Overcoming the immune response to permit ex vivo gene therapy for spine fusion with human type 5 adenoviral delivery of the LIM mineralization protein-1 cDNA.
METHODS
An animal study in immune competent rabbits and athymic rats was conducted.


OBJECTIVE
To develop an animal model for simulation of previous human Type 5 adenovirus (Ad5) exposure, to determine the impact of adenoviral pre-exposure on spine fusion induced with ex vivo Ad5-LMP-1, and to test strategies for overcoming any potential immune response.


BACKGROUND
Cells transduced with adenovirus containing the osteoinductive LMP-1 cDNA (Ad5-LMP-1) can induce spine fusion in rabbits. Because up to 80% of the human population has been exposed to adenovirus, immune responses to the vector may limit this strategy in humans. Few studies have modeled previous adenoviral exposure and tested strategies to circumvent it.


METHODS
Adult New Zealand white rabbits were injected with 10 or 10 viral particles of Ad5-LacZ. At 4 or 16 weeks after Ad5 injection, autologous buffy coats were prepared from peripheral blood, and 4 million cells per side were infected ex vivo for 10 minutes with Ad5-LMP-1 (multiplicity of infection = 4). Cells were implanted on a collagen matrix instead of an autograft for posterolateral lumbar arthrodesis. Unimmunized rabbits served as control subjects. Additional immunized rabbits underwent arthrodesis at 4 weeks with increased cell number (10 million) and viral dose (multiplicity of infection = 10), or with both parameters increased. The rabbits were killed at 4 weeks, and the spines were assessed by palpation and radiograph. A parallel study was performed in athymic rats using immunized rabbits for the donor cells.


RESULTS
All the unimmunized rabbits had solid spine fusions. None of the rabbits arthrodesed 4 weeks after Ad5 pre-exposure achieved fusion. At 4 weeks after Ad5 exposure, increasing the multiplicity of infection to 10 did not overcome the immune response (0/3 fused), but increasing the cell number to 10 million (2/3 fused) or increasing both cell number and multiplicity of infection (3/3 fused) did overcome the immune effects. Delaying arthrodesis until 16 weeks after Ad5 pre-exposure also overcame the immune response (3/3 fused). Similar results were seen in the athymic rat ectopic implant model, suggesting that the immune effect was mediated by humoral antibodies rather than a T-cell response.


CONCLUSIONS
Two model systems were developed that simulate previous exposure to human Ad5 and could separate the cellular and humoral components of the response. There was a dose-dependent inhibition of ex vivo Ad5-LMP-1 gene transfer to cells from animals previously exposed to human Ad5. Data suggested that the inhibition of Ad5 infection was caused by humoral antibodies rather than a T-cell-based response. Minor modifications in the gene transfer protocol, such as doubling the viral dose or number of cells infected, or increasing the infection time, could overcome the immune response for an ex vivo approach.
Kim
Hak-Sun
HS
Department of Orthopaedic Surgery, Emory Spine Center, Decatur, GA 30033, USA.
Viggeswarapu
Manjula
M
Boden
Scott D
SD
Liu
Yunshan
Y
Hair
Gregory A
GA
Louis-Ugbo
John
J
Murakami
Hideki
H
Minamide
Akihito
A
Suh
Daniel Y
DY
Titus
Louisa
L
D048868:Adaptor Proteins, Signal Transducing; D000256:Adenoviridae / Q000235:genetics / Q000276:immunology*; D000818:Animals; D000914:Antibodies, Viral / Q000097:blood; D000917:Antibody Formation; D002113:Calcification, Physiologic / Q000187:drug effects; D002352:Carrier Proteins / Q000008:administration & dosage* / Q000096:biosynthesis / Q000235:genetics; D002460:Cell Line; D003598:Cytoskeletal Proteins; D018076:DNA, Complementary / Q000008:administration & dosage / Q000276:immunology; D004195:Disease Models, Animal; D004306:Dose-Response Relationship, Immunologic; D005260:Female; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000008:administration & dosage / Q000037:antagonists & inhibitors / Q000276:immunology*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D060588:LIM Domain Proteins; D008161:Lumbosacral Region; D011817:Rabbits; D011859:Radiography; D051381:Rats; D011923:Rats, Nude; D013123:Spinal Fusion / Q000379:methods*; D013131:Spine / Q000000981:diagnostic imaging / Q000601:surgery; D016896:Treatment Outcome
D048868:Adaptor Proteins, Signal Transducing; D000914:Antibodies, Viral; D002352:Carrier Proteins; D003598:Cytoskeletal Proteins; D018076:DNA, Complementary; D047908:Intracellular Signaling Peptides and Proteins; D060588:LIM Domain Proteins; C484380:PDLIM7 protein, human