Transduction of rat pancreatic islets with pseudotyped adeno-associated virus vectors.
BACKGROUND
Pancreatic islet transplantation is a promising treatment for type I diabetes mellitus, but current immunosuppressive strategies do not consistently provide long-term survival of transplanted islets. We are therefore investigating the use of adeno-associated viruses (AAVs) as gene therapy vectors to transduce rat islets with immunosuppressive genes prior to transplantation into diabetic mice.


RESULTS
We compared the transduction efficiency of AAV2 vectors with an AAV2 capsid (AAV2/2) to AAV2 vectors pseudotyped with AAV5 (AAV2/5), AAV8 (AAV2/8) or bovine adeno-associated virus (BAAV) capsids, or an AAV2 capsid with an insertion of the low density lipoprotein receptor ligand from apolipoprotein E (AAV2apoE), on cultured islets, in the presence of helper adenovirus infection to speed expression of a GFP transgene. Confocal microscopy and flow cytometry were used. The AAV2/5 vector was superior to AAV2/2 and AAV2/8 in rat islets. Flow cytometry indicated AAV2/5-mediated gene expression in approximately 9% of rat islet cells and almost 12% of insulin-positive cells. The AAV2/8 vector had a higher dependence on the helper virus multiplicity of infection than the AAV 2/5 vector. In addition, the BAAV and AAV2apoE vectors were superior to AAV2/2 for transducing rat islets. Rat islets (300 per mouse) transduced with an AAV2/5 vector harboring the immunosuppressive transgene, tgf beta 1, retain the ability to correct hyperglycemia when transplanted into immune-deficient diabetic mice.


CONCLUSIONS
AAV2/5 vectors may therefore be useful for pre-treating donor islets prior to transplantation.
Craig
Anthony T
AT
Laboratory of Molecular and Cellular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland 20892, USA. anthonycraig3@gmail.com
Gavrilova
Oksana
O
Dwyer
Nancy K
NK
Jou
William
W
Pack
Stephanie
S
Liu
Eric
E
Pechhold
Klaus
K
Schmidt
Michael
M
McAlister
Victor J
VJ
Chiorini
John A
JA
Blanchette-Mackie
E Joan
EJ
Harlan
David M
DM
Owens
Roland A
RA
D000818:Animals; D002460:Cell Line; D000229:Dependovirus / Q000235:genetics* / Q000378:metabolism; D003922:Diabetes Mellitus, Type 1 / Q000235:genetics / Q000601:surgery / Q000628:therapy*; D005260:Female; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007515:Islets of Langerhans / Q000378:metabolism / Q000821:virology*; D016381:Islets of Langerhans Transplantation; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D051381:Rats; D017208:Rats, Wistar; D014161:Transduction, Genetic / Q000379:methods*