[Development of rAAV2-GAD-Ig for IDDM gene therapy].
OBJECTIVE
To develop rAAV2 containing cDNA of GAD-Ig fusion gene [GAD-Ig fusion gene is constructed by inserting glutamic acid decarboxylase(GAD) into N-terminal of murine IgG3 heavy chain(Ig)], and to study whether rAAV2 mediated GAD-Ig fusion gene expression could induce specific tolerance in IDDM animal model-nonobese diabetic (NOD) mice.


METHODS
GAD-Ig cDNA was amplified by PCR from the plasmid BSSK-GAD-Ig and inserted into an eukaryotic expression plasmid pSNAV to construct a recombinant expression plasmid pSNAV/GAD-Ig. Plasmid pSNAV-GAD-Ig was then transfected into the rAAV2 packaging cell-BHK-21 cells using LipofectAMINE TM 2000 to produce rAAV-GAD-Ig. Target gene expression in BHK-21 cells infected by rAAV-GAD-Ig was confirmed by RT-PCR, Western blot and ELISA respectively. Then rAAV-GAD-Ig was injected into intramuscularly NOD mice. Antigen-specific tolerance in NOD mice was detected by specific lymphocytic proliferation reaction to GAD antigen.


RESULTS
GAD-Ig gene was inserted into the genome of target cells. Target cells transfected by rAAV2-GAD-Ig could secret GAD-Ig. rAAV2-GAD-Ig induced specific tolerance in NOD mice.


CONCLUSIONS
rAAV2-GAD-Ig was obtained, which can be used in IDDM gene therapy in NOD mice.
Wang
Ren-Xi
RX
Laboratory of Molecular Immunology, Basic Medical Institute, Academy of Military Medical Sciences, Beijing 100850, China.
Wang
Jian-An
JA
Song
Lun
L
Han
Gen-Cheng
GC
Shen
Bei-Fen
BF
Wu
Xiao-Bing
XB
Li
Yan
Y
D000818:Animals; D019677:Artificial Gene Fusion; D002460:Cell Line; D049109:Cell Proliferation; D004274:DNA, Recombinant / Q000235:genetics*; D000229:Dependovirus / Q000235:genetics*; D003922:Diabetes Mellitus, Type 1 / Q000235:genetics / Q000276:immunology / Q000628:therapy*; D015870:Gene Expression; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000235:genetics; D005968:Glutamate Decarboxylase / Q000235:genetics* / Q000276:immunology; D007108:Immune Tolerance; D007074:Immunoglobulin G / Q000235:genetics* / Q000276:immunology; D007143:Immunoglobulin Heavy Chains / Q000235:genetics* / Q000276:immunology; D008214:Lymphocytes / Q000166:cytology / Q000276:immunology; D051379:Mice; D016688:Mice, Inbred NOD; D016133:Polymerase Chain Reaction; D011506:Proteins / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism
D004274:DNA, Recombinant; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D011506:Proteins; D012333:RNA, Messenger; D005968:Glutamate Decarboxylase