Recombinant Adeno-Associated Virus-Mediated Inhibitng The Expression Of Interleukin-4 In CD4～+ T Lymphocytes For Asthma Gene Therapy

Part 1The study on the selection of antisense oligonudeotides to inhibit the expression of IL-4 in asthmatic rats' CD4⁺ T cellsObjective: To investigate the effects of IL-4 antisense oligonudeotides of different sequences on the expression of IL-4 and IL-4 mRNA in CD4⁺ T lymphocytes of asthma rats.Methods: The CD4⁺ T lymphocytes from asthma rats were purified by negative selection with a magnetic cell sort. Through liposome, different antisense oligonudeotides (AS-1 to translation initiation, AS-2 to Exon-2, AS-3 to translation terminal) and blank control were transfected into the CD4⁺ T lymphocytes respectively. After 28 hours, IL-4 in supernatant of cells culture and IL-4 mRNA in T lymphocytes were detected by ELISA and semi-quantitative RT-PCR respectively. Results: RT-PCR showed IL-4/β-actin (relative ratio of absorbance): 0.2615 ± 0.1476 for AS-1, 0.2885 ± 0.1411 for AS-2, 1.1012 ± 0.3641 forAS-3, and 1.2068 ± 0.3836 for blank group respectively (F=22.597, P<0.01). The production of IL-4 in culture supernatant showed: 13.800±7.233 for AS-1, 15.329 ± 7.358 for AS-2, 52.643 ± 12.075 for AS-3, and 58.286± 14.100 for blank group respectively (F=34.976, P<0.01). The expression of IL-4 was significantly lower in the groups treated by AS-1 and AS-2 than that in the group treated by AS-3 and blank(P<0.01). Conclusion: IL-4 antisense oligonudeotides can inhibit the expression of IL-4 and IL-4 mRNA in CD4⁺ T lymphocytes of asthma rats; the inhibitory effects of threekinds of antisense oligonudeotides are different significantly. Part 2Construction and identification of adeno-associated virus-based plasmid carrying of antisense IL-4Objective: To construct a recombinant plasmid for rattus IL-4 RNA antisenseexpression which contains adeno-associated viral (AAV) inverted terminal repeats(ITR).Methods: An intermediary plasmid was constructed by inserting IL-4 cDNA inreverse order to the eukaryotic expression vector phMGFP which had been cleavedwith Xba I + EcoR V to remove the GFP gene. Then the sequenceCMV_p-asIL4-SV40_E was amplified from the intermediary plasmid by PCR. Therecombinant plasmid pasIL4 was constructed by replacing the viral replicable genesin the psub201 with the sequence CMV_p-asIL4-SV40E. pasIL4 was transfected intoCD4⁺ T lymphocytes, and later on IL-4 protein in supernatant of cells culture and IL-4mRNA in T lymphocytes were detected by ELISA and semi-quantitative RT-PCRrespectively.Results: pasIL4 was identified with double restriction enzymes digestion and DNAsequence analysis. The expression of IL-4 protein and IL-4 mRNA in CD4⁺ Tlymphocytes cells treated by pasIL4 was significantly decreased.Conclusion: The recombinant plasmid pasIL4 was constructed successfully. Theavailability of the pasIL4 should facilitate construction of a recombinantadeno-associated virus 2 (AAV)-based virions containing IL-4 antisense gene forpotential use in asthma gene therapy.Part 3Recombinant adeno-associated virus-mediated inhibiting ofinterleukin-4 expression in CD4⁺ T lymphocytes for asthma genetherapy in vitroObjective: To construct rAAV which contains IL-4 antisense gene, then assess theprocedure of rAAV construction and purification, and investigate rAAV-asIL4 potential use in asthma gene therapy.Methods: 293T cells were transfected with pasIL4, pXX2 and pXX680 using the calcium phosphate method to construct rAAV-asIL4. Southern blot were performed to detect the titer of rAAV-asIL4. The ability of antisense IL-4 gene mediated by rAAV vector (rAAV-asIL4) to infect CD4⁺ T lymphocytes in vitro was confirmed by RT-PCR for IL-4 mRNA in T lymphocytes and ELISA for supernatant of cells culture. Results: In vitro administration of rAAV-asIL4 resulted in significant reduction in he levels of IL-4 mRNA and protein expression compared with group A₀ or group C₀. Conclusion: We have gotten high titer rAAV, and it can inhibit the expression of IL-4 in CD4⁺ T lymphocytes. The results provide evidence for the potential utility of rAAV-asIL4 as an approach to gene therapy for asthma. Part 4Effects of recombinant adeno-associated virus-antisense IL-4 onasthmatic rats by tail vein deliveryObjective: To construct eukaryotic antisense IL-4 expressing vector of recombinant adeno-associated virus (rAAV-asIL4), and explore effects of this vector on asthma rats, for investigating rAAV-asIL4 potential use in asthma gene therapy. Methods: rAAV-2 containing antisense IL-4 gene was constructed. In vivo 24 rats were randomized into four groups: normal group (group N), asthma group (group A), rAAV-asIL4 treated group (group T), and rAAV-GFP treated group (group C). The rats of group T were administered rAAV-asIL4 on day 0 and day 14 by tail vein injection. rAAV-GFP was used as a control. Blood samples and bronchoalveolar lavage fluids (BALF) samples were collected. The production of IL-4 and IgE in BALF and serum were measured using ELISA kits respectively. IL-4 mRNA in lung tissues and lymphocytes were determined with RT-PCR. Histological sections of lungs, livers and kidneys were evaluated by staining with H&E.Results: In vivo, in the group T the levels of IL-4 mRNA, IL-4 proteins and IgE were lower than those of group A and group C respectively. The pulmonary histopathological changes were less in group T compared with group A and group C. Few pathologic lesions were found in livers and kidneys in the group T. Conclusion: The results of this study provide evidence for the potential utility of rAAV-asIL4 as an approach to gene therapy for asthma. Part 5Effects of recombinant adeno-associated virus-antisense IL-4 onasthma rats by airway deliveryObjective: To construct eukaryotic antisense IL-4 expressing vector of recombinant adeno-associated virus (rAAV-asIL4), and explore effects of this vector on asthma rats, for investigating rAAV-asIL4 potential use in asthma gene therapy. Methods: 28 SD rats were randomized into four groups: normal group (group N), asthma group (group A), rAAV-asIL4 treated group (Group T), and rAAV-GFP treated group (Group C). The rats of group T were intratracheally administered rAAV-asIL4 on day 1 and day 14. rAAV-GFP was used as a control. The nucleated cells and eosinophils in bronchoalveolar lavage fluids (BALF) were counted. The production of IL-4 and IgE in BALF were measured using ELISA kits respectively. IL-4 mRNA in lungs was determined with RT-PCR. Histological sections of lungs were evaluated by staining with H&E.Results: Although the number of nucleated cells and eosinophils were decreased in group T compared with group A and group C, there was no significantly difference among them. In rats administered with rAAV-asIL4, IL-4 mRNA in lung tissues, IL-4 protein and IgE in BALF were significantly decreased compared with those of group A and group C. The pulmonary histopathological changes were less in group T compared with group A and group C. Conclusion: The results of this study provide evidence for the potential utility ofrAAV-asIL4 as an approach to gene therapy for asthma.