Patent Application: US-80058510-A

Abstract:
the invention discloses an engineered oncolytic respiratory syncytial virus , ns1 gene deficient rsv , and its usage to treat breast 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 cotransfecting 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 . to identify whether δns1 rsv lacks ns1 gene , we infected vero cells ( ifn - f3 gene deficient cells ) with wt rsv and δns1 rsv ( moi = 5 ), ns1 protein were tested using ns1 specific antibodies by immunoblotting . as shown in fig2 a , ns1 protein was only visualized in wt rsv - infected vero cells , not δns1 rsv - infected cells , indicating that δns1 rsv lacks ns1 gene . δns1 rsv preferentially kills breast cells both in vitro and in vivo . mda - mb231 breast cancer cells and normal ccd - 1059sk ( human normal breast fibroblast ) were cultured in as indicated by atcc ( american type culture collection ) instruction , and then infected with wt and δns1 rsv ( moi = 5 ). changes in cell morphology were observed and viral replication was measured . fig2 b shows that δns1 rsv selectively induces cytopathic effect ( cpe ) in mda - mb - 231 breast cancer cells , and that δns1 rsv has a higher viral titer in this tumor cells than in ccd - 1059sk cells 24 h post - infection ( fig2 c ), suggesting that mda - mb - 231 cells efficiently support the propagation of δns1 rsv . to test if δns1 rsv also kills other breast cancer cell lines , we infected breast cancer cell lines t - 47d and mcf - 7 with δns1 rsv ( moi = 5 ). cpes were observed 48 h post - infection ( table 1 ), indicating δns1 rsv specifically kills breast cancer cells . to determine whether δns1 rsv infection induces tumor growth regression in vivo , mda - mb - 231 breast cancer cells were injected s . c . into the left and right flanks of 4 - 6 weeks old nude balb / c mice ( n = 6 per group ) and the resulting tumors were allowed to develop . viruses ( 1 × 10 10 pfu / ml ) 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 . δns1 rsv infection induces apoptosis in tumor cells , but not in normal human breast fibroblast ccd - 1059sk cells . to test the differential effect of δns1 rsv infection on apoptosis , mda - mb - 231 tumor cells and normal ccd - 1059 sk cells were infected with the indicated viruses ( moi = 5 ) and apoptosis was measured by the annexin v binding assay . fig3 a shows that δns1 rsv selectively induces apoptosis in tumor cells , compared to the cell spontaneous apoptosis shown in control . knockdown of the rsv ns1 gene allows the production of more ifn - 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