell sorter. For transduction and transplantation of stem cells i will follow the procedure described by Levasseur and coworkers.6 The sorted stem cells will be infected at an MOI of 30 for 4 hours. Lenti βAS3-transduced stem cells will be injected into anesthetized patients that were irradiated. Hematopoietic reconstitution with transduced HSCs will be monitored by analyzing the replacement of sickle hemoglobin polypeptides with normal human hemoglobin polypeptides using HPLC. The lenti/βAS3 vector copy number in transplanted human will be determined by an allele-specific assay as described by Levasseur and coworkers.6 Hemoglobins will be separated by isoelectric focusing (IEF) of hemolysates. Hemoglobin bands will then be quantitated using densitometry and will be further confirmed using tryptic digestion and analysis by mass spectrometry. Analysis of mRNA level will be performed on peripheral blood reticulocytes isolated from patients receiving transplant with the lenti-βAS3 vector. Reticulocytes will be stained with thiazole orange, purified on a FACScan flow cytometer and will be plated using limiting dilution, which will be used for RNA isolation and subsequent RT-PCR analysis.
Hematologic indices and histopathology will be performed as per standard procedure.
IV. INNOVATION SIDES
Since bone marrow mobilization protocols lead to a rapid increase in leucopoiesis, which ultimately can result in death, they are not safe for human sickle cell patients and are associated with vascular crisis. In order to address this, the test protocol, originally demonstrated in mice, that do not include mobilization of bone marrow or cytokine stimulation of stem cells for human will be implemented. 8,9 The mouse made as models for the discoveries of this human disease have been tested and experimented. The recent stem cell procedure where the lentiviral vectors were utilized for the globin gene transfer going to the mononuclear cells of the bone marrow has resulted to correction of the sickle cell anemia among the mouse models. This innovative study is a great advance in the field of gene therapy for the hemoglobinopathies. However, the transduction procedures are not used in the humans yet. 6
The equivalent treatment for humans is when the hematopoietic stem cells or HSCs will be mobilized along with the granulocyte colony stimulating factor (G-CSF), or even te other stem cell factors. The mobilization of the bone marrow HSCs going into the peripheral blood is effective as they are successfully engrafted to their recipients. However, the other cells G-CSF can not be done with the same procedure as the HSCs for patients with sickle cell disease. 6There was a reported death when the G-CSF was mobilized with the patient with age of 47. The cases for those patients that were using mobilized G-SCF were more severe.9
V. IMPLEMENTATION PLAN and DISCUSSIONS
In order to achieve our aim, I will obtain all the mandatory regulatory clearances such as biosafety and ethical clearance before the start of the work. And I will first like to produce the antisickling lentiviral vector for human stem cell transduction. The lentivirus based vector system provides an efficient delivery system for delivering genes in animals and human. Next, I will obtain and prepare human bone marrow cells from the volunteered patients and use them to sort the hHSC. The hHSC preparation will next be subjected to the stem cell transduction with the earlier developed antisickling lentiviral vector for human and the transuded cell will be transplanted autologously in patients. The success of this stem cell therapy without causing leukopoiesis will be monitored using globin protein analysis, single cell mRNA analysis, hematologic indices and histopathology. We expect that the sickle cell gene therapy demonstrated in mouse models 10 will work effectively for humans.
VI. REFERENCES
Bunn, H. Franklin. "Pathogenesis and treatment of sickle cell disease." New England Journal of Medicine 337.11 (1997): 762-769.
Ye, Lin, et al. "Induced pluripotent stem cells offer new approach to therapy in thalassemia and sickle cell anemia and option in prenatal diagnosis in genetic diseases." Proceedings of the National Academy of Sciences 106.24 (2009): 9826-9830.
Yu, Junying, et al. "Induced pluripotent stem cell lines derived from human somatic cells." Science 318.5858 (2007): 1917-1920.
Hanna, Jacob, et al. "Treatment of sickle cell anemia mouse model with iPS cells generated from autologous skin." Science 318.5858 (2007): 1920-1923.
Romero, Zulema, et al. "β-globin gene transfer to human bone marrow for sickle cell disease." The Journal of clinical investigation 123.8 (2013): 3317-3330.
Levasseur, Dana N., et al. "Correction of a mouse model of sickle cell disease: lentiviral/antisickling β-globin gene transduction of unmobilized, purified hematopoietic stem cells." Blood 102.13 (2003): 4312-4319.
Larochelle, Andre, et al. "Identification of primitive human hematopoietic cells capable of repopulating NOD/SCID mouse bone marrow: implications for gene therapy." Nature medicine 2.12 (1996): 1329-1337.
Abboud M, Laver J, Blau CA. Granulocytosis causing sickle-cell crisis [letter]. Lancet.1998;351: 959.
Adler BK, Salzman DE, Carabasi MH, Vaughan WP, Reddy VV, Prchal JT. Fatal sickle cell crisis after granulocyte colony-stimulating factor administration. Blood. 2001;97: 3313-3314.
Pawliuk, Robert, et al. "Correction of sickle cell disease in transgenic mouse models by gene therapy." Science 294.5550 (2001): 2368-2371.
11 .Gabriel, A. and Przybylski, J. (2010). Sickle Cell Anemia: A Look a Global Haplotype
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