Therapeutic Hemoglobin Levels after Gene Transfer in β-Thalassemia Mice and in Hematopoietic Cells of β-Thalassemia and Sickle Cells Disease Patients

Abstract

Preclinical and clinical studies demonstrate the feasibility of treating β-thalassemia and Sickle Cell Disease (SCD) by lentiviral-mediated transfer of the human β-globin gene. However, previous studies have not addressed whether the ability of lentiviral vectors to increase hemoglobin synthesis might vary in different patients. We generated lentiviral vectors carrying the human β-globin gene with and without an ankyrin insulator and compared their ability to induce hemoglobin synthesis in vitro and in thalassemic mice. We found that insertion of an ankyrin insulator leads to higher, potentially therapeutic levels of human β-globin through a novel mechanism that links the rate of transcription of the transgenic β-globin mRNA during erythroid differentiation with polysomal binding and efficient translation, as reported here for the first time. We also established a preclinical assay to test the ability of this novel vector to synthesize adult hemoglobin in erythroid precursors and in CD34 + cells isolated from patients affected by β-thalassemia and SCD. Among the thalassemic patients, we identified a subset of specimens in which hemoglobin production can be achieved using fewer copies of the vector integrated than in others. In SCD specimens the treatment with AnkT9W ameliorates erythropoiesis by increasing adult hemoglobin (Hb A) and concurrently reducing the sickling tetramer (Hb S). Our results suggest two major findings. First, we discovered that for the purpose of expressing the β-globin gene the ankyrin element is particularly suitable. Second, our analysis of a large group of specimens from β-thalassemic and SCD patients indicates that clinical trials could benefit from a simple test to predict the relationship between the number of vector copies integrated and the total amount of hemoglobin produced in the erythroid cells of prospective patients. This approach would provide vital information to select the best candidates for these clinical trials, before patients undergo myeloablation and bone marrow transplant.

-thalassemias: paradigmatic diseases for scientific discoveries and development of innovative therapies

“…Over recent years, the techniques and tools to achieve transfer of a curative b-globin gene using lentiviral vectors have been significantly improved and have proved to be curative in several animal models for b-thalassemia. 163,164 As a result, clinical trials are in progress and several patients seemed to have been successfully treated with this approach. These encouraging results are now invigorating the field of gene transfer and cellular therapies.…”

Insulated Foamy Viral Vectors

“…Previous studies have identified problems with the retention of repeated elements within retroviruses as well as retaining a tandem repeat of the 250-bp cHS4 core. [55][56][57] To evaluate the fidelity of transfer of insulators during vector production and transduction a shuttle vector rescue (SVR) approach was used to isolate integrated vector proviruses and sequence the LTRs (Fig. 4a and b).…”

β-globin gene transfer to human bone marrow for sickle cell disease

“…These limitations have been overcome by embedding the exons encoding human γ-globin within the human β-globin gene 5′ promoter and 3′ enhancer elements (20,27,28). Breda et al (29) used an LV vector encoding the human hemoglobin (HBB) gene to increase the expression of normal HbA in CD34 + -derived erythroid cells from SCD patients; however, the expression level needed when the HBB gene is used would be higher than would be required for HBG1/2 gene expression to achieve therapeutic benefits in SCD patients.…”