Production of genetically stable high-titer retroviral vectors that carry a human γ-globin gene under the control of the α-globin locus control region

The ability to generate stable high-titer vectors that give rise to high levels of expression of transduced globin genes in erythroid cells is a prerequisite for effective retroviral-mediated globin gene therapy. The human β-globin gene with its immediate flanking sequences does not contain all the regulatory elements necessary for regulated high-level and position- independent expression in erythroid cells. The regulatory element known as the β-globin locus control region (βLCR) can provide a linked β-globin gene with these properties. However, addition of βLCR sequences to a retrovirus carrying a β-globin gene increases its genetic instability. We have developed a new generation of retroviral vectors in which a human γ- globin gene is placed under the control of the αLCR, the major regulatory element of the α-globin gene cluster. We demonstrate that these retroviruses are genetically stable in producer cell lines and can be produced at high titers that exceed 5 x 10⁶ colony-forming units (CFU)/mL. In addition, we show that the transduced γ-globin gene can be expressed in the adult erythroid environment of mouse erythroleukemia (MEL) cells at a level comparable to that of a single endogenous β(maj)-globin gene. These retroviruses can also transduce primary murine bone marrow progenitor cells as efficiently as retroviruses that carry the neomycin resistance (neo') gene. This new generation of globin retroviral vectors may prove useful for gene therapy of human β-globin gene disorders such as sickle cell disease and β-thalassemia.