Baculoviruses As Vectors For Gene Therapy Reversing Intervertebral Disc Degeneration

Objectives To determine the feasibility baculoviruses as vectors forgene therapy reversing intervertebral disc degeneration.Methods The current study include following parts: To comparemorphologic changes of nucleus pulposus (NP) cells and extracellar matrix(ECM) from intervertebral discs of young and adult rabbits, to test theefficacy of the baculovirus-mediated marked gene GFP transfer to normaland degenerative rabbit intervertebral disc cells and the expression of thetransgene in vitro and in vivo and, determine manipulating effect ofbaculovirus-mediated therapeutic gene sox9 transfer to degenerativeintervertebral disc cells from human in vitro and rabbit in vivo. ⑴ Thestructure and arrangement of the Nucleus Pulposus (NP) cells and ECM inyoung and old rabbits were investigated both the tissue and ultrastructure scaleby using confocal scanning microscopy and transmission electron microscopy.⑵ The intervertebral discs cells of rabbit cultured in monolayer were treatedwith six different doses of baculovirus carrying the green fluorescence proteingene (Ac-CMV-GFP). Transgene expression was analyzed by fluorescencemicroscopy and flow cytometry. The AcMNPV/GFP virus then was injecteddirectly into the intervertebral discs of eight rabbits;at 7,13,20 and 28 daysafter injection, the nucleus pulposus tissues of injected discs were evaluatedimmediately by fluorescence microscopy for GFP expression. ⑶ Rabbitnucleus pulposus cells were isolated and cultured from Lumber intervertebraldiscs of ①Young health rabbits, ②model rabbits, ③adult rabbits.Nucleus pulposus cells were treated with 200MOI Ac/CMV/GFP for 48 hours,The percentage of GFP-positive cells was analyzed by flow cytometry and thelevel of GFP expression was semi-quantified using HPIAS2000 imagineanalysis software. ⑷ Baculovirus delivery vectors expressing Sox9protein were constructed using the Bac-To-Bac Baculovirus ExpressionSystem (Gibco BRL). The cultured rabbit NP cells were infected with thevectors. Immunohistochemical analyses were performed to assess sox9expression. ⑸ Intervertebral disc degeneration induced by needleanulotomy, in a rabbit model, three test conditions were studied ① PBS wasinjected directly into L12,L23. ② Ac/CMV/GFP virus was injected directlyinto L34,L45. ③ The Ac/CMV/Sox9 virus was injected directly into L56,L67. After 5 weeks, the injected discs were evaluated histologically and thecontents of Ⅱcollagen was detected using Immunohistochemical staining.Results ⑴ The young rabbit discs were Thompson's gradeⅠ. TheNP cells from the young rabbit discs, which were round and contain numerouslarge inclusion bodies, often formed clusters with a diameter of 266±167μmand a density of 14.9±4.3 per high power(×40);The collagen fibril washomogeneous. The adult rabbit discs were Thompson's grade Ⅲ, the NPcells from adult rabbit discs were round or oval shape and no inclusion bodiesof large size, formed less clusters or were singly diffusedly distributed, withthe diameter of clusters to be 94±42 μm (single cell excluded) and densityto be 8.0±2.4 per high power (×40). Collagen fibril often cross-linked andarranged irregular. ⑵ A dose of Ac-CMV-GFP at an MOI of 200 wasfound to achieve the highest transduction ratio (approximately 87% of NPcells) and long-term expression without any toxicity to the cells. In vivo assaydemonstrated that Ac-CMV-GFP could also mediate GFP expression in rabbitintervertebral disc cells without inducing any symptoms. The GFP expressionlevel at 7 days after transduction was significantly greater than at 21 and 28days post treatment. ⑶ The percentage of GFP-positive cells of threeexperimental groups were 84.4±3.4, 82.9±5.7, 81.8±5.5 respectively, Thefluorescence intensity values of three experimental groups were 0.0054±0.0029, 0.0052±0.0032, 0.0056±0.0031 reprectively. ⑷ The recombinantBaculovirus plasmids were cut by EcoR I and XbaI respectively, both 6.5kbvector fragments and 1.9kb interest gene fragments appeared in agaroseelectrophoresis . The productions of PCR confirmed the hSox9 presence ofrecombinant Baculovirus. The expression of hSox9 in rabbit NP cells wasverified by immunohistochemical staining. ⑸ In the rabbit model,intervertebral disc cells treated with Ac/CMV/Sox9 in vivo maintained aprimary morphologic and the contents of Ⅱcollagen was significantly higherin tissues treated with Ac/CMV/Sox9 than Ac/CMV/GFP and PBS. There wasno significant difference between Ac/CMV/GFP and PBS (P>0.05).Conclusions Baculovirus can transfer exogenous genes into rabbitNP cells at a high efficiency and safely both in vitro and in vivo andtransducibility rate is relatively insensitive to disc degeneration grade.Ac/CMV/Sox9 can efficiently increase Ⅱcollagen synthesis of NP cellsfrom rabbit. The results suggest that baculoviruses might be a promisingvector for gene therapy of degenerative disc disease.