We have begun to make a transgenic pig model for sickle cell disease (SS disease) to facilitate studies for sickle cell pathology and evaluations of new drug and gene therapies. Previous attempts to make transgenic models using the mouse have failed to produce the clinical symptomatology of SS disease, in spite of the production, in some cases, of significant amounts of human sickle hemoglobin (HbS) and evidence for intracellular polymerization of hemoglobin. The failure of the transgenic mice to develop significant sickle cell disease pathology appears to be due to differences in the vascular anatomy and physiology between mice and humans. On the other hand, the pig provides an excellent model for human circulatory physiology, significantly, for our purposes, evincing cognate responses to vaso-active compounds, similarities in capillary bed density, cerebrovascular diseases and certain red cell structural properties. The technology to produce transgenic farm (full-size) pigs is also well established. To produce transgenic animals with high level globin transgene expression, a genetic regulatory region called by beta-globin locus control region (LCR), usually located upstream of the beta-globin gene cluster, has been shown to be required in cis. We have prepared DNA constructions containing the functional portions of the human beta-globin LCR (mini-LCR) linked to the human alpha-2-globin gene or a modified human sickle beta-globin gene with additional mutations to promote polymerization (beta SAD, containing additional mutations Antilles and D Punjab) for microinjection into the pigs. At the same time, to facilitate the maintenance and clinical analysis of the transgenic pigs we have begun to develop the NIH--minipig as a transgenic system. Using a regimen of purified FSH, significant superovulation has been obtained (40) eggs/pig). Following breeding, fertilize eggs have been recovered and procedures for microinjection, surgical reimplantation and maintenance of pregnancy are being developed to extend the transgenic technology to this more conveniently-sized pig variety. Once established, the transgenic pig model that produces high levels of human sickle hemoglobin and demonstrates SS disease pathology, would greatly accelerate our efforts to understand and treat the disease.
