IL-4RA Gene Transfer By Recombinant Retrovival Vector Prevents The Development Of Experimental Asthma

Asthma is a chronic inflammatory disease, approximately 25-30% ofasthmatics require long-term theraphy with inhaled steroids. Long-termmanagement of asthma with steroid theraphy is not always effective and may leadto severe adverse effects. Thus, development of a safe long-lasting therapeuticagent remains a high medical necessity. Current theory suggests that thedevelopment and clinical manifestations of asthma are driven by a dysregulated, Thelper 2 lymphocyte (TH2) biased immune response to an antigen. Inhaledantigen provokes the activation of CD4+ TH2 and the production of specificcytokines (IL-4, IL-5, IL-9 and 13) Cytokine expression results in the recruitmentof eosinophils, leading to chronic airway inflammation, mucus hyperproductionand airway hyperresponsiveness (AHR). Although asthma symptoms can becontrolled acutely, there is a need for a theraphy that address the underlyingimmune dysfunction and provide continuous control of chronic airwayinflammation. The TH2-type cytokines, IL-4 and IL-13 have been demonstrated toplay a crucial role in asthma pathogenesis. The closely related TH2 cytokines,IL-4 and IL-13, share many biological functions that are considered important inthe development of allergic airway inflammation and airway hyperresponsiveness(AHR). The overlap of their functions results from the IL-4Rα-chain forming animportant functional signaling component of both the IL-4 and IL-13 receptors.Using neutralizing Ab to IL-4 administered during sensitization, he developmentof airway eosinophilia, AHR, and allergen provocation are reduced. However,administration of Ab to IL-4 during secondary allergen challenge only partiallyreduces the response. More recently, inhibition of airway eosinophilia and AHRusing a soluble IL-13R fusion protein that specifically binds and neutralizes IL-13has been shown. Moreover, administration of IL-13 or overexpression of IL-13 inthe airway can induce airway eosinophilia, mucus production, and AHR. IL-4exert its biological effects by binding to the 140-kDa IL-4Rα-chain thatcomplexes with the 64-kDa common γ-chain. Formation of the receptorheterodimer initiates signal transduction by activation of STAT6 (via the IL-4Rα-chain), resulting in the gene transcription that is critical for many of theIL-4-induced effects associated with the allergic response. The IL-4Rα-chainalso forms the functional signaling component of the IL-13R heterodimer (IL-13Rα1/IL-4Rα) and largely explains the overlap of IL-4 and IL-13 biologicalfunctions.Mutagenesis studies have identified the regions of IL-4 important forinteraction with the IL-4Rα-chain. Mutations of arginine 121, tyrosine 124 andserine 125 in the C terminus region of the human protein result in that IL-4mutants have the same capacity to bind to the IL-4Rα-chain as native IL-4, buthave no signaling activity and are antagonists of both IL-4 and IL-13 inducedresponses.Similarly mutation of amino acids glutamine 116 and tyrosine 119 toaspartic acid in murine IL-4 produce a mutant protein (Q11D/Y119D)that bindswith high affinity to the IL-4Rα-chain without inducing signal transduction.Because the activity of both of cytokines can be inhibited by a mutant IL-4protein, IL-4 receptor antagonist (IL-4RA), continual IL-4RA theraphy might bebeneficial in treatment of chronic asthma. To explore the utility of long-term genetheraphy for the treatment of asthma we used a recombinant retrovirus vector todelivery and provide sustained expression of IL-4RA in vivo.The total RNA was extracted from mouse spleen cell suspension after PHAstimulation, and IL-4RA was amplified by RT-PCR. The cDNA of IL-4RA wasidentified correctly by endonucleases cleavage and sequencing. The fragment wasthen subcloned into pIRES and pLNC-laz vector. The recombinant vector(pLNC-IL-4RA) was transfected into BHK-21 cells followed the detection of theexpression of IL-4RA by ELISA. The IL-4RA was obtained by detection ofIL-4RA from the supernatant of BHK-21. The recombinant retroviral vector wastransfected into PA317 cells for package by liposome transfection, after 2-weeksof G418 selection, G418-resistant PA317 colonies were obtained and amplified.The supernatant of cell culture was harvested and was used to infect NIH3T3 cellsto measure the viral titer of recombinant retrovirus. Results showed that thehighest titer of viral supernatant was 1×104CFU/ml.The mouse asthmatic model mimicked the basic feature of people's bronchialasthma, so we selected mouse as animal model. The OVA has powerful immuneantigenicity and used as common antigen in preparing asthmatic model. In ourexperiment we selected female BALB/C as model and challenged with mistinhalation with OVA.The mice were divided into 5 groups randomly, blank control, asthma model,asthma model administered by pLNC-laz, asthma model administered byglucocorticoid and pLNC-IL-4RA. Groups of mice (ten mice per group perexperiment) were sensitized by i.p. injection of 10 μg of OVA emulsified in 40 mgof aluminum hydroxide in a total volume of 200 μL on days 7and 14.On days 15,mice were challenge via the airways with OVA (5% saline) for 7 days, 20 min aday, blank control were sensitized by i. p. injection of 200 μL pH:7.4 PBS on days7and 14, On days 15, challenge via the airways with OVA (5% saline) for 7 days,asthma model was administered by pLNC-laz and pLNC-IL-4RA on days 12, for3 days. On days 15, challenge via the airways with OVA (5% saline) for 7 days,asthma model administered by glucocorticoid was administered by glucocorticoidon days 15, for 7 days. 24 hours after the last challenge mice were sacrificed bybloodletting, serum OVA-specific IgE levels,haemal Eosinophils count weremeasured and pneumonic tissues were obtained for further analysis. Cytokinelevels in the bronchoalveolar lavage fluid (BALF) were measured by ELISA. Thenumber and type of inflammation cells in the airways were assessed inBALF.Lung were fixed by immersion in 10% formalin, histological sections oflungs were prepared and stained with H&E, the expression of STAT6 wereindentified by immunohistochemical, IL-4RA (include 3'-LTR of retrovirus) wasamplified by PCR from pneumonic tissue genome and identified by sequencing.The results were as following: 1: By observing the changes of action ofasthma model, eosinophil increasing in BALF, the levels of IgE raised, lots ofinfiltration inflammatory cells were detected in pneumonic tissues sections stainedwith H&E, hyperplasia of cap-shaped cell existed, mucus membrane broke. Allthese indicated that asthma model in mice had been established successfully. 2:These changes significantly reduced in asthma models treated with glucocorticoidand pLNC-IL-4RA and not in asthma models treated with pLNC-laz. Statisticallysignificant difference (P<0.01). 3: The effect of pLNC-IL-4RA and glucocorticoidon IL-4 and IL-13 induced expression of STAT6 was analyzed byimmunohistochemical analysis, the expression of STAT6 in pneumonic tissues ofasthmatic mice was higher than that of blank control, and it was reduced aftertreatment with glucocorticoid and pLNC-IL-4RA. 4: The levels of IL-4, IL-5,IL-13 of BALF was measured by ELISA, the levels of IL-4, IL-5, IL-13 of BALFwas undetectable in mice that received OVA challenge alone, following OVAsensitization/challeng, significantly elevated levels of IL-4, IL-5, IL-13 of BALF,administration of pLNC-IL-4RA and glucocorticoid significiantly inhibited theelevations in BALF of asthmatic mice IL-4, IL-5, and IL-13 levels, statisticallysignificant difference (P<0.01), administration of pLNC-laz had no significantlyeffect on BALF cytokine levels of asthmatic mice (P>0.05). 5: SerumOVA-specific IgE levels were measured following challenge by ELISA. Thelevels of OVA-specific IgE of Serum in mice that received OVA challenge alonewere more lower than that received OVA sensitization/challenge, Statisticallysignificant difference (P<0.01), pLNC-IL-4RA and glucocorticoid significiantlyinhibited the elevations of IgE levels in Serum of asthmatic mice (P<0.01).Administration of pLNC-laz had no significantly effect on Serum OVA-specificIgE levels of asthmatic mice (P>0.05). There is no significant defference betweenadministration of pLNC-IL-4RA and glucocorticoid above all (P>0.05). 6:IL-4RA (include 3'-LTR of retrovirus) was amplified by PCR from pneumonictissue genome and identified correctly by sequencing.Conclusion: pLNC-IL-4RA was integrated to pneumonic tissue genomesuccessful, we show that retrovirus mediated delivery of IL-4RA to airways ofmice reduced airways eosinophilia triggered by either IL-13 or IL-4. Furthermore,IL-4RA delivered by retrovirus, expressd in the airways of mice followingallergen sensitization, significiantly inhibited development of airways mucusproduction and reduced the levels of asthma-associated TH2 cytokines in theexperimental mouse of allergic asthma. Thus, gene theraphy can be a potentialtherapeutic option to treat and control chronic airways inflammation andasthmatic symptoms.Asthma is a global health problem that results from a complex interplaybetween genentic and environmental factors.Inhaled β-adrenergic agonists andsteroid treatments are effective, but are costly,require daily dosing and depend onpatient compliance and coordination. The short half-life of many cytokine-baseddrugs requires repeat and frequent dosing which can result in anti-drug antibodiesthat block therapeutic effectiveness over time. Over a third of patients takingrecombinant interferon will develop neutralizing antibodies that diminish itsefficacy after 1-2 years of theraphy. Extensive studies of the pathophysiology ofchronic airway inflammation and reversible increase in airway resistance haveuncovered immunologic pathways that might be countered by gene transfer. Suchtherapies could be long-lived and might overcome the limitations of oral andinhaled medications. Gene transfer to bronchial epithelium has been used inexperimental model. Doing so can be expected to yield long-lasting protectionfrom bronchospastic challenge and reduce dependence on inhaled and oralmedications.