Reversal of diabetes in the rat by injection of hematopoietic stem cells infected with recombinant adeno-associated virus containing the preproinsulin II gene.
OBJECTIVE
To study the effect of injecting hematopoietic stem cells containing the preproinsulin gene II (rI2) via recombinant adeno-associated virus (rAAV) into normal and streptozotocin-diabetic rats.


METHODS
rI2 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 rI2 in various tissues. The pLP-1 recombinant plasmid containing rI2 (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 rI2 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 rI2 in liver, spleen, thymus, brain, and heart at week 1 only. In group 2, rI2 was seen in the thymus up to 6 weeks; in diabetic animals (group 3) rI2 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 rI2 into rat hematopoietic stem cells; (2) normal animals remained euglycemic after injection of stem cells containing rI2 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 rI2.
Shah
Rita
R
Department of Surgery, Indiana University School of Medicine, Indianapolis, USA.
Jindal
Rahul M
RM
D000818:Animals; D001786:Blood Glucose / Q000378:metabolism; D015139:Blotting, Southern; D000229:Dependovirus / Q000235:genetics*; D003921:Diabetes Mellitus, Experimental / Q000097:blood / Q000628:therapy*; D005260:Female; D015967:Gene Expression Regulation, Viral; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000821:virology*; D007328:Insulin; D008297:Male; D011384:Proinsulin / Q000235:genetics*; D011498:Protein Precursors / Q000235:genetics*; D051381:Rats; D011920:Rats, Inbred WF; D011994:Recombinant Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors
D001786:Blood Glucose; D007328:Insulin; D011498:Protein Precursors; D011994:Recombinant Proteins; C013737:preproinsulin; D011384:Proinsulin