J Korean Soc Spine Surg.  2001 Dec;8(4):437-446.

Bone Forming Gene Therapy (Immune Animal Model in Ex Vivo Gene Therapy for Spinal Fusion with Type 5 Adenoviral Delivery of the LIM Mineralization Protein-1 cDNA)

• Kim HS
• Park HW
• Lee HM
• Moon SH
• Park JO
• Ha JW
• Kim NH
• Kang YH
• Boden SD

Abstract

STUDY DESIGN: In vivo study to determine the immune effects to adenoviral vector 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 response 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-betaGal to total 24adult 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 3immunized 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 16weeks, but some bone formation was observed at 16 weeks. Group IV, V. None of them immunized rabbits had bone formation. The anti-Ad5 Ig and neutralizing Ab were detected and peaked at the 4weeks and significantly 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 human posses the Ab against adenovirus, it will be mandatory to overcome immune response in adenoviral vector gene therapy.

Keyword

Spine fusion; Lim mineralization protein; Gene therapy

MeSH Terms

Adenoviridae
Animals*
Antibodies
Antibodies, Neutralizing
Arthrodesis
DNA, Complementary
Genetic Therapy*
Humans
Models, Animal*
Osteogenesis
Rabbits
Spinal Fusion*
Spine
Transgenes
Antibodies
Antibodies, Neutralizing
DNA, Complementary

Figure

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  Fig. 1. Kinetics of adenovirus specific antibody response according to viral dose and time sequence. NZW rabbit were immunized with i.v. injections of non-virus, 1×108, 1× 109, 1× 101 0, 1× 101 1 v.p. of Ad5-ßGal(CMV promotor). At the indicated times after administration, serum anti-adenovirus immune globulin were measured. Era bars indicated the standard deviation of the mean(n=5).

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  Fig. 2. Inhibitory effects of immune-rabbit sera on adenovirus mediated in vitro infectivity. A293 cell were infected with Ad5-CMV-GFP △ E 1 at an 10 particle per unit(p.f.u.) per cell(10 M.O.I.), in the presence of serial dilution of serum, and the fluorescence emission at 538 nm recorded after infection. Titers are compared to the GFP fluorescence relative to the total recorded in absence of serum. The number of % GFP fluorescence infected with adenovirus with immunized sera was significantly smaller than that infected with adenovirus with non-immunized sera in 1:50, 500, 2,000 dilution.

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  Fig. 3. Inhibitory effects of immune-rabbit buffy coat on an adenovirus mediated in vitro infectivity using same method of figure 2. The number of % GFP fluorescence infected with adenovirus with immunized buffy coat was significantly smaller than infected with adenovirus with non-immunized buffy coat in 1:50 dilution in 1× 108 Ad5-ßGal and 1× 1 09 A-5ßGal. However, there was no significant difference between 1:500 dilution in 1× 108 Ad5-ßGal and 1× 109 ßGal immunized buffy coat.

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  Fig. 4. Comparison of plain radiography 4 weeks after immunization according to immunized viral doses. A is the 1× 108 v.p. immunized rabbit, B 1× 109 v.p., C 1× 1010 v.p., D 1 × 1011 v.p. Generally, the bone formation were not observed and small bone formation could be observed at the 1× 108 v.p. immunized rabbit. It represents that recent any viral doses effect strong inhibition

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  Fig. 5. Comparison of plain radiography 16 weeks after immunization according to immunized viral doses. A is the 1× 108 v.p. immunized rabbit, B 1× 109 v.p., C 1× 1010 v.p., D 1× 1011 v.p. Generally, the more bone formation were observed in small viral doses(A, B) It represents that large viral doses effect more strong inhibition in bone formation.

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  Fig. 6. Comparison of histology at 4 weeks and 16 weeks after immunization in 1× 108 Ad5-ßGal immunized group II. Left figure is the histology of 4 weeks after immunization. Right figure is the histology of 16 weeks after immunization. The lots of bone formation were observed in left, but right. It represents that time sequence blunted the impact of immune reaction.

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