452 MegaTAL Disruption of CCR5 To Protect Anti HIV CAR+ Lymphocytes from HIV Infection Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy S179[.]
Immunological Aspects of Gene Therapy I vaccines generated strong T cell responses, including GP-specific polyfunctional CD4+ and CD8+ T cells secreting IFNγ, TNFα, and IL2 BALB/c mice (n=10/group) were challenged with a lethal dose of a heterologous mouse-adapted Mayinga 1976 EBOV strain The two consensus DNA vaccines and and vaccine combinations were 100% protective in mice The matched vaccine afforded 90% protection Total GP-specific IgG levels were high in all surviving mice and low in unprotected animals suggesting that titers may correlate with therapeutic efficacy As all DNA vaccines were immunogenic and no antigen interference was observed, we next investigated immunogenicity of the DNA vaccines in rhesus macaques following a DNA-DNA prime-boost immunization regimen Macaques (n=5/group) received by IM-EP delivery:1) matched vaccine (4mg), 2) 3-construct formulation administered at a single injection site (2mg/vaccine), 3) 3-construct formulation delivered at separate injection sites (2mg/vaccine), or 4) 3-construct formulation combined with IL12 adjuvant (2mg/vaccine) Each immunization was followed by subsequent DNA boosts at month intervals All groups had high GP-specific total IgG titers post-1st boost immunization that continued to increase over subsequent boosts and detectable T-cell responses Overall, these studies demonstrate that a DNA-DNA prime-boost regimen can effectively induce seroconversion in NHPs and further studies will serve to evaluate protective immunogenicity This approach not only serves as a tool to better understand the immune responses associated with protection against EBOV infection, but also has potential for translation towards human applications 451 Successful Gene Transfer in Passively Immunized Mice With Immunologically-Inert AAVrh.10 Vectors Ruchita Selot,1 Balaji Balakrishnan,1 Sabna Cheemadan,2 Sudha Govindarajan,3 Rupali Gadkari,3 N Srinivasan,3 Giridhara R Jayandharan.1,2 Department of Biological Sciences and Bioengineering, Indian Institute of Technology, Kanpur, Kanpur, India; 2Department of CSCR/Haematology, Christian Medical College, Vellore, India; Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India Background: The development of an AAV vector that can bypass pre-existing immunity in humans is crucial for universal application of this vector system Among the diverse AAV serotypes, AAVrh.10 has shown rapid and sustained transduction at both adult and neonatal stages The low cross-reactivity of AAVrh.10 has also been reported to help vector re-administration In the present study, we evaluated if disruptions of immunogenic epitopes predicted on AAVrh.10 could lead to evasion of pre-existing neutralizing antibodies and result in enhanced gene expression Methods: The immunogenic T cell and B cell epitopes on AAVrh.10 capsid were predicted by ElliPro server (Immune Epitope DataBase) Site directed mutagenesis was performed on the epitopic regions predicted with highest probabality on AAVrh.10 capsid The generation of the mutated immunologically inert (i2) AAVrh.10 vector was confirmed by DNA sequencing Highly purified stocks of self complementary AAVrh.10 wild type (WT) and AAVrh.10 i2 vector was generated by polyethyleinimine based triple transfection protocols The efficiency of AAVrh.10i2 vector transduction in immunized mice was tested in C57BL/6 mice that were pre-treated with pooled immunoglobulin (n=6, 2mg IVIG/animal) After 24 hours, both mock-control and treated animals were injected with WT-AAVrh.10 and AAVrh.10-i2 vectors expressing luciferase as the transgene The luciferase activity was documented by serial bioluminescence imaging in small animal imaging system A Taqman based quantitative PCR was used for estimation of viral genome copy number in the gDNA isolated from liver tissue of experimental mice Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy Results and Discussion: A month after gene transfer, AAVrh.10-i2 (6 out of animals) mutant vectors had considerable transgene expression in the presence of IVIG while gene expression was completely neutralized in all the mice injected with WT-AAVrh.10 vectors This partial rescue in luciferase expression in the presence of IVIG was ~27 to 64 fold in comparison to WT vector administered animals To further evaluate their long-term transduction potential, C57BL/6 mice that did not receive IVIG were followed up further Seventy-two days post vector administration, animals that received AAVrh.10-i2 vector showed a 6.6 fold increase in hepatic luciferase expression when compared to the WT-AAVrh.10 vectors This was further corroborated by an increase in viral genome copies (2.07 vs 0.64) in the liver tissue of AAV-rh.10 i2 vector administered mice Further studies are needed to generate optimal AAVrh.10 vectors with complete immune evasion potential The development of such AAV vectors with an immunologically-inert phenotype augurs well for their potential use in human gene therapy 452 MegaTAL Disruption of CCR5 To Protect Anti-HIV CAR+ Lymphocytes from HIV Infection Thor A Wagner,1,3 Malika Hale,2 Guillermo Romano,2 Iram Kahn,2 Jaya Sahni,2 Andy Scharenberg,2,3,4 David J Rawlings.2,3,4 Center of Global Infectious Disease Research, Seattle Children’s Research Institute, Seattle, WA; 2Center for Immunity and Immunotherapies and Program for Cell and Gene Therapy, Seattle Children’s Research Institute, Seattle, WA; 3Pediatrics, University of Washington, School of Medicine, Seattle, WA; 4Immunology, University of Washington, School of Medicine, Seattle, WA Introduction: More than 30 million people are infected with HIV Antiretroviral therapy (ART) dramatically decreases mortality, but HIV-infected individuals on ART have an increased risk of malignancies, cardiovascular disease, neurologic disease, and shortened life expectancy Therefore a cure for HIV remains an important treatment goal A previous Phase II randomized clinical trial of anti-HIV Chimeric antigen receptor (CAR)-expressing T-cells was partially effective We hypothesize that a limitation of the previous strategy was that the anti-HIV CAR+ lymphocytes were susceptible to HIV infection Objective: Produce anti-HIV CAR expressing lymphocytes that are protected from HIV infection Methods: We designed novel anti-HIV CARs based on the scFV of a series of broadly neutralizing HIV antibodies A CCR5 megaTAL nuclease (an engineered homing endonuclease and TALEN chimera) was used to disrupt CCR5 as a means of protecting lymphocytes from HIV infection Two methods were used to produce CCR5-disrupted anti-HIV CAR+ lymphocytes 1) Primary PBMC were transduced with anti-HIV CAR using a lentiviral (LV) vector, CAR+ were cells sorted, and then transfected with CCR5 megaTAL mRNA 2) Alternatively, donor PBMC were transfected with CCR5 megaTAL mRNA followed by AAV delivery of the anti-HIV CAR with homology arms to induce homology directed recombination (HDR) of the anti-HIV CAR into the CCR5 locus Cells that underwent HDR were sorted and expanded CAR+ lymphocytes derived from each approach, were mixed with HIV-infected cell lines in the presence of ART or added to active HIV viral culture The reduction in the number of HIV infected cells was assessed by flow cytometry and PCR The reduction in replicating HIV was quantified by HIV capsid protein ELISA Results: LV delivery of anti-HIV CAR followed by sorting CAR+ cells and then CCR5 megaTAL transfection, resulted in CAR+ cells with up to 60% CCR5 disruption HDR, followed by sorting and expansion resulted in approximately 40% CAR+ cells Anti-HIV CAR+ lymphocytes upregulated cell surface CD137 and secreted IFNγ when mixed with HIV-infected target cells, killed a median of S179 Immunological Aspects of Gene Therapy I 75% (range 38-94%) of HIV-infected cells over 2-3 days, and reduced HIV DNA by approximately one log over days Beyond 72 hours of culture the anti-HIV CAR+ lymphocytes with CCR5 disruption resulted in much greater reduction in HIV than CAR+ lymphocytes without CCR5 disruption Conclusions: It is feasible to construct anti-HIV CAR+ T-cells that are protected from HIV infection This strategy warrants further study using in vivo models of HIV latency 453 Protection Against Lethal Dengue Challenge By IM Delivery of Synthetic, EPDelivered DNA Encoding Designed Anti-Dengue Neutralizing Antibodies Seleeke F Flingai,1 Emily M Plummer,2 Ami Patel,1 Sujan Shresta,2 Janess M Mendoza,3 Kate E Broderick,3 Niranjan Y Sardesai,3 Kar Muthumani,1 David B Weiner.1 Department of Pathology and Laboratory Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA; 2Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, CA; 3Inovio Pharmaceuticals Inc., Plymouth Meeting, PA Dengue virus (DENV) is the most important mosquito-borne viral infection in humans, with nearly 400 million infections occurring each year and a continually expanding geographic reach While vaccines are being developed, none are currently available that provide balanced protection against all DENV serotypes Advances in human antibody isolation have uncovered DENV neutralizing antibodies (nAbs) that are capable of preventing infection from multiple serotypes Yet delivering monoclonal antibodies using conventional methods is impractical due to high costs Engineering nucleic acid-based strategies for delivering monoclonal antibodies could tip the scale in the fight against DENV, as well as other emerging infectious diseases Here, we describe an approach to delivering cross-reactive neutralizing antibodies against DENV into the host circulation using DNA plasmid-mediated antibody gene transfer This approach, which we term DNA mAb (DMAB) delivery, generates biologically relevant levels of mAbs after a single intramuscular injection of antibody-encoding DNA followed by in vivo electroporation (EP) Since this approach allows for genetic tailoring of the exact features of the desired antibody, we incorporated Fc region modifications to a naturally occurring human anti-DENV neutralizing antibody to enhance antibody function in vivo We demonstrate that intramuscular delivery in mice of pDVSF-3 LALA, which encodes a human antiDENV1-3 IgG1 neutralizing antibody modified with a mutation that abrogates FcgR binding, produces anti-DENV antisera capable of binding and neutralizing DENV1-3 Importantly, mice receiving pDVSF-3 LALA, but not the unmodified pDVSF-3 WT, were protected from both virus-only disease and antibody-enhanced lethal disease These data establish this novel platform as a safe, effective means of delivering protective monoclonal antibodies to a host This work was supported by grants funded to DBW through the National Institutes of Health, the DARPA-PROTECT award, and Inovio Pharmaceuticals Inc 454 Prior AAV2-hIL10 Infusion Decreases AAV9-Dependent Immunotoxicity in Rat Brain Agnieszka Ciesielska,1 Piotr Hadaczek,1 Janine Beyer,1 John Forsayeth,1 Krystof Bankiewicz.1 Department of Neurological Surgery, University of California, San Francisco, San Francisco, CA Cerebral infusion of AAV9 vectors encoding non-self proteins results in the initiation of a robust immune reaction at the site of S180 injection due to the vector’s ability to transduce antigen-presenting cells in the brain [1] Infusion of AAV9 encoding a human gene, aromatic L-amino acid decarboxylase (hAADC), elicited an initial innate immune activation at the site of infusion that was succeeded by a robust adaptive immune response that included generation of hAADC antibodies, lymphocytic infiltration and cell-mediated elimination of transduced cells in the brain In this study, we investigated whether the potent anti-inflammatory cytokine, human interleukin-10 (hIL10) could suppress this immune response Four weeks prior to unilateral infusion of AAV9-hAADC into rat striatum, we infused AAV2-hIL10 bilaterally into striatum (control rats received bilaterally injection of AAV2-GFP) Six weeks after AAV9-hAADC injection, control rats revealed robust amphetamine-induced ipsilateral rotational behavior (956 ± 203 (S.D.) turns/1 hour) In while, in AAV2-hIL10-treated rats ipsilateral rotations were reduced by 66 % compared with control rats and this effect persisted for at least weeks This neuroprotective effect of hIL10 was evident histologically The number of surviving neurons (NeuN) was significantly increased in AAV2-hIL10-treated animals compared to AAV2-GFP-treated controls, and microglial and astrocytic activation was diminished Whereas, in control rats, a significant number of hAADC-positive astrocytes was observed (GFAP/hAADC double fluorescent staining), astrocytic transduction in AAV2-hIL10-treated rats was almost completely suppressed This somewhat puzzling result may be due to an effect of IL10 on astrocytic expression of an AAV9 receptor required for transduction However, our findings suggest that expression of IL-10 in the brain may be a useful adjunctive therapy in the treatment of neurological diseases in which the expression of a foreign antigen is unavoidable Ciesielska, A, Hadaczek, P, Mittermeyer, G, Zhou, S, Wright, JF, Bankiewicz, KS, et al (2013) Cerebral infusion of AAV9 vectorencoding non-self proteins can elicit cell-mediated immune responses Mol Ther 21: 158-166 455 Safety of Vaccination to Treat Cocaine Addiction with Capsid Proteins from a Disrupted Adenovirus Conjugated to a Cocaine Analog David F Havlicek,1 Lauren Beatty,1 Lucy Quach,1 Sebastien Monette,2 Bishnu P De,1 Jonathan B Rosenberg,1 Dolan Sondhi,1 Ronald G Crystal,1 Stephen M Kaminsky.1 Weill Cornell Medical College, New York, NY; 2Memorial SloanKettering Cancer Center, New York, NY Cocaine is a highly addictive drug with significant social, societal, economic and medical impact, yet there are no effective therapies for cocaine addiction An anti-cocaine vaccine that could evoke antibodies to bind the drug and prevent its transfer across the blood-brain barrier to its cognate CNS receptors is a promising strategy for treatment of cocaine addiction In preclinical studies, an anti-cocaine vaccine (dAd5GNE) consisting of a disrupted E1- E3- serotype adenovirus covalently conjugated to the cocaine analog GNE evoked high-titers of anti-cocaine antibodies that blocked cocaine from reaching the central nervous system of mice, rats and monkeys, abrogating cocaineinduced behaviors The objective of this study was to ascertain if vaccination of a clinical grade dAd5GNE formulated in Adjuplex™ adjuvant administered to mice and non-human primates (NHPs) could be achieved with a safety profile acceptable to move this therapy to humans Age-matched Balb/c mice (n=48 male, n=48 female for each group) were vaccinated monthly with a vehicle control, Adjuplex™ alone or dAd5GNE, (0.4 or mg) + Adjuplex™ At 1, 5, 13 and 26 wk, evaluation of gross and histopathology, complete blood count, and serum chemistry were performed in a blinded fashion No significant differences between Adjuplex™ control- and vaccine-treated groups were observed and no adverse effects could be attributed to the vaccine Some histiocytic inflammatory infiltrates at the site of injection were observed in mice for which doses of Adjuplex™ were Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy ... with CCR5 disruption resulted in much greater reduction in HIV than CAR+ lymphocytes without CCR5 disruption Conclusions: It is feasible to construct anti- HIV CAR+ T-cells that are protected from. .. Aspects of Gene Therapy I 75% (range 38-94%) of HIV- infected cells over 2-3 days, and reduced HIV DNA by approximately one log over days Beyond 72 hours of culture the anti- HIV CAR+ lymphocytes. .. Cerebral infusion of AAV9 vectors encoding non-self proteins results in the initiation of a robust immune reaction at the site of S180 injection due to the vector’s ability to transduce antigen-presenting