Proteasome inhibition enhances AAV-mediated transgene expression in human synoviocytes in vitro and in vivo.
To explore the potential applicability of recombinant adeno-associated virus (rAAV) vectors in the treatment of rheumatoid arthritis (RA), primary human fibroblast-like synoviocytes (FLS) derived from patients with RA were infected with rAAV encoding mouse IL-10 under the control of the CMV promoter. Addition of the proteasome inhibitor carbobenzoxy-l-leucyl-l-leucyl-l-leucinal (zLLL) to the cultures dramatically enhanced expression of the IL-10 transgene, in a dose-dependent manner. The increased expression was transient, peaking at 3 days and returning to near baseline by 7 days. The enhancement was observed even when zLLL was added 13 days after infection with rAAV. The effect of zLLL was not specific to either the mIL-10 transgene or the CMV promoter, as similar findings were observed using an rAAV construct encoding alpha1-anti-trypsin under the control of the chick beta-actin promoter or GFP, driven by the CMV promoter. Transgene expression could be repeatedly induced by reexposure to zLLL. Transgene mRNA levels increased in parallel with protein levels. Transgene expression could also be repeatedly induced in vivo by administering zLLL to SCID mice previously injected with rAAV-infected FLS. These data demonstrate that proteasome inhibition can dramatically enhance transgene expression in human RA FLS following infection with rAAV and suggest a possible approach to regulating synovial transgene expression in vivo.
Jennings
Kristi
K
William S. Rowe Division of Rheumatology, Children's Hospital Medical Center, Cincinnati, OH 45229, USA.
Miyamae
Takako
T
Traister
Russell
R
Marinov
Anthony
A
Katakura
Shigeki
S
Sowders
Dawn
D
Trapnell
Bruce
B
Wilson
James M
JM
Gao
Guangping
G
Hirsch
Raphael
R
D000818:Animals; D001172:Arthritis, Rheumatoid / Q000235:genetics / Q000378:metabolism* / Q000628:therapy; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D003587:Cytomegalovirus / Q000235:genetics; D000229:Dependovirus / Q000235:genetics*; D005260:Female; D005786:Gene Expression Regulation; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D016753:Interleukin-10 / Q000235:genetics*; D007976:Leupeptins / Q000494:pharmacology; D051379:Mice; D016513:Mice, SCID; D011401:Promoter Regions, Genetic / Q000235:genetics; D061988:Proteasome Inhibitors; D014176:Protein Biosynthesis / Q000187:drug effects; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013583:Synovial Membrane / Q000166:cytology*; D019076:Transgenes
D015853:Cysteine Proteinase Inhibitors; D007976:Leupeptins; D061988:Proteasome Inhibitors; D012333:RNA, Messenger; D016753:Interleukin-10; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde