Abstract
Aim: To study the effect of injecting hematopoietic stem cells containing the preproinsulin gene II (rI<sub>2</sub>) via recombinant adeno-associated virus (rAAV) into normal and streptozotocin-diabetic rats. Methods:rI<sub>2</sub>was transfected into rat hematopoietic stem cells using rAAV vector. Stem cells were injected by intravenous route into normal and STZ-induced diabetic rats to study blood sugar and expression of rI<sub>2</sub>in various tissues. The pLP-1 recombinant plasmid containing rI<sub>2</sub>(vLP-1) was engineered as previously described. Bone marrow from female Wistar-Furth rats was enriched for stem cells by using plastic adherence and monoclonal antirat CD3 and CD45 RA to deplete T and B cells. The remaining cells were exposed to vLP-1 (multiplicity of infection MOI = 50:1 or 100:1) for 2 h. Approximately ten million exposed stem cells were injected by intravenous route into each animal; there were four groups: normal animals at MOI 50:1 (group 1) or MOI 100:1 (group 2); group 3 animals (n = 9) were streptozotocin-induced diabetic animals at MOI 100:1. Animals that showed reversal of diabetes from group 3 were sacrificed for study of gene expression at weeks 1, 2, and 6, respectively. Control diabetic animals did not receive stem cells or virus constituted group 4. Expression of rI<sub>2</sub>was analyzed by RT-PCR and Southern analyses. Results: Despite introduction of insulin gene, groups 1 and 2 had blood sugar concentrations that remained within normal levels, while 3 of 9 animals in group 3 showed reversal of diabetes; using RT-PCR, group 1 expressed rI<sub>2</sub>in liver, spleen, thymus, brain, and heart at week 1 only. In group 2, rI<sub>2</sub> was seen in the thymus up to 6 weeks; in diabetic animals (group 3) rI<sub>2</sub> was seen in liver, bone marrow, spleen, thymus, and peripheral blood lymphocytes at week 2 and in thymus and lymphocytes at week 6. Conclusions: We have shown that (1) rAAV is a useful vector for transferring rI<sub>2</sub>into rat hematopoietic stem cells; (2) normal animals remained euglycemic after injection of stem cells containing rI<sub>2 </sub>despite identification in various tissues suggesting autoregulation, and (3) short-term reversal of diabetes was achieved in some animals by injection of stem cells containing rI<sub>2</sub>.
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