The New Self-Inactivating Lentiviral Vector for Thalassemia Gene Therapy Combining Two HPFH Activating Elements Corrects Human Thalassemic Hematopoietic Stem Cells

Abstract

To address how low titer, variable expression, and gene silencing affect gene therapy vectors for hemoglobinopathies, in a previous study we successfully used the HPFH (hereditary persistence of fetal hemoglobin)-2 enhancer in a series of oncoretroviral vectors. On the basis of these data, we generated a novel insulated selfinactivating (SIN) lentiviral vector, termed GGHI, carrying the A c-globin gene with the -117 HPFH point mutation and the HPFH-2 enhancer and exhibiting a pancellular pattern of A c-globin gene expression in MEL-585 clones. To assess the eventual clinical feasibility of this vector, GGHI was tested on CD34+ hematopoietic stem cells from nonmobilized peripheral blood or bone marrow from 20 patients with b-thalassemia. Our results show that GGHI increased the production of c-globin by 32.9% as measured by high-performance liquid chromatography ( p = 0.001), with a mean vector copy number per cell of 1.1 and a mean transduction efficiency of 40.3%. Transduced populations also exhibited a lower rate of apoptosis and resulted in improvement of erythropoiesis with a higher percentage of orthochromatic erythroblasts. This is the first report of a locus control region (LCR)-free SIN insulated lentiviral vector that can be used to efficiently produce the anticipated therapeutic levels of c-globin protein in the erythroid progeny of primary human thalassemic hematopoietic stem cells in vitro.

Molecular Imaging Studies on CD133+ Hematopoietic Stem Cells From Human Umbilical Cord Blood

“…This approach has also been used in the treatment of some solid tumors, most notably breast cancer, for which its value is still in dispute because of poor accrual in randomized trials and because it takes many years to obtain definitive answers, even from well-designed trials (Joshi et al, 2000). Hematopoietic stem-cell therapy is also being tried in sickle cell disease and thalassemia and recently in progressive multiple sclerosis, systemic scleroderma, severe systemic lupus erythematosus, and rheumatoid arthritis with a poor prognosis (Shichishima et al, 1983;Papanikolaou et al, 2011;Alderucciro et al, 2011).…”

Progresses towards safe and efficient gene therapy vectors

“…They also prevent transgene silencing by the chromosomal position effect [ 185 ]. The chicken hypersensitivity site 4 insulator (cHS4) has been the standard for improving transgene expression in several studies [ 186 ] [ 187 ] [ 188 ] [ 189 ]. This type of insulator enhances vector titers compared to LV vectors containing other types of insulators such as the locus control region [ 187 ].…”

Gene Addition Strategies for β-Thalassemia and Sickle Cell Anemia

“…They saw fetal Hb (HbF) production ranged between 45 and 60% of total Hb, and up to a threefold increase in total Hb content. Papanikolaou in 2012 [59] published a report of a gamma-globin virus without the LCR, but instead containing a gamma-globin promoter with a −117 point mutation associated with HPFH, the HS40 enhancer from the alpha-globin locus, the HPFH-2 enhancer, and the cHS4 insulator. They saw mild improvement of HbF synthesis compared to mock-transduced controls.…”