Sl. Mccune et al., RECOMBINANT HUMAN HEMOGLOBINS DESIGNED FOR GENE-THERAPY OF SICKLE-CELL DISEASE, Proceedings of the National Academy of Sciences of the United Statesof America, 91(21), 1994, pp. 9852-9856
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.