Sickle cell anemia (SCA) is characterized by repeated vascular occlusions and hemolysis, primarily resulting from the sickle-shaped RBCs formed under hypoxia. Treatment for the disease is mainly symptomatic. Bone marro_ transplantation, a curative modality, is limited to a few with matched donors and has potential side effects. Increasing expression of the """"""""anti-sickling' 7-globin in the RBCs by long-term administration of hydroxyurea reduces the frequency of sickling events. Gene therapy using the """"""""/-globin gene in hematopoietic stem cells (HSCs) can improve the survival of RBCs derived from the genetically modified HSCs permanently. Gene therapy for hemoglobinopathies with oncoretroviral vectors has suffered from problems of vector instability, low titers and variable expression. With the advent of better vectors, improved gene transfer techniques and a better understanding of stem cell and vector biology, gene therapy is going from the bench to the bedside, in disorders like SCID and hemophilia B. The recently developed, lentiviral vectors transduce the non-dividing HSCs and stably export large genomic fragments required for high-level regulated """"""""globin' gene expression. Self-inactivating (SIN) lentiviral vectors are even more advantageous: the viral long terminal repeat is deleted upon integration into cells, completely inactivating viral transcription, a feature ideal for the expression of a highly lineage-restricted gene, and additionally improves the bio-safety. We have recently shoven 1:- remarkably lineage-specific and long-term expression of GFP and a therapeutic correction of the murine erythropoietic porphyria in primary and secondary mice with SIN-lentiviral vectors. We would like to extend these results and examine the properties of SIN-lentiviral vectors in carrying the human y-globin gene and erythroid regulatory elements for gene transfer into HSCs, resulting in high-level, stable and sustained expression of ,/-globin in RBCs.
The aims of the study are: 1) Develop SIN-lentiviral vectors carrying the human y-globin gene and erythroid regulatory elements, and screen them in MEL cells for stable transmission and high-level expression. 2) Determine the efficacy, lineage-specificity and long term expression of these vectors in transgemc sickle mice. 3) Determine gene transfer and efficacy of these vectors in the RBC progeny of human SCA progenitor cells, using the unique model of human RBC production from normal and SCA progenitors developed in our laboratory. Together, these aims comprise a focussed research to produce sustained and therapeutic levels ofy-globin in human SCA RBCs, and form the basis of future preclinical studies.
Showing the most recent 10 out of 79 publications
