골 형성 유전자 치료 (제 5형 Adenoviral vector를 이용한 Ex Vivo 유전자 치료로 척추 유합 시도시 면역학적 모델)

Abstract

Study-Design: In vivo study to determine the immune effects to adenoviral encoding LMP-1 cDNA in rabbit. Objective: To quantify the immune effect of Ad5-LMP-1 in the rabbit during the therapeutic gene transfer. Summary-of-Literature-Review: One of the major limitations in the use of adenoviral vector for gene therapy is the immune response and it made the poor transduction efficiency when re-administrated. Adenoviral antigen plus those derived from transgene expression in transduced cell contribute to cellular, humoral and non-specific immune resonse constitutes barriers to successful gene therapy. Therefore, the animal immune model will be mandatory to study the immune impact. Materials-and-Method: We i.v. injected Ad5-βGal to total 24 adult NZW rabbits; 1x108, 1x109, 1x1010, 1x1011v.p. to each 6 rabbits allowed them to develop immune response. Six non-immunized animals were used as control Adenovirus antibodies were measured at 0, 4, 8, 16, 20 weeks. Group Ⅰ. 6 control rabbit underwent spinal arthrodesis at 4 weeks (n=3) and 16 weeks (n=3) with 4 million cells and MOI of 4. Group Ⅱ. 6 rabbit underwent spinal arthrodesis at 4 weeks after injection of 108 p.f.u virus (n=3) and 16 weeks (n=3). Group Ⅲ. six 109 immunized rabbits. Group Ⅳ. six 1010 immunized rabbits. Group Ⅴ. six 1011 immunized rabbits, underwent spinal arthrodesis at 4 and 16 weeks after injection. Total anti-Ad Ig and neutralizing antibody titer was measured on the 0, 4, 8, 16, 20 weeks after injection. Results: Group Ⅰ. All 6 non-immunized rabbits had solid spine fusions at 4 and 16 weeks. Group Ⅱ. All 3 immunized rabbits had not spine fusions at 4 weeks and all three had solid spine fusion at 16 weeks. Group Ⅲ. None of them (n=6) immunized rabbits had spine fusion at 4 and 16 weeks, but some bone formation was observed at 16 weeks. Group Ⅳ,Ⅴ. None of them imunized rabbits had bone formation. The anti-Ad5 Ig and neutralizing Ab were detected and peaked at the 4 weeks and significnatly dropped off 16 weeks after injection. Conclusion: This experiment revealed that a small dose of adenovirus elicited an enough immune response that inhibited the bone formation. Because majority of huan posses the Ab against adenovirus, it will be mandatory to overcome immune response in adenoviral vector gene therapy.