The purpose of this project is to produce a transgenic pig model for sickle cell disease. There is no naturally occurring non-human cognate to the disease and the availability of an animal model would greatly enhance our ability to study the disease and develop new therapies. The pig provides a generally excellent model for human circulatory physiology, with certain aspects of similarity to humans of particular relevance to the model such as analogous responses to vaso-active compounds, a similar response to extracellular hemoglobin, and other similarities in capillary bed density, certain red cell structural properties and the occurrence of cerebrovascular diseases. Previous attempts to make a transgenic mouse model for sickle cell disease have not produced satisfactory symptomatology, probably due to differences between murine and human vascular anatomy and physiology. In order to achieve the high levels of human sickle hemoglobin (HbS) necessary to produce the disease in pigs we are utilizing the most efficient construction for the production of normal human hemoglobin in transgenic lull size pigs that has been assembled for synthetic hemoglobin production from the LCR and human alpha and beta genes, and mutagenizing the included adult beta-globin gene to produce the sickle mutation and two additional mutations, Antilles and D Punjab, which promote sickle hemoglobin polymerization. In addition, to facilitate the maintenance and clinical analysis of the transgenic pigs we produce, we are developing a very small strain of pig, the Panepinto miniature swine, as a transgenic system. Using standard protocols developed for the superovulation of full size pigs, we have obtained significant superovulation (up to about 50 ovulations/pig) and recovery of fertilized ova (up to about 20/pig) following natural breeding. Control DNA constructions have been microinjected to develop procedures for the introduction of DNA, surgical reimplantation and maintenance of pregnancy which will be necessary to extend transgenic technology to this more conveniently sized breed. Our ability to understand and treat sickle cell disease should be greatly accelerated by the availability of an accurate transgenic pig model, readily amenable to clinical evaluation.
