Cancer Gene Therapy For B Cell Lymphoma Model Mice Using Adeno-assiociated Virus Vector Expressing MDA7/IL-24

BackgroundHuman malignant lymphomas are neoplasms arising from lymphocytes at various stages of differentiations, most NHLs (80-90%) are of B-cell origin. Variety approaches of lymphoma treatment are developed, such as chemotherapy, radiotherapy, immunotherapy, stem cell transplantation and so on. With modern treatment of patients suffering from NHL, the overall survival rate at 5 years is approximately 50-60%, it is not a cure. Furthermore, the mortality only decrease less than 5% in the past 30 years, such present approaches can not obviously reduce it. Relapse, acquired treatment resistance and treat-related complications from diverse medicine side-effect remain an important challenge to successfully treatment of B-cell malignancies. Melanoma differentiation-associated gene7/ interleukin-24 (mda-7/IL-24) is considered to be secreted by the immune system and melanocytes only. Actually, following researches present that the MDA7/IL-24 has multi-potent anticancer effect, it not only selectively induces apoptosis in cancer cells specially, but also has immunomodulatory and antiangiogenic properties, as well as stong antitumor bystander effects, making it an ideal candidate for cancer gene therapy. Efficient delivery system is also important for systemic gene therapy. Adeno-associated virus (AAV)-based vectors are nonpathogenic and less immunogenic than other vectors used for gene therapy. Especially, one of the serotype of AAV vectors, AAV type 8 has higher transduction efficiency in muscle than others by now as we know, so it seems more suitable to be used in the muscle directed lymphoma systemic gene therapy. Muscle directed systemic gene therapy is a promising molecularly treatment strategy. It is appealing because of its simply administration, large size and good capacity for protein synthesis, long-term gene expression, as well as target gene transduction of the cells can secrete proteins all over the body, that make it an optimal method to treat tumors not only the primary lesions but also the metastases. It is an alternative treatment strategy for lymphoma which is not in good outcomes administrated by conventional therapy or combining with other treatment modalities.Objective:The present study examined the feasibility of systemic gene therapy for lymphoma by using adeno-associated virus type 8 vector expressing MDA7/IL-24. We used an AAV8 vector for systemic delivery of mda7/IL-24 to culured cells and animal models to investigate the antitumor activity of secrected human MDA7/IL-24.Methods:(1) Contransfection and ultracentrifugation methods to make AAV vector type 8. (2) Transduce C2C12 cells to get the medium with IL-24 protein, then treat the lymphoma cell line by the medium and analysis the apoptosis induced by IL-24. (3) Inject A20 cell line(B cell lymphoma cell line of mouse)into caudal vein of mice to establishing the lymphoma model mice. (4) Devide the mice into two groups, and inject the AAV8-MDA7/IL-24 or AAV8-EGFP intramuscularlly to measure the tumor size and caculate the survival time. (5) Take out the serum of each mouse to measure the IL-24 level in vivo ervery week. (6) Detect the other cytokines such as IL-10, IL-6, TNFα, IFNγ, GM-CSF level in serum and analyze the changes of subpopulation of PBMC and splenocytes. (7) To sacrifice the mice to get the tumor tissue for frozen slides and to detect the apoptosis by TUNEL assay and CD31 staining to know the antiangiogenesis of IL-24.Results:(1) We produced AAV Vector type8 expressing the gene encoding human MDA7/IL-24 and assessed the secretion capacity of AAV8 Vector-Mediated MDA7/IL-24 in vitro and in vivo. (2) C2C12 were transduced with AAV8-MDA7/IL-24 and AAV8-GFP and changed medium after 24 hours. For another 72 hours, the medium were collected and measured the secreted IL-24 levels by ELISA assay. Significant discrimination presented between two groups(114.71±17.08ng/ml vs. 12.40±1.56ng/ml). (3) Normal mice were divided into two groups randomly, the equivalent viral genomes (1.5×10¹¹vg) of AAV8-MDA7/IL-24 and AAV8-GFP were administrated respectively by intramuscular injection at the right quadriceps of the mice. Obviously differences obtained in vivo. (1W:59.8±5.33ng/mlvs.7.76±2.38ng/ml; 2W: 71.36±5.69ng/ml vs. 16.38±2.55ng/ml; 4W: 100.92±6.12ng/ml vs. 13.76±3.52ng/ml,P<0.001)。(4) We established a lymphoma cells inoculated mouse model by intravenous injecting the caudal vein of BALB/C mice with the A20/LUC cells (luciferase expressing A20 cells). (5) Blood samples were then obtained from all animals every 7 days after lymphoma tumor cell transplantation and were subjected to ELISA. We found that following intramuscular injection of AAV8-MDA7, the concentration of MDA-7/IL24 in serum increased and reached a plateau (50-70ng/ml) one week after vector administration. Thereafter, plasma MDA-7/IL24 levels remained stable during the observation period. (4W:2.80×10⁷±1.53×10⁷ vs.1.78×10⁸±8.20×10⁷ photon/sec ; 5W:1.32×10⁸±2.30×10⁷ vs. 2.63×10⁸±6.50×10⁷ photon/sec, P<0.05). (6) Treatment group got longer survival time (65.6±5.8 vs. 47.4±4.8 days; P<0.03), and Lower lightness and smaller area of bioluminescence presented in AAV8-MDA7 treated mice after 4 weeks. (4W: 2.80×10⁷±1.53×10⁷ vs.1.78×10⁸±8.20×10⁷ photon/sec ; 5W:1.32×10⁸±2.30×10⁷ vs. 2.63×10⁸±6.50×10⁷ photon/sec, P<0.05).。(7) IL-24 induced the dose-dependent apoptosis in lymphoma cell line in vitro(30ng/ml:16.52±1.80%, P<0.05;50ng/ml:24.41±4.03%, P<0.01;100ng/ml:62.61±3.66%,P<0.001 VS. Control:10.43±4.08%)and in vivo(51.18±2.44% VS。7.01±1.80%,P<0.001). (8) IL-24 inhibited the angiogenesis in tumor tissue in vivo. (8.42±2.8 VS 15.25±4.8,P<0.03) (9) IL-24 increased the level of IL-6 and IFN-γin serum (IL-6: 137.17±22.4ng/ml VS 62.03±2.01ng/ml; IFN-γ: 43.07±15.36ng/ml VS 20.67±0.30ng/ml; P<0.01) one week afer injection. (10) CD3+CD8+ T cells and granulocytes in PBMC were increased in treatment group one and two weeks afer injection, while in splenocytes so did in macrophages and granulocytes(P<0.05)。Conclusion (1) The medium conditioned by AAV8-MDA7/IL-24 could induce the dose-dependent apoptosis in lymphoma cells (A20 cell line). (2) AAV8-MDA7/IL-24 injected into the quadriceps of the mice could secrete high level IL-24 in serum. (3) Suppression of tumor growth was observed in AAV8-MDA7/IL-24 treated mice. (4) Survival effect was also observed in AAV8-MDA7/IL-24 treated mice. (5) Tumor cell specific apoptosis and suppression of angiogenesis were detected in treated mice. (6) MDA7/IL-24 could stimulate some immune cells and anti-tumor cytokines increasing for modulating immune activities.SignificanceThese results suggest that AAV8 mediated systemic expression of MDA7/IL-24 is useful for treatment of lymphoma and make pave a way for human lymphoma therapies.