RECOMBINANT HUMAN HEMOGLOBINS DESIGNED FOR GENE-THERAPY OF SICKLE-CELL DISEASE

Two human hemoglobins designed to inhibit the polymerization of sickle hemoglobin (Db S; alpha(2) beta(2)(S)) have been produced. Mutations that disrupt the ability of Hb S to form polymers were introduced into the normal human beta-globin gene by site-specific mutagenesis. These mutations affect the axial and lateral contacts in the sickle fiber. The recombinant hemoglobin designated anti-sickling hemoglobin 1 (Hb A S1) contains the mutations beta 22 glutamic acid to alanine and beta 8 0 asparagine to lysine. Hb AS2 has the same beta 22 glutamic acid to a lanine mutation combined with beta 87 threonine to glutamine. Human al pha- and beta(AS)-globin genes were separately fused downstream of bet a-globin locus control region sequences and these constructs were coin jected into fertilized mouse eggs. Transgenic mouse lines that synthes ize high levels of each anti-sickling hemoglobin were established and anti-sickling hemoglobins were purified from hemolysates and character ized. Both AS hemoglobins bind oxygen cooperatively and the oxygen aff inities of these molecules are in the normal range. Delay time experim ents demonstrate that Hb AS2 is a potent inhibitor of Hb S polymerizat ion; therefore, locus control region beta(AS2)-globin gene constructs may be suitable for future gene therapy of sickle cell disease.