Evaluation of optimal expression cassette in retrovirus vector for β-thalassemia gene therapy

Trials of retroviral vector-mediated human β-globin gene transfer were hampered by low titers, unstable vector transmission, and low-level expression of transferred gene. With the goal of optimizing the retrovirally encoded human β-globin gene expression cassette for gene therapy of β-thalassemia, we generated 3 series of vector constructs (a total of 12 constructs) and investigated the effects of the proximal promoter, 3′-enhancer, and derivatives from the β-locus control region or α-major regulatory element on virus titer, vector transmission stability, and gene expression. The virus titers for 9 of the 12 vector constructs ranged between 2.8×104 cfu/mL and 1.0×106 cfu/mL. We found that proviral DNA was intact in most G418-resistant murine erythroleukemia (MEL) cell clones for 5 vector constructs, while obvious genetic instability was observed for 4 other vector constructs. MEL cells harboring the intact provirus were induced to differentiate, and human β-globin gene expression was analyzed with RNase protection assay. The percentage of human β-globin transcript relative to endogenous murine α-globin transcript were 101.8±64.3% (n=10), 40.1±28.7% (n=4), 31.1±31.9% (n=12), 52.4±11.2% (n=12), and 53.6±8.6% (n=12) for the 5 constructs, respectively, demonstrating the development of optimized retroviral vectors for β-globin gene therapy with murine erythroid cell lines as a model. Unexpectedly, we also documented that the point mutation 8700(C→T) in DNase I hypersensitive site 2 (HS2) core fragment might contribute to low-level expression of the human β-globin gene, based on a comparison of results from transfected and transduced MEL cells and sequence analysis of proviral DNA.