RETROVIRAL TRANSFER OF ANKYRIN FOR SPHEROCYTOSIS

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The current challenges in gene therapy with the hematopoietic stem cell as target include achievement of high transduction efficiency, long-term engraftment of transduced cells, and long-term expression of the transgene. Toward development o methods to achieve these aims, we plan to study stem cell transduction by a retroviral vector containing the ankyrin gene as a model for gene therapy and correction of hereditary hemolytic anemias. We-will use the nb/nb mouse as our hemolytic anemia model and study the impact of stem cell cycle status and low dose (100 cGy) host irradiation (minimal myeloablation) on engraftment of transduced cells. Hereditary hemolytic anemias, including thalassemia and sickle cell anemia, affect large populations worldwide, and result in significant morbidity and reduced survival. There are several naturally occurring inherited hemolytic anemias in mice which are analogous to the human disorder, hereditary spherocytosis. One such mutant, the nb/nb mouse, exhibits marked deficiency in ankyrin, a 210 kDa protein that anchors the red cell membrane skeleton to the lipid bilayer. This application proposes to transfer by retroviral vector the cDNA for normal ankyrin to marrow progenitor cells from nh/nb mice to correct the erythrocyte defect and improve the anemia. The murine/human hybrid cDNA consists of the human ankyrin gene promoter, most of the coding sequence of the murine domains for band 3 and spectrin binding, and the alternatively spliced (band 2.2) version of the human regulatory domain. The following objectives will be pursued: 1) to compare expression obtained with pG1-Ank to pG1-Ank/rev that contain ankyrin cDNA in the forward or reverse orientations between the LTRs, 2) to examine differentiating erythroid cells derived from both normal and nb/nb transduced marrow progenitors, 3) to engraft the transduced marrow progenitors in minimally myeloablated normal recipients with modification of the cytokine incubation time to optimize engraftment, and 4) to transduce bone marrow cells from the nb/nb mouse and engraft these cells in minimally myeloablated nh/nb recipients to improve the hemolytic anemia. The methods developed, including insertion of the cDNA encoding a large protein, the use of non-myeloablative procedures for transplant of genetic diseases, the ability to incubate stem cells in cytokines yet preserve engraftment, and the achievement of tissue-specific gene expression have direct relevance to the development of gene therapy approaches to inherited human disorders.