190 Immune Response and Growth Factor Formation After Direct Intra Articular Injection of AAV2 or AAV5 IGF I in an Equine Model Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The Amer[.]
IMMUNE RESPONSES TO CELL AND GENE THERAPY Immune Responses to Cell and Gene Therapy 190 Immune Response and Growth Factor Formation After Direct Intra-Articular Injection of AAV2- or AAV5-IGF-I in an Equine Model Kyla Ortved,1 Bettina Wagner,1 Alan Nixon.1 Cornell Unversity, Ithaca, NY Figure Oncolytic virus can replicate in cancer cells increased several ten thousand folds, the inserted gene can also be replicated with increase of several ten thousand folds The effect of CTGVT is not 1+1=2 but equal to 50-100 (the figure is omitted) Therefore, they both should go through to carried out the CTGVT stategy The CTGVT (OV-gene) is a revolution of gene therapy and oncolytic viral therapy Furthermore, we still made the modification of the CTGVT by the use of two gene strategy, the CTGVT-DG (double gene), bucause two genes may have synergetic or compensative effect between them By the of CTGVT-DG strategy, all the xenograft tumor of many cancers could be essentially completely eradicated Figure Complete eradication of the colorectal cancer by ZD55TRAIL+ZD55-IL-24 due to their synergetic effect The CTGVT-DG is the top antitumor effect of CTGVT In the past years, we always used the OncoAd (OV from adenovirus) as vector, however, the OncoPox (OV from vaccinia virus of Poxvirus) has many superiority by its replication in cytoplam and never integration to chromsome of genome, OncoPox will never induce cancer and is the first virus to be i.v injected to human Thus, from 2011, we developed the strategy of the combination of CTGVT-DG and OncoPox, then double targeting OncoPox by double deletion of TK and B18R of vicinia virus (Tk-, B18R- OncoPox, vvDD) and harboaring double genes (vvDD-gene1-gene2) was innovated The antitumor effect of our vvDD-gene1-gene2 will have much much better antitumor effect than that of the current most antitumor drugs, the OncoPox-GM-CSF which published a paper in Nature in 2011 and Nat Med in 2013, and also much better antitumor effect than that of OncoHSV-GM-CSF (OV from Herpe Sinplox Virus) which Amgen paid billion USD to BioVex The higher atntiumor effect of our constructed drugs is due to that they have double genes for cancer theapy A patent of OncoPox-gene1-gene2 is in application S72 Intra-articular viral vectors expressing therapeutic transgenes are potential treatments for OA AAV is a common vector used for gene transfer but concerns regarding neutralizing antibodies (NAb) and T cell responses have been expressed The immune response to IA vectors remains largely unknown We hypothesized that AAV2- or AAV5-IGF-I would result in IGF-I formation but would induce NAbs and cytotoxic T cell responses 12 healthy horses were assigned to treatment (AAV2 or AAV5) or control (saline) groups Middle carpal (MC) joints were injected with 5e11vg in 3ml of DPBS Each horse had clinical evaluations and arthrocentesis on days 0,4, 7, 14, 28 and 56 Synovial fluid was analyzed for total protein (TP) and nucleated cell counts (NCC), NAbs, cytokines including IL-10, IL-6, IFN-γ, sCD14, and CCL2, and IGF-I concentration Serum was evaluated for cytokines and NAbs Blood was collected prior to injection, at day 14 and day 56 PBMCs were isolated using Ficoll centrifugation Cells were stimulated with PMA/iono, media, AAV2 or AAV5 At 48h cells were harvested, fixed, permeabilized, and stained for CD4 or CD8 and for IFN-γ, IL-4 or IL-10 Cells were analyzed using a flow cytometer with fluorescence gates set according to isotype control staining Cells were also plated in 96-well plates Supernatant was collected at 48h for cytokine secretion analysis Injection of AAV2- or AAV5-IGF-I did not induce greater articular inflammation compared to saline TP levels were slightly increased in AAV5-IGF-I joints at days 14 and 28; however, these increases were not significant (fig 1) NCCs were significantly increased in AAV5-IGF-I joints on day 14 (3375 ± 957.73 cells/mL; p