957 Simultaneous Administration of Multiple Oncolytic HSV 1 Vectors Expressing Different Immunostimulatory Genes Provides High Antitumor Efficacy in a Poorly Immunogenic Tumor Model Molecular Therapy[.]
OTHER DNA VIRUSES 956 Hormonal Milieu Enhances the Efficacy of Oncolytic Herpes Viral Therapy in Prostate Cancer Amit Bhargava,1 Brendon M Stiles,1 Michael Mullerad,1 Niraj J Gusani,1 Prasad Adusumilli,1 Teresa H Kim,1 Yuman Fong.1 Department of Surgery, Memorial Sloan-Kettering Cancer Center, New York, NY Introduction: In prostate cancer, androgens upregulate cellular proliferation and protect cells from apoptosis These cellular responses to androgens produce a milieu that is theoretically conducive to viral replication Therefore, we hypothesized that oncolytic herpes simplex virus type-1 (HSV) therapy of androgensensitive prostate cancer cells would be more efficacious in the presence of dihydrotestosterone Materials and Methods: NV1066 is an oncolytic HSV with single deletions in ICP-0, ICP-4 and γ134.5 The virus carries a transgene for enhanced green fluorescent protein (EGFP), under the control of a cytomegalovirus promoter LNCaP (androgen-sensitive human prostate cancer) cells were grown with either dihydrotestosterone (DHT, 3μM) or vehicle Cells were infected at multiplicity of infections (MOI; number of viral plaqueforming units per cell) of 0.01, 0.1 and Cytotoxicity was determined by lactate dehydrogenase release assay, EGFP expression by flow cytometry, and viral titers by plaque assay Apoptotic fractions were determined by Hoescht staining Results: Cell kill was significantly greater in LNCaP cells infected in the presence of DHT By day after infection at MOI 0.01, 80% of cells were killed compared to 7% in the no hormone group (p < 0.01, Figure 1) Viral growth was significantly increased in LNCaP cells infected at MOI 0.01 in the presence of hormone when compared to the hormonefree cells, with peak values of 2.3 x106 and x 104, respectively (p < 0.03, Figure 2) As measured by EGFP expression, cell-to-cell spread of virus was enhanced in LNCaP cells grown in the presence of DHT By day after infection at MOI 0.01, 93% of live cells expressed green fluorescence compared to 65% in the no hormone group (p < 0.01) Infection with NV1066 at MOI produced half the rate of apoptosis in the presence of DHT compared to cells not exposed to androgens Conclusions: Dihydrotestosterone creates a cellular environment conducive to prostate cancer cell growth While standard therapies aim to block these effects, oncolytic HSV therapy benefits from them, resulting in increased viral growth and cell kill This may translate into improved clinical results in the treatment of prostate cancer S368 957 Simultaneous Administration of Multiple Oncolytic HSV-1 Vectors Expressing Different Immunostimulatory Genes Provides High Antitumor Efficacy in a Poorly Immunogenic Tumor Model Yasushi Ino,1,3 Yoshinaga Saeki,2 Ennio A Chiocca,1 Robert L Martuza,1 Tomoki Todo.1,3 Molecular Neurosurgery Laboratory; 2Molecular NeuroOncology Laboratory, Massachusetts General Hospital/ Harvard Medical School, Boston, MA, United States; 3Department of Neurosurgery, University of Tokyo, Tokyo, Japan The use of replication competent, oncolytic herpes simplex virus type (HSV-1) vectors is a promising strategy for cancer gene therapy The vectors are genetically engineered to replicate and spread selectively within the tumor In the course of destroying tumor cells through a direct cytopathic effect, the vectors also induce tumorspecific immune responses Previously, a combination with a defective HSV-1 vector expressing mouse interleukin 12 (IL-12) or the soluble form of B7.1 (B7.1-Ig) enhanced the antitumor immune response elicited by G207, a multimutated oncolytic HSV-1 The use of replication-competent vectors for transgene expression should cause amplified gene delivery leading to improved antitumor efficacy and allow continuous generation of a high-titer, homogenous vector stock To test this theory, we used the replication-competent HSV1 vector, HsvQuik1, as the backbone to express various immunostimulatory genes Like G207 and MGH-1, HsvQuik1 has a deletion in both copies of the γ34.5 gene and an inactivation of the ICP6 gene The use of BAC plasmids and two different recombinase systems allowed us to construct multiple replication-competent HSV-1 vectors simultaneously in a relatively short time A desired transgene was inserted into the ICP6 locus and controlled by the immediate-early 4/5 promoter Three replication competent HSV-1 vectors, vHSV-B7Ig, vHSV-IL12 and vHSV-IL18, expressing B7.1Ig, IL-12 and IL-18, respectively, plus a null-expressing control vector (vHSV-null) were created These vectors also contained the GFP marker gene driven by the ICP6 promoter The four vectors demonstrated a similar in vitro replication capability on Vero cells They also showed a similar cytopathic effect on Neuro2a murine neuroblastoma cells in culture, killing approximately 70% of cells by day four at a multiplicity of infection of 0.1 A/J mice with bilateral subcutaneous tumors of syngeneic, poorly-immunogenic Neuro2a were used to test the efficacy in vivo When vectors were inoculated directly into the left tumor at 3x105 pfu (days and 3), each of the three vectors expressing an immunostimulatory gene was significantly more efficacious than the control vector on both the inoculated and the non-inoculated tumors, although no difference was observed among the three When vHSV-IL18 (1x105 pfu) was combined and simultaneously inoculated with one or two other vectors (total 3x105 pfu), the combination with both vHSV-B7Ig and vHSV-IL12 was the most effective in inhibiting the tumor growth on the inoculated side as well as the contralateral side, leading to Molecular Therapy Vol 7, No 5, May 2003, Part of Parts Copyright ® The American Society of Gene Therapy OTHER DNA VIRUSES cure of bilateral tumors in of animals The antitumor effect on the contralateral side by these three vectors combined was not manifested in athymic mice, indicating that it requires T cell mediated immune responses These findings suggest that the expression of immunostimulatory molecules can augment the antitumor action of oncolytic HSV-1 vectors via enhancement of antitumor immune responses The antitumor efficacy may be further improved by combination of multiple vectors expressing different transgenes 958 Immunological Dissection of Transgene Product-Mediated Responses Following Intracerebral Injection of a HSV-1 Amplicon Vector William J Bowers,1,3 Sean Hurley,2 Michael A Mastrangelo,3 Jeffrey Senfield,2 Howard J Federoff,1,3 John A Olschowka.2 Neurology, University of Rochester School of Medicine and Dentistry, Rochester, NY; 2Neurobiology and Anatomy, University of Rochester School of Medicine and Dentistry, Rochester, NY; Center for Aging and Developmental Biology, University of Rochester School of Medicine and Dentistry, Rochester, NY Immunological considerations related to vector safety and gene product expression become apparent with the development and application of direct gene transfer systems for the study and/or treatment of chronic CNS diseases In the current study, we examine the immunological influence of the transgene product in inflammatory response induction Transgenic mice that over express an E coliderived β-galactosidase (lacZ) transgene from birth within their skeletal muscle (LacZ Tg) were successively backcrossed into the C57Bl/6 genetic background and used to assess immunological effects of amplicon-delivered lacZ in an animal model tolerized to this “self” protein LacZ Tg and C57Bl/6 control mice were transduced with either 1.5 μl of vehicle (sterile saline) or a lacZ-expressing amplicon (HSVlac) packaged using a helper virus-free system Animals were sacrificed at 2, 7, or 21 days post-transduction for either mRNA analysis (using real-time quantitative RT-PCR) or combined immunocytochemistry (ICC)/stereological quantification Quantitative RT-PCR analyses of the pro-inflammatory cytokines IL-1β, TNFα, and IFN-γ, the CC chemokines MCP-1 and MIP1α, the CXC chemokine IP-10, and the adhesion molecule ICAM1 elucidated distinct expression level profiles as a function of time in the HSVlac-injected LacZ Tg versus control mice IL-1β, TNFα, ICAM-1, MCP-1 and IP-10 mRNA levels were significantly elevated above saline controls at Day in both LacZ Tg and control mice Importantly, ICAM-1, MIP-1α, TNF-α, and IP-10 mRNA levels were significantly enhanced in HSVlac-injected C57Bl/6 mice on Day as compared to LacZ Tg mice, suggesting that the lacZ transgene product does participate in inflammatory processes in naïve animals Interestingly, a delayed INF-γ mRNA expression profile was evident in LacZ Tg mice where IFN-γ mRNA levels were greatest at Day 7, suggesting that an exacerbated TH1 response had been elicited in these animals By 21 days, all comparative measures between the control and LacZ Tg mice had returned to baseline levels Together these findings suggest that transgene products play a significant role in inducing immune responses that may limit transduction efficiency and more importantly could impact therapeutic safety in the settings of both immunological naiveté and tolerance 959 Derivation and Use of Composite Herpesvirus Amplicon Vectors for Gene Therapy Niza B Frenkel,1 Gabriela Kotlliroff,1 Ronen Borenstein,1 Orna Sharabani Yosef.1 Tel Aviv University, Tel Aviv, Israel of selected transgenes The composite ampliocn systems consist of (i) defective genomes with multiple reiterations of amplicon units containing a DNA replication origin, packaging signals and the added transgene(s) and (ii) helper viruses which supply trans acting replication functions as well as additional foreign genes of choice The vectors can be employed as: 1.composite amplicons fortified by helper viruses, which are mutated to decrease viral replication for vector safety 2.amplicons devoid of helper viruses, prepared ex-vivo in packaging cell lines We describe three types of vectors derived from different herpesviruses: the HSV-1 amplicon with wide host range and efficient targeting of epithelial and neuronal cells in solid tissues; amplicon-6, which employs the CD46 receptor targeting most efficiently lymphocytes, but can also enter other cells and lastly Tamplicon–7, which employs the CD4 receptor targeting mostly CD4+ T cells Several features of the herpesvirus amplicons make them advantageous for gene transfer: (i) large transgene capacity (ii) efficient replication / gene expression due to sequence reiterations (iii) no integration into the host genome, avoiding potential insertional mutagenesis (iv) single cycle vectors that not spread into the neighboring cells and tissues We will exemplify in different cells with the various amplicon vectors carrying GFP and B-Gal markers We will show the ability of producing cell associated vs cell free ampllicon vectors, employing sample genes Furthermore, as examples of oncolytic vectors we will show composite HSV-1 amplicon vectors containing toxic genes, in combination with mutant oncolytic HSV helper viruses carrying the deletion in the g134.5 genes and the virion host shutoff (ul41, vhs) functions providing oncolytic capabilities The vectors have been tested in normal cells and in the U87 human glioblasoma maltiforme tumor cells and H1299 lung carcinoma cells Overall the amplicon vectors have high gene expression capability and reduced replication capacity increasing safety for their potential use in human gene therapy 960 Examination of the Pathological Mechanism of the Microtubule Associated Protein Tau Using an HSV Amplicon Based Neuronal Gene Delivery System Sean E Lawler,1 Martin Ingelsson,2 Jean C Augustinack,2 Yoshinaga Saeki,1 Bradley T Hyman,2 E Antonio Chiocca,1 Richard Wade-Martins.3 Neurosurgery, Massachusetts General Hospital, Charlestown, MA; 2Center for Aging, Genetics and Neurodegeneration, Massachusetts General Hospital, Charlestown, MA, United States; 3The Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, United Kingdom The microtubule associated protein tau is the major component of intracellular neurofibrillary tangles observed in a wide range of neurodegenerative disorders These include Alzheimer’s disease, frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP17), progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD) A genetic link between tau and FTDP17, PSP, CBD and also late onset Parkinson’s disease (PD), has been established but the factors causing tangle formation and neurodegeneration remain poorly understood Elimination or prevention of tangle formation in these disorders may represent an important therapeutic approach Developmentally regulated tau splicing leads to the expression of six isoforms in human adult brain Alternative splicing of exon 10 generates isoforms containing either or C-terminal microtubule binding repeats, normally expressed in a 1:1 ratio Approximately twenty different tau point-mutations are associated with FTDP17, some of which lie in the intronic 5¢ splice site sequence of exon 10, Herpesvirus amplicon vectors are disabled viral vectors that can be employed in dividing and non-dividing cells for efficient transfer Molecular Therapy Vol 7, No 5, May 2003, Part of Parts Copyright ® The American Society of Gene Therapy S369 ... Pathological Mechanism of the Microtubule Associated Protein Tau Using an HSV Amplicon Based Neuronal Gene Delivery System Sean E Lawler ,1 Martin Ingelsson,2 Jean C Augustinack,2 Yoshinaga Saeki ,1 Bradley... Developmentally regulated tau splicing leads to the expression of six isoforms in human adult brain Alternative splicing of exon 10 generates isoforms containing either or C-terminal microtubule binding...OTHER DNA VIRUSES cure of bilateral tumors in of animals The antitumor effect on the contralateral side by these three vectors combined was not manifested in athymic mice, indicating that it requires