Patent Application: US-201313790278-A

Abstract:
the invention discloses an engineered oncolytic respiratory syncytial virus , ns1 gene deficient rsv , and its usage to treat lung cancer by killing cancer cells with in vitro and in vivo evidences .

Description:
the respiratory syncytial virus ( rsv ) was used in this study . the ns1 gene was deleted by the removal of 122 to 630 nt in the antigenomic cdna using reverse genetics approach , resulting in the joining of the upstream nontranslated region of ns1 to the translational initiation codon of ns2 . the δns1 rsv was recovered through co - transfecting vero cells with the ns1 - deficient viral cdna clone and expressional plasmids encoding n , p , m2 - 1 and l . alternatively , the engineered virus could be any other viruses with the deletion of similar ns1 gene . δns1 rsv preferentially kills nsclc cells both in vitro and in vivo . nsclc cells and wi - 38 normal human diploid lung cells were infected with wt or δns1 rsv ( moi = 5 ). changes in cell morphology were observed and viral replication was measured . fig2 a shows that δns1 rsv selectively induces cpe in a549 cells , and that δns1 rsv has a higher viral titer in a549 cells than in wi - 38 cells 24 hr after infection ( fig2 b ), suggesting that a549 cells efficiently support the propagation of δns1 rsv because they are defective in producing and responding to ifn . dr . bose &# 39 ; s group reported that wt rsv kills prostate cancer cells [ 11 ]. to test if the higher titer of wt rsv also kills lung tumor cells , we infected different lung tumor cell lines and normal wi - 38 and nhbe cells with high dose of viruses ( moi = 10 ), and checked cpe 24 hr post - infection . as shown in table 1 , δns1 rsv , but not wt rsv , preferentially kills nsclc cells . these experiments were done on cell lines in vitro , but proof of efficacy requires demonstration in vivo . to determine whether δns1 rsv infection induces tumor growth regression in vivo , a549 cells were injected s . c . into the left and right flanks of 4 - 6 weeks old nude balb / c mice ( n = 4 per group ) and the resulting tumors were allowed to develop . viruses were locally injected into the tumors three times and the sizes of the tumors were measured using digital calipers . fig2 c , d show that δns1 rsv infection caused regression in tumor growth versus controls . to test the safety of locally administered viruses , the virus titer in various organs of infected mice was determined by plaque assay and rt - pcr assay . as shown in fig2 e , f , the viruses specifically localize to tumors . cell cycle dysregulation is a critical feature of tumor cells . the inhibition of cell cycle is a potential therapeutic target for the control of tumor cell proliferation . to test whether δns1 rsv induces cell cycle arrest , we infected a549 cells with the indicated viruses at an moi of 5 . analysis of propidium iodide ( pi ) staining by flow cytometry clearly revealed that virus infection did not significantly affect tumor cell cycle , but the appearance of a sub - g1 ( apoptosis ) peak was considerably elevated in δns1 rsv - infected cells ( fig3 ). δns1 rsv infection induces apoptosis in tumor cells , but not in normal human bronchial epithelial cells . to test the differential effect of δns1 rsv infection on apoptosis , a549 cells and nhbe cells were infected with the indicated viruses ( moi = 5 ) and apoptosis was measured by the annexin v binding assay . fig4 a shows that δns1 rsv selectively induces apoptosis in tumor cells , compared to the cell spontaneous apoptosis shown in controls , which was verified by immunoblotting ( fig4 b , c ). recent research reports demonstrated that p53 participates in rsv - induced apoptosis [ 12 ]. to determine if p53 is required for δns1 rsv - induced apoptosis , p53 - deficient nsclc h1299 were tested . fig4 d shows that δns1 rsv infection induced apoptosis in h1299 cells , indicating that p53 protein is not an exclusive factor required for development of apoptosis in δns1 rsv - infected tumor cells . we found that δns1 rsv triggered apoptosis in lung cancer cells through mitochondrial pathway ( fig4 b - c ). to test the effects of viral infection on mitochondrial δψm , we measured mitochondrial δψm in a549 cells upon viral exposure ( moi = 5 ). as shown in fig5 a , b , ns1 prevented loss of mitochondrial δψm in response to viral infection . we further confirmed mitochondrial δψm results by transmission electron microscopy . mitochondria in vehicle - treated a549 cells exhibit a characteristic electron - dense matrix , in contrast to the swollen mitochondria with a loss of electron density in the matrix of δns1 rsv - infected cells . cells infected with wt rsv show less mitochondrial alteration than δns1 rsv - infected cells . ifn - β did not significantly affect mitochondrial morphology upon δns1 rsv infection ( fig5 c ). 1 . molina , j . r ., et al ., non - small cell lung cancer : epidemiology , risk factors , treatment , and survivorship . mayo clin proc , 2008 . 83 ( 5 ): p . 584 - 94 . 2 . jemal , a ., et al ., cancer statistics , 2008 . ca cancer j clin , 2008 . 58 ( 2 ): p . 71 - 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