Gene Therapy (2001) 8, (Suppl 1), S1–S13  2001 Nature Publishing Group All rights reserved 0969-7128/01 $15.00 www.nature.com/gt

QUASISPECIES AND EVOLUTION PANDEMIC SPREAD: Robert G. Webster1•2, Yi Guan2, Scott Krauss1, Kennedy Esteban Domingo Shortridge2, Malik Peiris2 Centro de Biologfa Molecular "Severo Ochoa" 1) Dept. Virol Mo! Biol, St. Jude C R Hosp., 332 N. (CSIC-UAM). Cantoblanco, 28049 Madrid, Spain Lauderdale, Memphis, Tennessee, and 2) Dept Microbiol, Hong Kong Univ., SAR China

High mutation rates and quasispecies dynamics are a Influenza virus continues to evolve, and new antigenic drift hallmark of RNA . Recent studies with foot-and• variants emerge constantly, giving rise to yearly epidemics. In mouth disease virus (FMDV) have revealed rapid addition, strains to which most humans have no coevolution of antigenicity and cell tropism of virus appear suddenly, and the resulting pandemics vary from populations in cell culture and in vivo. Remarkable serious to catastrophic. Studies in aquatic birds over 25 years expansions of host cell tropism occurred in a clonal have established that they play an important role in the natural population of FMDV as a result of prolonged cytolytic history of influenza viruses. In the past four years there have replication in BHK-21 cells. Reversion of mutants with been two different transmissions of viruses to humans in Hong Kong. The first genetic lesions associated with decreases in viral , occurred in 1997, when 6 of 18 infected humans died of H5Nl influenza virus. In March and analysis of the mutant spectra of revertant 1999, H9N2 influenza viruses were isolated from two children populations, have indicated the presence of memory in Hong Kong and unconfirmed reports of an additional five in . Memory can be durable cases have been reported from Southern China. The re• and memory levels are fitness-dependent. Use of FMDV emergence of H5Nl influenza viruses in poultry markets in mutants covering a 106 - fold range in relative fitness 2001 resulted in slaughter of all of the poultry in Hong Kong values has permitted progress in understanding the and re-evaluation of the role of poultry markets as the site for mechanisms of viral by accumulation of of influenza viruses. Although no human cases mutations and by enhanced mutagenesis (virus entry into of H5Nl were detected in 2001 the presence of multiple error catastrophe). These observations may find reassortants containing gene segments related to HSNl/97 application in improved diagnostic procedures and in the influenza viruses was of great concern. Sooner or later these viruses will acquire development of new antiviral strategies. the ability for human to human transmission and it is urgent to be able to detect such viruses as early as possible. The long-term goal is to define the molecular markers of such transmission.

Protection from SIV vaginal challenge using Sabin EMERGENCE OF NIP AH VIRUS. SherifR. Zaki, M.D., Ph.D. vectors. S. Crotty, CJ. Millel", B.L. Lohman~ M. Neagu, L. Compton#, Centers for Disease Control & Prevention, 1600 Clifton Rd., N.E., D. Lu#, F. X.-S. LO#, L. Frittl, J.D. Lifson , and R. Andino•. Dept MS-G32, Atlanta GA 30333 Microbiology & Immunology, UCSF; #California Regional Research Center, Dept Pathology, UCD; "Depts Pathology, Microbiology, & Immunology, School of Veterinary Medicine, and An outbreak of severe febrile associated with high Center for Comparative Medicine, UCD;® Retroviral Pathogenesis mortality rates was reported in Peninsular Malaysia beginning in Laboratory, AIDS Program, SAIC Frederick, Frederick, MD. late September 1998. By mid-June 1999, more than 265 The Sabin live poliovirus vaccine is one of the best human . It encephalitis cases, including l OS deaths, had been reported in produces long lasting immunity and herd immunity; it is easy to Malaysia, and 11 cases of encephalitis or respiratory illness with experimentally manipulate; it has a proven safety and efficacy record in one death had been reported in Singapore. Pathologic, electron over I billion vaccines; it is cheap to produce and distribute; and most importantly, it produces a potent mucosa! immune microscopic, serologic, and genetic studies indicate that this virus, response. These beneficial characteristics have provided the bases for the optimism on the now known as Nipah virus, belongs to the family current efforts for the poliovirus eradication program. The WHO wild and is most closely related to the recently discovered Hendra virus. poliovirus eradication effort has been very successful, and we are Hendra and Nipah viruses are likely representatives of a new genus hopeful that wi ld poliovirus can be eliminated. In the other within the family Paramyxoviridae. hand, with respect to AIDS vaccine, the capacity of poliovirus to Like Hendra virus, Nipah virus is unusual among the generate a strong mucosa! is particularly important given that greater than 90% of HIV-I infections worldwide have been paramyxoviruses as it exhibits an extended host range, with natural sexually transmitted. Any strategy to control the AIDS pandemic must and experimental infections and fatal cases occurring in swine, include a vaccine that prevents sexual transmission of HIV-I. We have humans, cats, and dogs. The emergence of this disease can be developed a live poliovirus based vaccine vector system, making it attributed to a number of factors including environmental factors, possible to insert gene fragments derived from various into the animal husbandry and deforestation. Recent studies including viral full poliovirus . This vaccine vector system is able to stimulate potent immune responses directed against desired isolation have implicated fruit bats as a possible reservoir for viral pathogens both in mice and . Now we generated a series of new Sabin-SIV viruses Nipah virus. containing SIV gag, pol, env, nef, and tat in overlapping fragments. Nipah virus is a newly recognized disease, with a These Sabin-SIV recombinants were then inoculated into seven spectrum of microscopic morphological changes involving the macaques as a candidate S I V vaccine. All monkeys made substantial CNS in humans and the in pigs. Widespread anti-SIV serum and mucosa! immune responses. Vaccinated macaques, and twelv endothelial cell infection, vasculitis, and CNS parenchymal cell e control macaques, were challenged vaginally with pathogenic SIV mac25 I. Strikingly, 4 of 7 vaccinated animals exhibited substantial infection play an essential role in the fatal outcome of infection in protection against the vaginal SJV challenge. In two of the seven SabRV• humans and appear to be central to the pathogenesis of this SIV vaccinated monkeys we found no virological evidence of SIV emerging infectious disease. infection following challenge, indicating that these two monkeys were completely protected. Three of 6 control animals developed clinical AIDS by 48 weeks post-challenge. In contrast, all 7 vaccinated monkeys remain healthy as judged by all clinical parameters. These results demonstrate the efficacy of SabRV as a potential human vaccine vector. Abstracts S2 THE CORRELATES OF PROTECTIVE IMMUNITY MEASLES VACCINES-ISSUES OLD AND NEW. D. Griffin, F. AGAINST HIV AS DETERMINED BY PASSIVE Polack, H. Robinson, A. Valsamakis, S. Hoffman. W. Harry WITH HUMAN NEUTRALIZING Feinstone Dept of Molecular Microbiology & Immunology, Johns MONOCLONAL IN PRIMATES Hopkins Bloomberg School of Public Health, Baltimore, MD The Edmonston strain of measles virus (MV), isolated in 1954 by Flavia Ferrantelli1.2, Weidong Xu' ·2, Regina Hofmann-Lehmann 1•2, John Enders, is the source of most of the current measles vaccine Pei-Lin Li u, Lisa Ca vacini'-3, Marshall Posner2·3, Hermann Katinger4 strains. Wild type MV was attenuated primarily by multiple Gabriela Stiegler4 , Harold McClure5 and Ruth M. Ruprecht '· 2 passages in tissue culture, mainly chicken cells, a host that is not normally susceptible to MV infection. The molecular basis of 'Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115, attenuation is not fully understood, but sequence changes exist 2 3 throughout the genome, most notably in the genes for the Harvard Medical School, Beth Israel Deaconess Medical Center, hemagglutinin (H) surface glycoprotein and the large polymerase 4Institute of Applied Microbiology, Vienna, Austria and 5Yerkes . Normal sites of MV replication are endothelial cells, Regional Primate Research Center, Emory University, Atlanta, GA epithelial cells and and the vaccine is assumed to have similar target cells, but to replicate and spread less efficiently in Passive immunization with synergistic combinations of humans (approximately 5% of immunized infants develop a rash). The primary problem human monoclonal antibodies with this otherwise safe and effective (mAbs) directed against vaccine is the inability to immunize young infants, due in large conserved of the HIV envelope completely protected part to interference with virus replication by maternal . In 13 out of 16 rhesus monkeys challenged intravenously or orally developing countries measles often occurs prior to the age of with chimeric simian-human immunodeficiency virus (SHIV) routine immunization (9-15 mos) and 1/3 of the 1 million measles deaths each year occur in infants under the age of 1 year. Deaths strains; partial protection was seen in another two. A high are usually due to secondary infections associated a MY-induced degree of protection was seen among orally challenged suppression of cellular immune responses. Immune suppression is neonates. A quadruple combination of human mAbs isolated associated with failure to produce IL-12 and skewing of the responses toward type 2 cytokine production. Clinically significant from HIV clade B-infected individuals neutralized all 20 immune suppression is not a feature of the vaccine when the primary HIV clade C strains tested between 97.5% and 100%. vaccine is given in the standard dose (3000 pfu) although the type Because we only used mAbs with well-defined of immunity induced is similar to natural disease. A current goal is specificities, our primate studies also yielded key information to develop a measles vaccine that can be given to young infants who still have maternal antibody. Killed MV vaccines induce only for designing AIDS vaccines: the correlates of immune short-lived protective immunity, no cellular responses, poor quality protection. Vaccine strategies that can evoke antibody antibody and predispose to more severe immune complex• responses to epitopes recognized by the mAbs we used could be mediated disease (atypical measles). To improve immune responses we have explored the use of DNA vaccines, alphavirus• impmtant components of successful AIDS vaccines. Also, vectored vaccines and MV engineered to express IL-12 and each of passive immunization with synergistic combinations of these has provided information on induction of protective neutralizing human mAbs may be effective in preventing immunity to MV shows some promise for future vaccine development. maternal HIV transmission when given as post-exposure Funded by the NIAID prophylaxis at birth and as prophylaxis against milk-borne and the Gates Foundation transmission. RCAS VECTORS: NEW APPLICATIONS AND OLD RECOMBINANT NEWCASTLE DISEASE VIRUS AS A PROBLEMS. Eugene Barsov, Jangsuk Oh, Holly VACCINE VECTOR Zheng, Andrea L. Ferris and Stephen H Hujlhes. HIV Program, Peter Palese, Takaaki Nakaya, Jerome Cros, Man-Seang Park, NCI-Frederick, P.O. Box B, Frederick, MD 21702. Yurie Nakaya, Hongyong Zheng, Ana Sagrera, Enrique Villar, and Adolfo Garcia-Sastre There are several blocks to the replication of avian sarcoma leukosis viruses (ASL Vs), including the RCAS vectors, in Department of Microbiology, Mount Sinai School of Medicine, mammalian cells. The first is at the membrane of the cell: New York, NY 10029 mammalian cells do not express receptors that are efficiently recognized by any of the known ASLV envelope subgroups. We A complete cDNA clone of the Newcastle disease virus (NOV) have overcome this block in two ways, either by expressing a vaccine strain Hitchner B 1 was constructed, and infectious cloned receptor (usually tva, the receptor for subgroup A) in the recombinant virus (rNDV /BI) was generated by reverse genetics target cell and by creating RCAS vectors that express and techniques. The influenza A/WSN/33 virus hemagglutinin (HA) incorporate either the ecotropic or the amphotropic MLV gene was inserted into the recombinant virus between the P and M envelope. Both of these solutions have particular advantages: we genes. The rescued virus (rNDV/81-HA) efficiently expresses the have created lines of transgenic mice that express the subgroup A HA protein on the surface of infected cells and the HA was also receptor in a tissue-specific manner which makes it possible to incorporated into NDV particles. rNDV/B 1-HA virus replicates direct the RCAS vectors to particular tissue types slightly slower and to lower titers than rNDV /B 1 in embryonated in vivo. The alternative approach (using chicken eggs and shows increased attenuation properties when MLV derived envelopes) makes it compared to the parent virus. Recombinant NDV/ B 1-HA is possible to use the RCAS vectors with a variety of mammalian nonpathogenic in mice and induces a strong humoral antibody cells; however, the vectors are still replication defective in response against influenza virus as well as NDV. Finally, this mammalian cells. We are continuing to study the additional recombinant virus provides complete protection against a lethal blocks to ASL V replication in mammalian cells. The second dose of influenza virus challenge, demonstrating the potential of block (a block to assembly) can be overcome using elements recombinant NOV as a vaccine vector. derived from other . A third block to replication is still under investigation.

To make the RCAS vector system easier to use, we have recently developed a new shuttle vector, RSVP (B.CAS Shuttle Yector flasmid) which makes it much easier to molecularly clone both w1integrated and integrated viral DNA. We have also developed an RCAS-based gene trap vector (pGT GFP) that can be used with either avian or mammalian cells.

Gene Therapy Abstracts S3 Replicative and semi-replicative retroviral vectors: new TARGETING STRATEGIES FOR REPLICATION• powerful gene transfer vectors for cancer gene therapy COMPETENT VECTORS David Klatzmanni., Georg Holzer', Stephane Trajevski1, Nori Kasahara, MD PhD Sunkary Solly', Elizabeth Nelson2, Milaine Grisoni2 'Biologie et Therapeutique des Pathologies Immunitaires, Institute for Genetic Medicine, University of Southern California CNRS/UPMC ESA 7087 Hopital Pitie-Salpetriere, 83 bd de 2250 Alcazar Street, CSC-240, Los Angeles, CA, 90033 USA ' l'Hopital, F-75651 Paris, FRANCE Conventional replication-defective murine leukemia virus 2 Genopoi:etic S.A., 22 rue Esquirol, 75013 Paris, FRANCE (ML V)-based retroviral vectors have generally proven inadequate for in _vivo gene transfer. While ML V-based replication-competent The bottleneck of cancer gene therapy lies in poor gene retrovirus (RCR) vectors containing inserts have been transfer efficiency. We reasoned that because tumors are made of descnbed prev10usly, such vectors usually exhibited rapid constantly dividing cells, they would be efficiently transduced only rearrangement and transgene deletion within 1 or 2 replication with vector systems allowing transgene propagation. We thus cycles. By altering the insert position, we have developed ML V• designed three, fully or semi-replicative, retrovirus-derived vector based RCR vectors that can maintain trans 0 ene insert stabilitv over 0 systems: one inherently replicative vector, one defective vector multiple serial replication cycles, and we have demonstrate·d that propagated by a helper retrovirus, and two defective "yin-yang" these vectors can achieve highly efficient replicative spread and transgene delivery in vivo within solid tumors in animal models. vectors which can generate infectious defective retroviral particles In contrast to a number of oncolytic viruses in development as by transcomplementation. We obtained very efficient transgene cancer therapeutics, ML V-based RCR vectors replicate without propagations in vitro with each of these systems. In vivo, we could lys1s of the host cells and can spread via direct cell-to-cell budding, transduce >85% tumor cells with replicative vectors, compared to hence may be less likely to elicit robust immune responses that

STRATEGIES TO IMPROVE THE UTILITY OF Genetically Retargeted Adenovirus Vectors for Human Gene CONDITIONALLY REPLICATIVE ADENOVIRUS Therapy. Peter W. Roelvink, David A. Einfeld, A. Rosanna (CRAds). Schroeder, Masaki Akiyama, Imi Kovesdi, C. Richter King and Thomas J. Wickham. GenVec Inc, 65 West Watkins Mill David T. Curiel, M.D., Director, Division of Human Gene Rd, Gaithersburg, MD 20878, USA Therapy, Gene Therapy Center, Increasing the tissue selectivity of adenovirus (Ad) vectors for The University of Alabama at Birmingham, Birmingham, gene therapy has the potential to make these vectors safer, to reduce humoral and CTL response against the vector, and to better AL, USA. enable their systemic administration. The development of these vectors requires the ablation of native receptor binding and the CRAds represent an exc1tmg new approach for addition of tissue-specific ligands. The ability of the adenovirus cancer therapy. Clinical results employing CRAds as single vector to bind to the native Coxsackie and Adenovirus Receptor (CAR) has been an obstacle to the development of targetable modality agents, however, have been disappointing. On this adenovirus vectors in human gene therapy and cancer treatment. basis, we have sought to improve CRAd efficacy by Recent studies have resulted in the identification of a conserved addressing critical biologic steps relevant to their oncolysis receptor binding site on the fibers of the CAR binding . We have mutated the AB loop of the Ad5 fiber, and physiology. In this regard, specific steps to improve the introduced a HA tag into the HI loop of the fiber. This resulted in a infectivity and lateralization of CRAds have been vector that no longer binds to CAR and, instead, is directed to a endeavored. These maneuvers have been shown to allow pseudoreceptor in a cell line that was created to allow for the dramatic enhancements in the oncolytic potency of CRAd production of mutant Ad vectors. We demonstrate that it is feasible to redirect an Ad vector to a new receptor. Some residual agents. transduction (<2%) of cell lines is observed with the singly ablated vector. In vivo evaluations of these singly ablated vectors demonstrated that, whereas transduction of the liver is I 0-fold reduced, transduction of the lung and muscle tissues is unaffected. This data suggests that the continued interaction of penton base with offers a surrogate route of entry into tissues in vivo. We therefore improved our Ad vectors further by removal of the RGD sequence from the penton base protein. This resulted in the prototype "doubly-ablated" vector, that has no affinity for the fiber receptor and the av-integrins. The role of the CAR and integrins in vector bio-distribution, , and, possibly, in vivo immune responses to the vector, will be discussed. Results of studies towards redirecting the ablated vectors to new receptors will be preselted. We will show the first results obtained with these vectors in vitro and in vivo and discuss configurations that are presently being considered for application in human (cancer) gene therapy.

Gene Therapy Abstracts S4 REPLICATING MEASLES VIRUSES ENTERING CELLS ONCOL YTIC POLIOVIRUS RECOMBINANTS FOR THE THROUGH TARGETED RECEPTORS TREATMENT OF MALIGNANT GLIOMA Roberto Catlaneo, Molecular Medicine Program, Mayo Clinic, Melinda Merrill, Elena Dobrikova, and Matthias Gromeier Rochester, MN 55905, USA Dept. of Microbiology, Duke University, Durham, NC 27710

Paramyxoviridae, including , measles and Newcastle Poliovirus CNS infections exclusively affect lower motor disease virus can have therapeutic effects in certain malignancies. neurons. We deciphered the molecular principles that underlie We are developing the Edmonston B strain of measles virus (MV), poliovirus organ- and cell-type specificity. While contributing to a safe human live vaccine, as a replicating vector for the selective the understanding of a unique neurological syndrome, insight into elimination of tumor cells. We show that the MV envelope can be the molecular biology of neuropathogenesis has also modified to elicit through certain cell surface . set the stage for a promising new avenue towards cancer therapy. Towards this aim specificity domains were added to the We discovered the poliovirus receptor CD155 to be expressed in extracellular (carboxyl) terminus of the attachment H protein. Recombinant MV in which the standard H gene was replaced with structures involved in development, matching one coding for a hybrid protein were then constructed. MV poliovirus' predilection for this CNS compartment. Developmental displaying a growth factor (EGF or IGF-1) or a single chain expression ofCD155 physiologically subsides towards partum, but antibody against human carcinoembryonic antigen (scAbCEA) ectopic upregulation is observed in malignancy. Tropism for a replicated to titers approaching those of the parental strain in tumor antigen prominently displayed in glial malignancy renders primate cells expressing a natural MV receptor. Moreover, these poliovirus a prime candidate for the engineering of oncolytic viruses efficiently entered rodent cells expressing the agents with superb targeting abilities for this type of neoplasm. corresponding human growth factor receptor, or CEA, To harness poliovirus for therapeutic use, we eliminated respectively. MV-H/EGF and MV-H/scAbCEA caused extensive neuropathogenicity through exchange of the cognate internal syncytia formation and cytolysis in these cells, whereas the ribosomal entry site (IRES) with its counterpart from . parental MV failed to infect. Taking advantage of a factor Xa The resulting chimeric PVS-RIPO exhibits a cell type-specific recognition site engineered in the hybrid H proteins, the propagation defect in cells of neuronal derivation. Consequently, displayed domains were cleaved off from virus particles, and intraspinal inoculation of PVS-RlPO in non-human primates failed specific entry and cytotoxicity were abrogated. We now aim to to induce symptoms of . HRV2 IRES function is uninhibited weaken or eliminate cell entry via the natural MV receptors, the in glial tumors and thus permits efficient viral oncolysis without ubiquitous regulator of complement activation CD46 and the B• collateral damage to healthy neuronal components of the CNS. and T- cell specific protein SLAM. We have identified residues This approach is the first example of targeting tumor-specific influencing the interactions with one or the other receptors, and we are producing recombinant viruses with combinations of these H gene expression at the level of translational control. This principle protein mutations. Recombinant MV which maintain the oncolytic has shown great promise in preclinical trials in experimental properties of wild type strains but gain specific cell entry may have animals. A prototype poliovirus-rhinovirus chimera is currently applications in the treatment of different malignancies. being prepared for clinical trials in patients with glioblastoma REFERENCES: J. Virol. 74:9928 and 75:2087; Blood 97:3746 multiforme. and 98:October 2001.

EXPLOITING TUMOUR SPECIFIC Drug-reguiatable conditionally replication-competent SIGNALING DEFECTS WITH VESICULAR STOMATITIS adenoviral vectors Heung Chong*, Anja Ruchatz*, David VIRUS. John C. Bell, Centre for Cancer Therapeutics, Ottawa Regional Cancer Centre, 503 Smyth Road, Ottawa, Ontario, Riddle*, Dragan Jeremovic*, Tim Clackson+, Victor M. Rivera+, Canada, KI H I C4 Richard G. Vile* *Molecular Medicine Program, Mayo Clinic, Rochester, Minnesota +Ariad Pharmaceuticals, Cambridge, are circulating factors that bind to cell surface receptors activating a signaling cascade ultimately leading to a Massachusetts number of biological responses. Two of the outcomes of interferon signaling are tightly linked: ( 1) an antiviral response and (2) Replication-competent adenoviral vectors are potentially far induction of growth inhibitory and/or apoptotic signals. Cancer more efficient than replication-defective vectors. However, for cells, gain a survival advantage over their normal counterparts by reasons of safety, there is a need to restrict both acquiring mutations in growth inhibitory or apoptotic pathways spatially, by limiting replication to certain cell types, and and, in the case oflnterferons, would do so at the expense of critical antiviral defense mechanisms. While on the one hand, temporally. In order to control replication temporally, we describe tumour cells gain a significant growth advantage by mutating here a pair of drug-inducible conditionally replication-competent interferon response genes, on the other hand; they will be more adenoviral vectors in which replication competency is controlled susceptible to virus infection. Our approach has been to use by the rapamycin dimerization system. In one vector, Ad(Zl2-I• Vesicular Stomatitis Virus (VSV), to exploit this tumour specific ElaElb19k), the El genes are placed downstream of the sequence viral susceptibility for therapeutic advantage. We have examined recognized by one of the transcription factors. The second vector, the spectrum of tumour cell types which harbour interferon defects in both cell lines and primary biopsy material. In tumour bearing AdC4, expresses the two transcription factors which constitute the animal models, VSV is an effective therapeutic whether drug-regulatable system. Co-infection of a number of cell lines by administered locally or systemically. We are studying a series of the vector pair, in the presence of the dimerizer, leads to El attenuated interferon inducing VSV mutants which have an expression and a marked increase in viral replication, as shown by improved safety profile in pre-clinical studies. The bio• Southern blots and replication assays. Furthermore, expression of a distribution, efficacy, physical stability, immunogenicity and reporter gene from a replication-defective vector, Ad-GFP or Ad• growth characteristics of these mutants will be discussed. GM-CSF, can be augmented by up to 18-fold by co-infection with the replicating vector pair and the dimerizer. Similar results are obtained when the vectors are directly injected into subcutaneous HTI080 xenograft tumors in nude mice. We believe that vectors based on this principle will be a useful additional tool to enhance efficiency and safety of gene delivery for anti-cancer therapy.

Gene Therapy Abstracts m S5 MONITORING THE IN VIVO EXPRESSION KINETICS OF CONDITIONALLY REPLICATING HERPES SIMPLEX VIRAL ON COL YTIC VIRUSES. Kah-Whye Peng and Stephen J VECTORS FOR CANCER THERAPY. Robert L. Martuza, M.D., Russell, Molecular Medicine, Mayo Clinic, Rochester MN 55905 Higgins Professor of Neurosurgery, Harvard Medical School, Rational clinical development of oncolytic viruses is problematic Chief, Neurosurgery Service, Massachusetts General Hospital because their spread and elimination cannot easily be monitored in vivo. For example, using existing (nontrackable) oncolytic viruses Herpes viral vectors offer unique advantages as vectors for tumor there is no satisfactory way to measure the efficiency of tumor cell therapy. They are large (153kb), fully sequenced, stable, grow in transduction, the changing levels of viral gene expression over time or the timing of virus elimination. Quantitative data most mammalian cell types and can be constructed either as a describing the in vivo kinetics of oncolytic viruses in individual replication-competent or a replication-defective vector. The genes patients is needed to provide a rational basis for dose escalation associated with neurovirulence have been defined and can be and repeat dosing decisions in early phase clinical studies, and in deleted or altered to make the virus non-virulent. Anti-herpetic the longer term to facilitate the development of safer, more agents such as acyclovir and others exist which can abrogate an effective personalised oncolytic virotherapy programs. We infection. Efficient growth has been demonstrated in many types therefore generated trackable oncolytic measles viruses expressing inert (nonimmunogenic, nonfunctional, accurately measurable) of human malignancies. The initial vector which we have taken soluble marker , including the beta chain of human into human trial for malignant glioma is 0207, a multimutated chorionic gonadotropin, the extracellular domain of vector with deletion of both ICP34.5 and genes and with a lacZ carcinoembryonic antigen, and insulin C-. The marker disruption disabling the ICP6 gene. Data will be shown peptides did not compromise virus replication and they were demonstrating efficacy of 0207 in several human nervous system expressed concordantly with the measles 'structural and nonstructural proteins. In vivo and ex vivo kinetics of the tumors in animal models and demonstrating safety in several trackable viruses could be easily followed by measuring the including primates. The results of the Phase I clinical trial concentrations of the virally encoded marker peptides in serum or of 0207 for the treatment of malignant gliomas will be presented in tissue culture fluid. When mice bearing human tumor along with longer term follow-up of several patients since the xenografts were challenged with the trackable viruses, distinct published report and plans for a Phase II trial. More recently, kinetic profiles of marker gene expression could be correlated with herpes vectors have been explored for the treatment systemic distinct therapeutic outcomes. For example, (i) absent/insignificant marker gene expression indicated resistance to cancers as well. Such studies have exploited the ability of these virus infection and was predictive of treatment failure, (ii) transient vectors to kill prostate cancer cells while sparing normal nerves high-level marker gene expression indicated susceptibility to virus required for bladder and sexual function. These pre-clinical infection and virus-mediated cell killing, and predicted a studies will be discussed in consideration of a clinical trial in successful therapeutic outcome with tumor regression, and (iii) prostate cancer. Studies involving , persistent high-level gene expression indicated susceptibility to virus infection but resistance to virus-mediated cell killing and was intraarterial therapy, the use of herpes vectors to stimulate an anti• accurately predictive of partial or total treatment failure. In tumor immune response, the use of promoter-specific vectors, conclusion, the use oncolytic viruses expressing inert soluble interactions with radiotherapy and , and further marker polypeptides should greatly facilitate the rational vector development will be presented. development of effective, individually tailored cancer virotherapy.

Systemic administration of replication-selective adenoviruses. SELECTIVELY REPLICATING ADENOVIRUSES D. Kirn. Viral and Gene Therapy Program, Imperial Cancer Research L. Johnson, S. Laquerre, J. Shen, A. Perez, G. Habets, M. Giedlin, Fund, Molecular Oncology Unit, Hammersmith Hospital S. Freeman, and T. Dubensky, Onyx Pharmaceuticals, 3031 Replication-selective viruses and bacteria are being developed for the Research Drive, Richmond, CA 94806. treatment of cancer. d/1520 (Onyx-0 I 5 or CJ- I 042, Pfizer) is an EIB-55kD gene-deleted replication-selective adenovirus that was the ONYX-OJ 5 is an adenovirus (Ad) deleted of the EIB 55-kDa first genetically-engineered, replication-selective virus to enter trials in humans. We used a staged clinical development approach that gene that selectively replicates in cancer cells having a non• proceeded from intratumoral (i.t.) to intraperitoneal (i.p.), intra• functional p53 pathway. ONYX-015 has been studied in over 300 arterial (i.a.) and eventually intravenous (i.v.) administration. Over patients in a wide array of tumor types, and has successfully 230 cancer patients have been treated to date, including 174 i.t. (head demonstrated proof of concept in Phase II clinical trials in head and neck, pancreatic, GI met. to liver), 16 i.p. (ovarian), 31 i.a. (hepatic artery for colorectal met. to liver), 10 i.v. (lung met., any and neck cancer, in combination with chemotherapy. A Phase III carcinoma). d/1520 has been well-tolerated by all routes of trial has been initiated, and other indications, including hepatic administration, including doses of up to 2xl0 12 particles i.a. and arterial infusion ofONYX-015 in combination with chemotherapy, 2x I 013 particles i.v .. Common toxicities included fever (typically for treatment of liver-predominant gastrointestinal carcinoma, have grade 2-3 i.a./ i.v.), asthenia and injection site pain (i.t.). Clinically• relevant hepatotoxicity due to d/1520 was not demonstrated, also yielded very encouraging results. although transient grade 1-2 transaminitis was documented in some We are exploring diverse means to enhance the directed tumor patients following i.v. infusion at doses 2'.2xl0 12 particles. cytotoxicty of the selectively replicating adenovirus platform. A Reproducible evidence of viral replication was obtained (within .'.SI 0 procedure, termed bio-selection, was used to select for Ad5 days) following I) i.t., i.a. and i.v. administration, 2) in head and neck and colorectal cancer patients but not in pancreatic (i.t.) or ovarian variants that efficiently and selectively kill human cancer cells. A (i.p.) carcinomas; no patient had replication documented >IO days second approach substituted therapeutic for viral genes after treatment. Single agent-induced objective tumor regressions were within the adenovirus E3 region, while retaining the native Ad demonstrated in head and neck cancers (15-20%) but not in promoters. Thirdly, a new virus, ONYX-411, has been developed pancreatic, colorectal, ovarian or metastatic lung tumors. Evidence for potential synergy with chemotherapy has been obtained from that propagates selectively in tumor cells having a deregulated head and neck (i.t.) and colorectal cancer patients (i.a.). IL-I, IL-6, pRb-signaling pathway. Disruption of the RB tumor suppressor IL-I 0, interferon-gamma and TNF levels all increased acutely pathway and the resulting deregulated E2F activity is a hallmark of following i.a. and/or i.v . administration. Given the clear documentation of safety and feasibility with this approach following human tumor cells. Tumor-selective viral replication was intravascular administration, but the lack of significant single agent accomplished in ONYX-411 by combining a mutant ElA gene efficacy to date with d/1520, we carried out preclinical studies of (d/922/47), whose products are significantly impaired in their intravenous treatment with a significantly more potent adenovirus ability to bind and inactivate pRb, with substitution of the native d/922/947 (EIA mutant) in nude mouse-human tumor xenograft models; antitumoral effficacy was significantly supreior to dll 520 in viral EIA/E4 region promoters with the human E2F-l promoter. all models, and equivalent to or even superior to wildtype adenovirus This strategy to combine alterations in ElA with E2F-dependent i.v. The replication-selective virus approach has promise as a regulation of early viral gene expression has yielded oncolytic systemic treatment for cancer. viruses that are both safe and highly selective for the loss of an intact pRb-pathway in human cancers.

Gene Therapy Abstracts S6 PROSTATE-TARGETED ADENOVIRUS. Daniel Henderson, REOVIRUS THERAPY OF METASTATIC CANCER MODELS Ph.D.,. Calydon, Inc., Sunnyvale, CA IN IMMUNE-COMPETENT MICE Safety of administrating wild-type Ad5 either intratumorally and Patrick W. K. Lee, Faculty ofMedicne, University of Calgary, intravenously was demonstrated at intermediate doses (1_07 to 109 Calgary, AB, Canada particles) over 40 years ago. None of the trea!ed patients h_ad significant side-effects. Safety and efficacy will be the maJor Reovirus selectively replicates in and destroys cancer cells with issues as adenovirus doses escalate from l 0 11 to 1015 particles. an activated Ras signaling pathway. We have previously reported CV706 is a replication-competent adenovirus attenuated I 00: I that direct intratumoral injection of reovirus resulted in tumor compared to the wild-type Ad5 in PSA- cells. CV706 has regression in mice. The application ofreovirus therapy to completed Phase 1/11 clinical trials in men with local prostate metastatic cancer models would be the next challenge. The cancer. No safety issues were encountered in the use of CV706 for objective of this study is to examine the effects of intravenous local therapy. CV787 is a replication-competent adenovirus (i.v.) reovirus treatment in metastatic models ofimmune• attenuated 10:000: I compared to the wild-type Ad5 in PSA- cells, competent mice. First, the maximum tolerated i.v. dose ofreovirus an unprecedented therapeutic index fo~ a cy_totox_ic agent as 8 measured in vitro. CV787 is capable of ehmmatmg distant mouse in C3H mice was determined to be 5X10 plaque forming units (PFU)/mouse. Using immune-competent C3H mice implanted with xenograft tumors with a single intravenous injection. Synergy of ras-transformed C3H-I0Tl/2 cells (C3 cells) at the hind flank, i.v. prostate cancer-specific Adenovirus varia_nts has be_en 8 demonstrated when combined with convent10nal therapies administration ofreovirus (4 times 5XI0 PFU's) resulted in including radiotherapy and chemotherapy. Synergy of CV706 significant reduction of tumor volumes. Combined treatment of reovirus with cyclosporine A (50 mg/kg) or cisplatin (3.0 mg/kg) with radiation for local prostate cancer therapy has decreased the further reduced the tumor size. To determine if reovirus therapy curative single-dose at least 50-fold in vivo. In addition, sy~ergy could be applied to experimental metastasis animal models, C3-L5 of CV787 and docetaxel for systemic therapy has opened a smgle• cells were introduced intravenously into C3H mice, which induced dose curative therapeutic window in excess of 1,000: l in vivo. rapid metastasis in the lung. Subsequent i.v. treatment with This progress, coupled with screening and immunoap~eresis to 8 control and remove pre-existent antibody to adenov1rus may reovirus (4 times 5XI0 PFU's) resulted in significant enhancement of survival rate of these animals. We also examined overcome the hurdles and achieve clinical benefits with systemic intravenous administration of adenovirus-based therapeutics. the effect ofi.v. reovirus therapy in the Lewis lung carcinoma metastasis mouse model, in which removal of the primary tumor Ongoing clinical trials of agents such as CV706 and CV7~7 will resolve the issues of safety and efficacy and hopefully pomt to a invariably leads to rapid metastasis in the lung. We found that i.v new mode of cancer chemotherapy, one that includes the use of reovirus treatment resulted in significant reduction in tumor burden targeted cytolytic adenoviruses. in these animals (based on the number of lung metastatic foci and lung weight). In conclusion, i.v. treatment ofreovirus is effective in metastastic cancer models of immune-competent animals.

ATTENUATED MEASLES VIRUS THERAPY FOR SUCCESSFUL GENE TARGETING TO THE CORONARY LYMPHOMA ARTERIES OR MYOCARDIUM Deanna Grote, Stephen J Russell, Roberto Cattaneo, Richard Vile Stefano E Branzoli, Timothy O'Brien, Henry D Tazelaar, and Adele Fielding Christopher GA McGregor; Mayo Clink, Rochester, MN Mayo Clinic, Molecular Medicine Program, Rochester, MN Our aim was to achieve in vivo both specific gene targeting of We have investigated the cytoreductive potential of several the coronary vasculature an~ consistent "'.idespre~ highly effici_ent derivates of the Edmonston-B vaccine strain of measles virus transfection of the myocardmm by studymg chemical and physical (MV) in models of indolent and aggressive lymphoma in severe delivery systems for gene transfer to the transpl~nted heart. . combined immunodeficient (SCID) mice. Both intratumoral and Syngeneic rat heterotopic heart transplantation was used. Five intravenous injections of MV-Nse, a strain of MV rescued from groups were studied (n=6 in groups 1~). In_group 1, the Ad~13- 9 cloned DNA, resulted in significant retardation of tumor growth Gal (3x10 ) was delivered to the heart m modified Krebs solution compared to that in control mice treated with UV-inactivated MV, A by Langendorff perfusion for 20 minutes at 3 7°C. Group 2 _was a control without insert. In group 3, Ad-13-Gal (3x109) was dehvered in which all tumors inexorably regressed. In some cases, complete in the same way in modified Krebs solution B. Group 4 was a tumor regression occurred after treatment with MV-Nse. The control. In Group 5 (n=3) Ad-13-Gal (3xl09) was delivered in oncolytic potential of intratumoral injection of MV-Nse was not University of Wisconsin (UW) so_lution at 4°C. All hearts were abrogated by the presence of high levels of passively transferred examined 7 days after transplantation. Two unambiguous patterns anti-MV antiserum. The possibility that infection of tumor cells of transgene distribution were ~v!dent. In group I ~Krebs A) with MV might stimulate an anti-tumor immune response was transfection occurred charactenst1cally m the media of the suggested by the up-regulation of highly immunostim~latory heat coronary arteries. The number of positively staining vessels. was shock proteins after infection of the lymphoma cells with MV-Nse 10-55% (mean 32%, median 42%). Rare (0.6%) myocytes stamed. in vitro. The MV vaccine strain, Moraten, which is currently The 13-Gal content was 6.6±7.1 ng/mg. In group 3 (Krebs B), produced commercially and widely used as a safe and effect!ve transfection was highly efficient and entirely myocardial. The vaccine, was also found to be oncolytic. As a result of our mun~e number of positively staining cells was 30-95% (mean 62.5%, studies, a phase one pilot study to investigate the safety of this median 84%). No vessels stained. The 13-Gal tissue content was approach in patients with non-Hodgkin's l~mpho~a is ~ow _open. 574±332 ng/mg. 13-Gal expression was not detecte~ in controls._ In Group 5 (cold UW perfusion) only myocardial transfect1on Current studies in our laboratory are focusmg on mvest1gat1on of occurred and that in 14 - 42% of cells (mean 28%, median 28%). the role in the in MY-induced tumor regression, The 13-Gal content was 44.0±34.2 ng/mg. 13-gal expression was not the possibility of targeting MV entry to _CD20 positive cell~ and detected in control Groups 2 and 4. evaluation of the strain-related determmants of MV mediated For the first time, efficient targeted gene transfection to the oncolysis. coronary arteries has been achieved by chemical ~odification ?fa normotherrnic cardiac perfusion system Highly efficient myocardial transfection was also achieved.

Gene Therapy Abstracts S7

BIOENGINEERING THE ONCOL YTlC POTENTlAL OF ONCOTROPIC VECTORS DERIVED FROM THE REOVIRUS AUTONOMOUS PARVOVIRUS MVM(p). Earl G. Brown, Hong Liu, Jean L. Mbisa, John Bell, and David Nathalie Clement, Karim El Bakkouri, Chiat Cheong, Thierry Velu, Stojdl. Annick Brandenburger Centre for Research in Biopharmaceuticals and Dept. of Universite libre de Bruxelles: IRlBHN-IBMM, rue profs Jeener et Biochemistry, Microbiology and [mmunology, University of Brachet. B-6041 Gosselies, BELGIUM Ottawa, Ottawa, Ontario, Canada KlH 8M5. The preferential expression of autonomous parvoviruses in To be therapeutically effective an must possess tumour cells and their oncolytic activity have attracted attention to differential ability to replicate in normal versus transformed cells. The the potential use of these viruses as vectors for cancer gene therapy. majority of tumor cells are defective in their interferon response thus Moreover they are non-pathogenic in adult animals and they seem increasing their susceptibility to infection by some viruses. Viral to be associated with low or no immunogenicity. Other interesting infection induces interferon an antiviral cytokine that acts through the features are their episomal replication and high stability. induction of several responsive genes including the dsRNA dependant MVM/IL2 vectors were elaborated for tumour cell targeted gene protein kinase, PKR. PKR inhibits by phosphorylation of transfer that could induce an anti-tumour immune response. The the eucaryotic initiation factor eIF2a and is also involved in stress original problems encountered with these vectors, low titres and induced apoptosis. In this paper we demonstrate that the differential contamination with wild-type virus, have been partially alleviated ability ofreovirus to replicate in PKR-/- cells can be engineered using by integrating helper sequences into the host to genetic reassortants that contain novel combinations of genes from prevent recombination and to allow for the amplification of vector stocks through serial infections. parental TlL and T3D reoviruses. We examined the effect ofPKR on Based on the structure of spontaneously occurring defective reovirus infection using PKR knock-out (PKR -/-) murine embryo particles, we have derived second-generation vectors lacking all VP fibroblasts (MEF). Although both TIL and T3D grew to higher titres sequences downstream the insertion site for transgenes. Matched in cells lacking the PKR gene, TIL virus grew to higher titres than helper plasmids were also constructed that have no homology with T3D in either PKR -/- or PKR +/+ cells. The genetic basis for the the vectors in the right part of the genome. With this sy stem helper• increased ability of TlL to grow in each cell type was determined free vector stocks have been produced, but still at low titres. using TlL x T3D reassortants. The difference in yield in wild type With MVM/IL2 stocks obtained in a first generation packaging MEF (PKR +/+) segregated primarily with the Ml gene whereas the cell line, we could show that MVM/JL2 infection of K-1735 difference in yield in PKR -/- MEF was associated with other genes melanoma cells strongly reduces their ability to form tumours in that did not include the Ml gene. This indicates that the ability of the vivo. We could also show that MVM/IL2 infected cells implanted PKR gene to inhibit reovirus infection is dependent on the Ml gene. in one flank could prevent the development of non-infected Reassortant viruses containing the T3D Ml gene with the Ll, L2, M3 tumours on the opposite flank. This antitumour effect was observed and S2 genes of Tl L, possessed > 10 fold greater differential at a very low multiplicity of infection, replication abilities in PKR-/- versus normal cells than either parental virus.

GENETICALLY ENGINEERED VESICULAR STOMATITIS BASIS FOR ATTENUATION OF NOVEL VSVs HAVING VIRUS FOR USE IN GENE THERAPY: APPLICATION FOR REARRANGED GENOMES. THE TREATMENT OF MALIGNANT DISEASE E. Brian Flanagan, L. Andrew Ball , Trenton R, Schoeb, and Gail Marilyn Fernandez, Siddharth Balachandran, Mercedes Porosnicu, W. Wertz. Microbiology Dept. , Univ. of Alabama Med. School, Dubravka Markovic and Glen N. Barber Birmingham, Alabama 35294, Department of Microbiology and Immunology and Sylvester Comprehensive Cancer Center, University of Miami School of Medicine, Miami, Florida, USA, 33136. Vesicular stomatitis virus (VSV) is the prototypic virus of the Rhabdoviridae. The genome is non-segmented negative-sense Our laboratory has shown that vesicular stomatitis virus, VSV, a RNA with 5 genes having the order 3'NPMGL5'. Transcripti on is relatively non-pathogenic, negative-stranded RNA virus, can controll ed by the di stance of a gene from the single 3' promoter selectively induce the cytolysis of malignant cells, through the such that promoter proximal genes are transcri bed at higher levels induction of apoptotic cell death. VSV oncolytic activity was than those in promoter distal positi ons. effective, in vivo, in tumors defective in p53 function and in myc or We recovered infectious viruses having novel gene orders by ras transformed tumors. Furthermore, VSV caused significant manipulation of a genomi c cDNA. We constructed viruses having tumor regression when administered at sites distal from the tumor, the nucleoprotein gene, N, moved from first in the gene order to when delivered intravenously, or against syngeneic tumors in second or fourth, to down-regulate expression, and havin g the immunocompetent hosts. The simple genetic constitution of VSV, glycoprotein gene, G, encoding the major neutralizing epitopes, lack of any known transforming properties, well studied moved forward to fi rst or third fo r over-expression. Viruses with immunobiology and the ability to genetically manipulate this virus the gene orders 3'GNPML5 ', 3'PMGNL5', and 3'QPMNL5' affords an ideal opportunity to further enhance the oncolytic differed in gene expression, replication in cell culture, and potential of this generally innocuous organism. To examine these immunogenicity. Lethality in mice decreased in a stepwise manner possibilities, we constructed recombinant VSV s that carried either each time the N gene was moved away from the promoter. the suicide gene HSY-TK or the cytokine IL-4 to determine if the We investigated the basis of this attenuation. C57Bl/6 mice were oncolytic activity of the virus could be augmented. Importantly, inoculated intranasall y with the rearranged viruses and the viral we determined that viruses expressing TK or IL-4 were not only load, severity of neuropathogenesis, serum cytokine, and lgG viable but could synthesize their heterologous gene products to extremely high levels, In addition, the engineered viruses exhibited isotype profiles were determined. The viruses differed in their more potent oncolytic activity in vivo, than the wild-type virus and tissue localization, pathology and the type and levels of cytokines were found to stimulate specific anti-tumor CTL responses. induced. These findings indicate that gene rearrangement alone Collectively, our data demonstrate that VSV could provide a can alter the viral tropism and the cytokine response to infection. promising and exciting approach to cancer therapy and may be Furthermore, gene rearrangement is stable after repeated passages useful as an effective oncolytic agent against a wide variety of and since homologous recombination has not been observed malignant disease that harbor a diverse array of genetic defects. among the Rhabdoviridae this method provides an approach for the development of live attenuated vaccines and vectors.

Gene Therapy Abstracts S8 VACCIN1A VIRUS VECTORS FOR CANCER GENE THERAPY. Istvan Fodor, Tatyana M. Timiryasova, Bing Chen, COLON CANCER TARGETING REPLICA TING Nadja Fodor, and Daila S. Gridley. Loma Linda University, Loma ADENOVIRUSES: ROLE OF P300 IN TRANSACTIV A Tl ON Linda, California OF VIRAL PROMOTERS CONTAINING TCF SITES Christophe Fuerer, Michele Brunori, Maddalena Malerba, and Vaccinia virus (VV) mediated delivery of therapeutic genes Richard lggo, may represent novel potential strategies for malignant tumor therapy. We examined the biological effect of attenuated Swiss Institute for Experimental Cancer Research (ISREC), recombinant VV in glioma cells. Infection of human and rat Epalinges, Switzerland. glioma cells in vitro with VV carrying wild-type p53 induced significant growth inhibition and apoptosis irrespective of the Despite important advances in understanding the molecular basis endogenous p53 status of the cell lines. In an in vivo experiment, of cancer, few treatments have been devised which rationally target injection of vector VV or rVV-p53 into the C6 glioma xenografts known causal oncogenic defects in tumour cells. Selectively established in nude mice reduced tumor growth. The oncolytic replicating viruses have a major advantage over non-replicating effect was significantly greater with rVV-p53. Low doses (102- viruses to target these defects because the therapeutic effect of the 103 PFU) ofVV expressing cytokines (IL-2 and IL-12) activated injected virus is augmented by virus produced within the tumour. immune system of treated mice and inhibited glioma growth. To target colon tumours, we have developed replicating Combination of virus-mediated and p53 therapy adenoviruses that express the viral rep! ication (E2) genes from a of C6 tumors resulted in synergistic antitumor response providing promoter controlled by the Tcf4 transcription factor. Tcf4 !s a promising adjunctive strategy for treatment of glioma and constitutively activated by mutations in the adenomatous polypos1s potentially other highly aggressive malignant cancers. We also coli (APC) and (l-catenin genes in virtually all colon tumours, and found that cells infected with PUV-inactivated, replication• constitutively repressed by Groucho and CtBP in normal tissue. deficient VY effectively expressed wt p53 gene inducing cell Viruses with Tcf4 sites in the E2 promoter show greater than apoptosis. To generate recombinant VY new methods of DNA 100-fold selectivity of viral replication for cells with APC and ~• packaging and homologous recombination were developed using catenin mutations. Unfortunately, the viruses only show wild type PUV-inactivated helper VY that produce 90-100% recombinant activity in 50% of colon tumour cell lines. A possible explanation viruses. Both lytic and PUV-inactivated VV vectors are safe in for this is that ElA represses p300, which is a cofactor for Tcf• immunocompromised nude mice. Construction of conditionally dependent transcription. Mutation of the p300 binding site in EI A replicating VV strains will further increase the safety of the relieves this repression in transient transfection assays, but in the oncolytic VY-mediated cancer gene therapy protocols. context of the viral genome, the deletion does not lead to increased Sponsored by the US Department of the Army and NMTB. The view, opinions and/or finding contained in this report are those of the authors and E2 protein levels or increased viral activity. On the contr~ry, ~he should not be construed as a position, policy, decision or endorsement of the tip300 mutation reduces the cytopathic effe~t and repl'.cat10n federal Government or the National Medical Technology Testbed, Inc. efficiency of the virus. Different changes will be reqrnred to expand the range of colon tumours which can be targeted by our viruses.

PHASE I/II TRIAL OF ONYX-015 IN COMBINATION PV701, A STRAIN OF NEWCASTLE DISEASE VIRUS, WITH CHEMOTHERAPY IN PATIENTS WITH DEMONSTRATES BROAD SPECTRUM ONCOLYTIC ADVANCED SARCOMAS ACTIVITY AND KILLS TUMOR CELLS THAT HAVE A Evanthia Galanis, Scott Okuno, Brad Lewis, Andre Oliveira, FUNCTIONAL DEFECT IN THE INTERFERON Antonio Nascimento, Jeff Sloan, John Edmonson, Britta ANTIVIRAL RESPONSE Randlev, Margaret Uprichard, Joseph Rubin William S. Groene, Pete T. Buasen, Candice S. Duhon, Grace P. ONYX-015 is a provisionally replication competent McDaniel, Anthony R. Welch, Beth A. Incao, M. Scot Roberts, adenovirus with oncolytic activity in cells with malfunctioning Harvey Rabin and Robert M. Lorence. p53. Sarcomas represent a rational target for this approach Pro-Virus, Inc., Gaithersburg, MD, USA given the high frequency ofp53 mutations (40-75%) and mdm- 2 amplification (I 0-30%). Therefore, we undertook a phase I/II PV701, a naturally attenuated, replication-competent, oncolytic study ofONYX-015 (days 1-5 q mo administered strain of Newcastle disease virus, is in clinical testing as an intratumorally under radiographic guidance) in combination intravenously (IV) administered anticancer agent. In pre-clinical with MAP (mitomycin-C, doxorubicin, cisplatin) chemotherapy studies, the range of PV701 's cytolytic activity was evaluated in patients with advanced sarcoma. ONYX-15 dose escalation in vitro using a panel of human tumor cells and in vivo using human was performed from 109 pfu/dose to I 0 10 pfu/dose. Four patients have been treated so far. No serious toxicity attributed tumor xenografts in athymic mice. In vitro, 80% (43/54). of tumor cell lines were 2-4 orders of magnitude more sensitive to ONYX-015 was encountered. Immunohistochemistry of the than normal cells to PV701 mediated killing. In athymic mice, metastatic lesions prior to treatment showed that 3/4 patients had mutant p53, while one patient also had mdm-2 IV doses of 6E+07 and 6E+08 plaque forming units (PFlJ) amplification. Adenoviral replication was detected in 2/4 resulted in complete regressions in 90% of subcutaneous Hfl080 fibrosarcoma xenografts. Even at doses of 6E+05 PFlJ patient biopsies on d 5 of the 1'1 cycle, by in situ hybridization, (-1000-fold below the IV maximum tolerated dose), a significant despite the significant increase in the titre of neutralizing Abs. Both patients were treated at the highest dose level, and had p53 rate of partial regression was observed. No tumor regressions mutations. ONYX-015 viral DNA was detected by quantitative were observed using UV-killed PV701. Treatment of normal PCR in the peripheral blood in 3/4 patients on d 5, 1st cycle. cells (keratinocytes and fibroblasts) with neutralizing antibodies In summary, intratumoral administration ofONYX-015 in to interferon (IFN) resulted in increased PV701 cytotox,ctty combination with MAP chemotherapy appears to be well demonstrating that normal cell resistance to PV701 is IFN tolerated with no significant toxicity due to ONYX-015 being dependent. The effect of exogenous IFN was tested on tumor encountered. Viral replication in sarcoma tissue was and normal cells. Unlike normal cells, most tumor cells demonstrated in patients with p53 mutations treated at the evaluated were resistant to the antiviral effects of IFN. In highest dose levels, and there was evidence ofviremia on day 5 summary, PV701 displayed a broad spectrum of potent oncolytic of the 1st cycle. Ongoing accrual at the highest dose level will activity both in vitro and in vivo and its tumor selectivity is based assess efficacy. (Supported by UOl CA69912-05) on a functional defect in the IFN-mediated antiviral response.

Gene Therapy Abstracts m S9 REPLICATION COMPETENT AVIAN LEUKOSIS VIRUS: COMPARING THE RATE OF CELL ENTRY OF WILD-TYPE A NOVEL SYSTEM FOR STABLE POLYPEPTIDES AND VACCINE STRAINS OF MEASLES VIRUS DISPLAY. Pranaj D. Khare, Kah-Whye Pen_g,_ Stephen J. Russell and Mark. Federspiel. Molecular Med1cme ~rogram, Mayo Clinic Rochester, Rochester, MN 55905 Anthea L Hammond, Richard K Plemper, Roberto Cattaneo Molecular Medicine Program, Mayo Foundation, Rochester, MN Murine leukemia viruses (MLV) pave bee_n widely _used f.or disJ)lay peptides, growth factors ap.d smgle c):lam antibodies o~ its Measles virus (MV), a member of the Paramyxoviridae, remains surface envelope (SU) glycoprotems. The displayed polypeptides a leading cause of death particularly in infants in developing have been used in part to redirect _the virus tropism. However, a significant drawback of polypepl!de display on. MLV envelope countries. An effective vaccine based on the Edmonston strain glycoproteins is the weak, noncovalent mteracl!on between. the (MV-Edm) exists; this virus was produced by repeated serial viral SU and transmembrane (TM) glycoprotem~ that results 1µ a passage in non-permissive cells, resulting in its attenuation. MV• significant Ios_s of SU and the d1spfayed polypeptide from the viral particles. Avian leukos1s viruses (ALVJ o_ffer ~everal advantag~s Edm efficiently infects cells via the ubiquitous receptor CD46. as a platform for the gener_ation of polxpepl!de display and for tlie1r Most wild-type strains of MV in circulation preferentially infect selection against euk::u-yol!c targets. First, the SU_ano TM of ALY cells via a recently identified receptor SLAM (signaling envelope glycoprotems are covalently bound: httle or no SU 1s normally sbed from ALV particles. Second, replication-competent lymphocyte activation molecule). While the vaccine strain can also AL V can accommodate an additional -2.~ lcb in the genome efficiently utilize SLAM, entry of wild-type strains through CD46 providing ample room for encodmg a vanety of polypept1d1?S is not common and less efficient. mcluding single chain antibodies. Fmally, ALV could lie used m mouse tumor models since mammalian cells do not express receptors for ALV. . . We aim to characterize the interaction of wild-type versus For proof of J)rinciple, we beg!).n to ~est the ut1hty of AL V as a vaccine MV strains with their cellular receptors. To investigate polypeptide disp1ay platform by d1splaymg the 8 ammo fLAG epitope and 53 amino acid epidermal growth factor (EGF) hgand, whether differences in the rate of cell entry between wild-type and as N-terminal extensions of the subgroup A AL V [AL V(A)J SU vaccine strains and thus through SLAM and CD46 exist, we have glycoprotein. The genes encodmg tbe chtmenc env were established an assay to quantify the rate ofMV entry. In CD46- engineered into an mfectious mofecular clone of ALV(A), RCASBP(A). Viruses with the <:hi!]leric envelop_e glycoprote1~s positive Vero cells, MV-Edm enters rapidly, with maximal replicated at similar rates and to s1m1jar titers as w1IcFty~ virus m infection achieved after just 4 minutes at 37°C. We have an avian fibroblast cell line. Both w1ld-tyl)e and ch1!]1er!c v1mses investigated the effects of temperature on the rate ofMV-Edm incorporated similar levels of envelope glycoprotems mto viral partic1es and the FLAG and EGF polypeptides were displayed as entry and found it to be significantly reduced at 20°C. In BJAB assayed by immunoblots. Further passage of the ch1menc viruses cells expressing high levels of SLAM and low levels of CD46, in uninfected cells demonstrated that the FLAG and EGF entry ofMV-Edm is similarly rapid. Having established th~se polypeptides were stably displayed. Infectious ~e~rovirus 1mmunosorbent assay (!RISA) sho_wed the access1b1hty_ of parameters, we will compare the entry rate of a panel of wild-type displayed non-viral l)O!ypeptides on vmons. Moreover, the EGF MV strains in BJAB cells with that ofMV-Edm. displayed virions 6ind specifically to EGF-receptor (EGFR) expressing cells. Furthermore, the pretreatment of EGJ:R expressin_g cells with competitive ligand" and virions treated with Factor Xa protease abrogate the virions binding. Our data demonstrate that AL V provide an innovative platform for stable polypeptide display.

VESICULAR STOMA TITIS VIRUS INFECTS AND KILLS GENETIC RE-TARGETING OF ADENOVIRUS USING HUMAN LEUKEMIC CELLS WHILE SPARING NORMAL ARTIFICIAL FIBER KNOBS WITH DEFINED SPECIFITIES. BONE MARROW PROGENITORS. Brian D. Lichty, Harry Leif G. Lindholm, Boulanger P, Gunneriusson E, Hong SS, Atkins, Leigh Miller, Irina Frenkel, David F. Stojdl, John C. Bell. Magnusson M, Nygren PA, Strand P. Got-A-Gene AB, Goteborg, Ottawa Regional Cancer Centre, 3rd floor, 503 Smyth Rd Ottawa, Sweden. Ontario, CanadaKlH 1C4 For efficient and versatile use of Adenovirus (Ad) as an in vivo tumor therapy vector, modulation of the viral tropism is highly Vesicular stomatitis virus (VSV) is a cytolytic virus that is able to desirable. A novel method to genetically alter the Ad fiber tropism infect a wide array of cell types but whose replication is was developed in which the knob of the fiber gene was deleted exquisitely sensitive to the interferon anti-viral pathway. We have and replaced with an external trimerisation motif and the amino previously reported that many tumour cells have defects in their acid sequence RGD. The corresponding recombinant fiber retained interferon response and are susceptible to infection and killing by biological functions of the natural fiber, i.e. trimerisation, nuclear VSV. In this study we have further assessed the ability ofVSV to import, penton formation and showed correct ligand binding. The infect and kill human leukemia cells. We report the efficient recombinant virus AdS/FibR7-RGD yielded plaques on 293 cells, killing of virtually every leukemic cell line tested not only with a and showed an expanded tropism corresponding to fiber wild-type strain of VSV but also with attenuated mutants of VSV binding, which is useful for applications where gene transfer to and other RNA viruses. This is in sharp contrast to the remarkable cells Jacking CAR is needed. resistance to VSV displayed by normal bone marrow progenitors. However, fibers containing ligands dependent on disulphide ~ond We are able to purge bone marrow/leukemia cell line mixtures of formation, such as single chain antibody fragments, were neither the leukemic cells while not reducing the number of normal colony able to assemble into functional pentons in vitro in the presence of forming progenitors in the bone marrow. As well, we extend the penton base, nor to be rescued into infectious virions. This was due sensitivity of leukemia cell lines to patient samples and to improper folding of the new ligand in the . demonstrate that they are also more sensitive to VSV than are Through combinatorial protein engineering targeted to surface normal bone marrow progenitors. located residues of the Z domain, a three-helix bundle domain structure derived from the lg binding Staphylococcal protein A, libraries have been constructed from which Z variants with novel binding specificities, so called affibodies, have been selected using phage display. In the present work, functional viruses were constructed where each fiber carry one or two different functional affibodies. Affibodies seem uniquely suited for the construction of genetically retargeted Ad. In conclusion, functional, genetically re• targeted viruses can be made using ligands capable of correct folding in the mammalian cell cytoplasm.

Gene Therapy Abstracts S10 THE ASSOCIATION OF CLASS II HLA ALLELES AND RETROVIRUS REPLICATION TARGETED TO PROSTATE ANTIBODY LEVELS AFTER A SINGLE DOSE OF MEASLES TUMORS IMMUNIZATION Inna G. Ovsyannikova, Robert M. Jacobson, Robert A Vierkant, Christopher R. Logg*\ Aki Logg*, Robert J. Matusik\ Bernard H. Daniel J. Schaid, V. Shane Pankratz, Steven J. Jacobsen, and Bochneri, Noriyuki Kasahara* t Gregory A Poland. Mayo Vaccine Research Group, Mayo Clinic and Foundation, Rochester, MN Institute for Genetic Medicine*, Department of Pathology\ and Department of Urologi, University of Southern California School Polymorphic HLA class II genes encode cell surface glycoproteins of Medicine, Los Angeles, CA that play an important role in immune response to measles vaccine virus (MVV). We investigated the relationship of the HLA class II Departments of Urologic Surgery and Cell Biology\ Vanderbilt genes and MVV-induced Ab levels. A wide range of circulating University School of Medicine IgG Ab (EIA, BioWhittaker) responses to MVV have been observed in schoolchildren who received one dose ofMMR-II The inability of replication-defective viral vectors to efficiently vaccine (Merck) routinely administered at the age of 15 months. transduce tumor cells in vivo has prevented the successful We studied 242 subjects between the ages of5 and 12 years, where application of such vectors in genetic therapy of cancer. To no circulating measles virus had occurred in the community since address the need for more efficient gene delivery systems, we have our subject's births. Using PCR-SSP we performed HLA typing of developed a replication-competent retroviral (RCR) vector based genomic DNA in 72 MVV non-responders (NR) (IgG Ab upon murine leukemia virus (ML V). We have previously shown seronegative) and 170 combined seropositive normal (n=93) and that this vector is capable of transducing solid tumors in vivo with hyper-responders (n=77) (upper 10th percentile oflgG levels). high efficiency. The use of such a vector as a therapeutic agent, Analysis of individual allele frequencies between NR's and however, would not be feasible unless there were means to confine seropositive responders revealed that MVV NR's had an excess of replication to targeted tissues. In order to achieve tissue-targeted the DRBl *03 allele [odds ratio (OR) 2.22; p=.004). NR's were vector replication, we replaced various lengths of the more likely to carry the DQBI *0201 (OR, 1.90; p=.004) alleles enhancer/promoter of the U3 region with sequences from the compared to MVV responders. Statistically significant associations prostate-specific probasin promoter. We assessed the cell type• were also found between the DQAl *0201 (OR, 1.64; p=.05) and specificity of the replication of these vectors by infection of DQAl *0501 alleles (OR, 1,73; p=.02) and MVV nonresponse. The cultured cells from a variety of tissues, and the transcriptional less polymorphic DPA genes had a significant excess of the specificity of the hybrid promoters by luciferase assay. Our results DPAI *0201 allele (OR, 1.71; p=.04) in NR's compared to demonstrate that high transduction efficiency may be combined responders. In conclusion, HLA class II alleles have important with strict cell type-specificity in a replicating retroviral vector. associations with MVV Ab response. This information can The genomic stability of the vectors over 20 weeks of continuous increase our understanding ofMVV-induced immune response propagation was also examined, and no evidence of loss of based on genetic polymorphism of the HLA class II genes. prostate specificity was observed.

STRENGTH OF INTERACTION BETWEEN MEASLES SELECTIVE L YSIS OF TUMOR CELLS WITH A VIRUS HAEMAGGLUTININ AND FUSION PROTEIN DEFICIENCY IN THE INTERFERON TYPE I SYSTEM WITH INFLUENCES VIRUS GROWTH AND CYTOPA THICITY IN VIVO Richard K. Plemper, Anthea L. Hammond and Roberto Cattaneo Jovan Pavlovic, Jan Schultz and Karin Moelling Molecular Medicine Program, Guggenheim 18, Mayo Foundation Institute of Medical , University of Zurich, 8028 Zurich Measles virus (MY) is a clinically relevant member of the Viruses that selectively replicate in tumor cells with a given Paramyxoviridae family. Its envelope contains two types of genetic defect without affecting the surrounding normal tissue glycoproteins that are thought to form a multi subunit complex, represent good candidates as anti-cancer agents. In particular, which consists of covalently linked haemagglutinin (H) dimers and tumor cells with acquired defects in the interferon type I system non-covalently linked fusion protein (F) trimers. have shown to be highly sensitive to the cytopathic effect of To strengthen the potential ofMV as a vector for cytoreductive viruses. We employed the melanoma cell line Bl6-FIO, which gene therapy, we aimed to identify molecular requirements for lack a functional anti-viral defence gene, the interferon type I• efficient MV glycoprotein oligomerization, an early step of particle induced Mxl gene. These cells were injected into mice resistant assembly. When analyzing MVs with mutations in the H to influenza virus infection due to the antiviral activity of the Mx 1 membrane proximal domains we found mutant particles carrying protein. The virus chosen was an influenza A virus strain that an 8 amino acid extension at the H cytosolic tail with an altered shows a strong cytopathic effect on cultured mouse and human : cytopathicity was increased, surprisingly in cells but is non-pathogenic for humans and mice with intact conjunction with efficient particle assembly. Mutant particles innate immune system. Treatment of B 16-tumors with multiple replicated to a SO-fold higher titer than the corresponding parental injections of this virus strongly reduced their size and in some strain, Edmonston B. The phenotype is mostly based on a greater cases even tumor erradication was observed. Immunization of the infectivity to particle ratio. It is not sequence specific as it was mice against the same virus strain before tumor treatment had no reproduced after recovery of mutant particles carrying a different significant effect on the therapeutic efficiency. A similar extension at their H tail. Purified particles showed a higher content therapeutic strategy can be envisaged for human tumors like of matrix and fusion protein as compared to unmodified virus. Co• cutaneous lymphomas that often acquire mutations in the immunoprecipitations indicated that the strength of interaction of mutant H with the fusion protein is reduced as compared to interferon type I signalling pathway thereby becoming highly unmodified H. The reduced complex stability might facilitate susceptible to the cytopathic effects of viruses. conformational rearrangements necessary for membrane fusion. Indeed, the sensitivity of mutants to soluble receptor molecules is significantly increased in comparison with unmodified viruses. In conclusion, by studying the modified particles we identified the strength of glycoprotein interaction as a determinant for cytopathicity and efficiency of infection by measles virus.

Gene Therapy Abstracts S11 DEFECTIVE VECTOR/ REPLICATION-COMPETENT SELECTION OF A POL YPLOID MEASLES VIRUS: THE HELPER HSY COMBINATIONS FOR CANCER THERAPY GENOMES HA VE HEXAMERIC LENGTH Samuel D. Rabkin, Robert Martuza, Masahiro Toda, Tomoki Monika Rager and Roberto Cattaneo Todo. Molecular Neurosurgery Laboratory, Massachusetts Molecular Medicine Program, Guggenheim 18, Mayo Clinic, 200 General Hospital, 13th St., Bldg 149, Charlestown, MA 02129 First Street SW, Rochester, MN 55905, USA (HSY) defective vector / replication• Measles virus (MV) attachment to its cellular receptors is competent helper virus combinations are a novel vector system mediated by the envelope glycoprotein hemagglutinin (H). Display for cancer therapy. They couple the oncolytic activity of of different specificity determinants on MV H leads to efficient replication-competent HSY with high-level transgene expression entry through targeted receptors. Not all the displayed specificity from defective vectors. The defective vector genome contains no determinants are tolerated, however. When either one or two CD4 viral genes and consists of tandem repeats of the transgene• domains were displayed on H, they interfered with fusion-support expressing amplicon plasmid (-I 0-20 copies) for a total size of function. Nevertheless, the corresponding genes were transferred -I 50 kb. Defective vectors are generated in the presence of an to an infectious cDNA clone in place of the standard H gene and oncolytic replication-competent HSY vector, 0207, which is virus was recovered, although very inefficiently. Genome analysis efficacious in tumor therapy yet non-pathogenic. In vivo, of the recovered virus by RT-PCR indicated that variants with different tumor cells are likely infected by 0207 or the defective point mutations resulting in stop codons or purine insertions at a vector. Therefore, transgene expression is not terminated by HSY pseudo-editing sequence leading to a reading frame shift were replication as would occur with recombinant vectors. Defective selected for. In the case of stop codons virus populations consisting vectors have been used to express immunomodulatory transgenes only of the mutated variants were rapidly selected. Remarkably, in (i.e., IL-12, B7.1-Ig, GM-CSF) to augment the anti-tumor insertion mutants even after plaque purification a mixture of hybrid immune response induced by 0207 infection. In situ expression and shortened H proteins was detected. RT-PCR on purified virus of IL-12 or soluble B7.1-Ig in combination with 0207 was particles indicated the existence of two main populations of significantly better at inhibiting tumor growth than 0207 alone, genomes, one without changes and the other with the insertion in H occurred at doses well below the efficacious dose of 0207 alone, and a compensatory deletion in a purine run at the beginning of the and significantly enhanced the induction of tumor cell-specific L reading frame leading to a truncated polymerase. Both genomes CTL activity. This activity was not detected in athymic mice. are therefore necessary in order to provide a functional H protein Numerous manipulations of this system are possible, including as well as a functional L polymerase. In addition, both genomes can be efficiently replicated because their length is a multiple of the use of: (1) different HSY mutants as helper viruses; (2) six. Since infectivity of particles followed a single hit kinetics, we different genes, or multiple genes, expressed from the defective conclude that these particles package multiple genomes. We vector; and (3) mixtures of different defective vector and/or suggest that the requirement of hexameric genome length, helper virus combinations. combined with the plasticity of the MV envelope, drives the selection ofpolyploid MV.

REENGINEERING ADENOVIRUS REGULATORY ICP34.5 DELETED HERPES SIMPLEX VIRUS I WITH PATHWAYS TO ENHANCE ONCOL YTIC SPECIFICITY AND ENHANCED ONCOLYTIC AND ANTI-TUMOUR POTENTIAL: EFFICACY. M. Ramachandra, A. Zou, A. Rahman, M. DEVELOPMENT OF NOVEL REPLICATING VECTORS FOR TUMOUR THERAPY. Vaillancourt, M. Hom, S. Neugebauer, B. Sugarman, J. Howe, B. Faha, D. Johnson, H. Engler, W. Demers, R. Ralston, P. Shabram. Michael Robinson' , Binlei Liu', Jill Smith2, Robert Coffin'·' , BioYcx Ltd' Canji, Inc., San Diego, CA 92128 and University College London2, The Windeyer Institute, 46 Cleveland Street, London WIT 4JF. Recognition of the dependence of adenovirus replication on HSY in which the neurovirulence factor JCP34.5 is inactivated has been cellular regulatory pathways that are frequently disrupted in shown to direct tumour specific cell in tumour models both in vitro and in vivo. Such viruses have also been shown to be safe in Phase I clinical neoplastic transformation has led to development of novel trials by intra-cerebral injection in glioma patients. Previous work has used oncolytic adenoviruses for cancer therapy. Attempts have been serially passaged laboratory isolates of HSY-! which might be anticipated made to construct selectively replicating adenoviruses by totally or to be attenuated in human tumour cells as compared to more recent clinical partially deleting viral El b or Ela genes to render the vector isolates. In work aimed at producing viruses with enhanced anti-tumour dependent upon tumor cell functions for its replication. However, potential, we have deleted ICP34.5 from a number of HSY! clinical isolates such strategies frequently result in attenuation of the virus because and compared their replicative and cell killing potential in human tumour cell lines. Such ICP34.5 deleted clinical isolates showed greatly enhanced of the multifunctional nature ofElb and Ela proteins. To increase lytic capabilities in all tumour cells tested as compared to HSY I strain 17+, oncolytic activity while providing selectivity, we have constructed suggesting them to have great promise for cancer treatment. We have a virus containing intact Elb and a novel regulatory circuit which further engineered these viruses by deleting the gene encoding ICP47 and to couples viral replication to the functional p53 status of the cell. express GM-CSF, both aimed at maximising anti-tumour immune responses This regulatory circuit employs a dominant negative inhibitor of following intra-tumoural inoculation, virus replication and accompanying E2F-dependent promoters to control viral replication. To further cell death. Experiments comparing these viruses in in vivo tumour models have demonstrated considerable anti-tumour effects in a number of tumour enhance tumor cell killing and virus spread, the viral major late models, and demonstrate that ICP34.5 deleted HSY based on recent clinical promoter was used to drive temporal overexpression of a viral isolates of the virus have considerable potential for the treatment of human protein (E3-l l.6K) involved in cell lysis and virion release. The cancers, either through oncolytic effects alone, or through the co-delivery of reengineered virus showed selective replication in cancer cells anti-tumour or immune stimulating genes which might be expected to compared to normal cells in vitro. This virus also showed synergisc with the oncolytic effect. significantly enhanced efficacy compared to E 1b-deleted viruses such as d/1520 in human xenograft tumor models.

Gene Therapy Abstracts S12 STRATEGIES TO MAKE ADENOVIRUS-MEDIATED A TRACKABLE VACCINE STRAIN OF MEASLES VIRUS ONCOL YSIS DEPENDENT ON P53 MUTATIONS FOR INTRAPERITONEAL THERAPY OF OVARIAN 2, 3 Judith Roth, Philipp Koch, Jennifer Gatfield, Claudia Lenz• CANCER. Cynthia2 TenEyck1, Eva Galanis Kimberly Kalli , Stiippler, and Matthias Dobbelstein Lynn Hartmann, Ster,hen J Russell 1 & Kah-Whye Peng 1 lnstitut for Virologie, Universitiit Marburg, Germany. Phone: 49 1Molecular Medicine, Medical Oncology, 3Endocrine Research 6421 2864318. E-mail: [email protected] Unit, Mayo Foundation, Rochester, MN 55905, USA.

For cancer therapy, it would be highly desirable to design viruses In the US, epithelial ovarian cancer kills more women than any with cytotoxicity that depends on the p53 status of the infected cell. other gynecologic malignancy. Primary therapy is surgery to The adenoviral oncoproteins ElB-55 kDa and E4orf6 inactivate debulk the tumor load followed by chemotherapy. The clinical and destabilize the tumor suppressor protein p53. However, it is response rate is between 60-70% and for patients who relapse after controversial whether adenoviruses with a deletion in the EI B-55 the initial treatment, the prognosis is poor. There is an urgent need kDa coding region might selectively replicate in cells with mutant for innovative approaches for the treatment of advanced stage or or absent p53. To address the role of p53 in virus replication, recurrent disease. We are using the Edmonston B vaccine strain of amino acid substitutions were introduced into the N-terminal measles virus (MV-Edm) for tumor therapy. Infection of cells by portion of p53, leaving p53 transcriptionally active but resistant to MV-Edm results in formation of multinucleated syncytia that inhibition and degradation by adenoviral oncoproteins. eventually become non-viable. To enable us to follow the spread of Surprisingly, even strong overexpression of this p53-variant this replicating virus in vitro and in vivo, we have engineered a allowed the virus to replicate and spread as efficiently as in the soluble marker peptide CC-terminus of carcinoembryonic antigen absence of p53 proteins, both in tumor cells and in primary CEA) into the genome of MV-Edm. The recombinant virus was endothelial cells, and in the presence of pl4ARF. Thus, active p53 rescued and was used to infect a panel of human epithelial ovarian does not inhibit the growth of adenovirus. carcinoma cells. The engineered MV-CEA was able to spread As an alternative strategy to make oncolysis dependent on mutant efficiently in all cell lines tested. We could correlate virus titer p53, we have developed an adaptor protein that reactivates mutant with the amount of CEA released into the culture supernatant. p53 by binding simultaneously to the DNA of p53-responsive Cytotoxicity due to virus infection and cell-cell fusion was also promoters, and to mutant p53. When the adaptor was expressed in determined. The SKOV3.ipl cells were exquisitely sensitive to tumor cells that contain mutant p53, expression of p53-responsive MV-CEA infection. These cells were also grown as subcutaneous genes was strongly activated, and growth was inhibited. This xenografts in SCID mice. When the tumors were about 0.5 cm in strategy turns mutant p53 into an inhibitor of tumor cell growth and diameter, they were injected directly with active MV-CEA. The may enable gene therapy to eliminate cancer cells with virus has potent anti-neoplastic activity. Tumors in 9 out of 10 unprecedented specificity. Further, similar adaptors may be used to mice totally regressed. The control group of 10 mice that received render the expression of adenovirus-genes, and thus virus equivalent doses of UV-inactivated MV-CEA did not respond to replication itself, dependent on mutant p53. treatment and all mice had to be euthanized due to tumor burden.

NEW CONVENIENT METHODS FOR CONSTRUCTION OF REPLICATION-DEFICENT VECTORS OBTAINED BY PUV RECOMBINANT VACCINIA VIRUSES TREATMENT OF VACCINIA VIRUSES EFFECTIVELY Tatyana M. Timiryasova, Bing Chen, Nadja Fodor and Istvan EXPRESS FOREIGN GENES. Tatyana M. Timiryasova, Bing Fodor. Loma Linda University, Loma Linda, California Chen and Istvan Fodor. Loma Linda University, Loma Linda, California Recombinant vaccinia viruses (VV) are widely used as expression vectors in molecular biology, immunology, and are Generation of non-replicating recombinant vaccinia virus (VV) now under evaluation for gene therapy. The current techniques vectors from the replicating VV could be a convenient strategy to for inserting foreign DNA into the large VV genome are based provide safe vectors for recombinant vaccines and gene therapy. on either homologous recombination between transfer plasmids We analyze the gene expression by VV after treatment with and VV genomes, or direct DNA ligation and packaging using psoralen and UV-irradiation (PUV) that introduces limited cross• replication-deficient poxviruses as helpers. We describe new, linking of double-stranded DNA and renders the virus replication• convenient and efficient versions of these methods. In both deficient. We constructed recombinant VV expressing reporter methods, VV DNA "arms" obtained by Not! digestion, and genes or tumor suppressor p53 under the control of early synthetic psoralen-UV-inactivated (PUV) VV used as a helper. In the new promoters. The inactivation ofVV was checked by virus titration homologous recombination method, DNA "arms" and intact on the CV-I cells. Cells infected with PUV-inactivated viruses transfer plasmid were used for co-transfection. In the direct DNA of PEIL promoter ligation method, foreign DNA was ligated into the unique Not! containing luciferase gene under control 5 6 site of VV DNA. Generation of recombinant viruses in both expressed the reporter at 1.4 x I 0 RLU/10 cells. The same methods was carried out in cells infected with a replication• promoter was used to control the expression of human wild-type deficient, PUV-inactivated helper VV. The effectiveness of these p53 cDNA. The recombinant VV-p53 produced high yield ofp53 new techniques was demonstrated by construction of protein, which was demonstrated by ELISA and Western blotting recombinant VV that produced 90-100% recombinant viruses of extracts from the cells infected with PUV-treated VV-p53 carrying and expressing E. coli ~-galactosidase or firefly virus. Approximately 40% and 88% ofC6 cells infected with non• luciferase. An important feature of these strategies is that any VV replicative VV-p53 underwent apoptosis on day 2 and 7, strain can be used as a helper virus after PUV-inactivation. respectively, while 80% of cells infected with non-replicative control virus (rVV-15) remained viable on day 7. In an ex vivo experiment, infection of C6 glioma cells with non-replicating VV• p53 inhibited the tumor growth progression in nude mice. Replication-deficient non-cytopathic VV vectors can be obtained from any attenuated or virulent VV strain and can be used for over-expression of foreign genes in a great variety of mammalian and human cells.

Gene Therapy Abstracts S13

OF DOGS AND MEN: CANINE DISTEMPER VIRUS RESIDUES REQUIRED FOR MEASLES VIRUS AS MODEL SYSTEM FOR MEASLES VIRUS HEMAGGLUTfNIN BINDING TO ITS RECEPTORS Veronika von Messling, Adele Fielding and Roberto Cattaneo Sompong Vongpunsawad, Urs Schneider, and Roberto Cattaneo Molecular Medicine Program, Mayo Foundation, Molecular Medicine Program, Mayo Foundation, Rochester, MN 200 1st Street SW, 55905 Rochester, MN 55905

Canine distemper virus (CDV) and measles virus (MV) are We aim to characterize cell entry of measles virus (MV), an members of the morbillivirus family. The course ofCDV infection enveloped negative-strand RNA virus of the Paramyxoviridae in ferrets or dogs corresponds to the course of MV infection in family and the genus Morbillivirus. Receptor usage by wild type humans. Due to these similarities, it may be possible to test clinical and vaccine MV strains appears to differ: the pathogenic wild type protocols aiming at the treatment of human diseases in ferrets or strains recognize preferentially the signaling lymphocyte• dogs. activation molecule (SLAM; also known as CD150), whereas the Since no system to generate recombinant CDV was available, we live attenuated Edmonston strain enters cells preferentially via the established one based on the vaccine strain Onderstepoort. To ubiquitous regulator of complement activation CD46. To identify address the question of what molecular characteristics cause residues on the MV cell-attachment protein hemagglutinin (H) , specific regions or complete proteins were exchanged important for viral receptor binding, we performed systematic against the respective sequences found in virulent strains. After mutagenesis of the H protein. We initially concentrated on the C• characterization of the recombinant viruses in vitro, a ferret model terminal half of this protein, and excluded residues conserved in a was established to examine the effects of the exchanges in vivo. related Morbillivirus (canine distemper virus). We introduced 45 The animals infected with the virulent strains developed first signs two-to-four amino acid mutations, verified the expression of the of disease between 8 and l O days after infection with I 03 tissue corresponding proteins, and functionally tested the mutants by co• culture infectious doses, demonstrating that this model is suitable transfection with the other MV glycoprotein F. Functional tests for our studies. The next step will be the assessment of the measured the fusion efficiency of the H protein mutants in CD46- pathogenicity of a first set of recombinant viruses. expressing or SLAM-expressing cells. Mutants that selectively In the context of a strategy to use MV as therapy against non• lost fusion-specificity depending on one receptor but not on the , we are currently exploring the possibility of a other were identified. On the basis of this information, single similar approach in dogs. Regression of lymphoma in dogs after mutants corresponding to all positions mutated in the natural infection with CDV has been described, creating a situation first vectors were produced. Remarkably, one single residue comparable to that in humans. Furthermore, the fact that dogs are mutant specifically and selectively lost SLAM-dependent fusion vaccinated against CDV the same way that humans are against MV support, while maintaining CD46-dependent fusion competence. makes the canine model especially meaningful. Two mutants with reduced CD46-dependent fusion and strong SLAM-dependent fusion were also identified. We are combining these mutations and transferring them in an infectious cDNA clone to verify function in the context of viral infection.

DEVELOPMENT OF RECOMBINANT VESICULAR USE OF SINDBIS VIRUS VECTOR FOR TUMOR GENE STOMATITIS VIRUS AS A NOVEL TARGETING GENE THERAPY DELIVERY SYSTEM Jie Zhang 1, Ilya Frolov2 and Stephen J. Russell1 1Molecular Medi• cine, Mayo Foundation, Rochester MN. 2Molecular Microbiology, Michael A. Whitt, Himangi R. Jayakar, Shelley Wright, Gina Washington University School of Medicine, St. Louis, MO Tanzer, Matt Bauler, Clinton S. Robison, and Mitchell S. Steiner GTx, Inc., 3 N. Dunlap, Memphis, TN 38163 We are developing a novel approach to cytoreductive cancer gene therapy employing a hyperfusogenic mutant of the gibbon Vesicular stomatitis virus (VSV) is a nonsegmented, negative• ape leukemia virus (GALV) envelope glycoprotein that potently strand (NNS) RNA virus and is the prototype for the induces cancer cell fusion. We reasoned that a Sindbis Rhabdoviridae family. The recent development of"reverse containing the GAL V envelope cDNA should facilitate the genetic" systems for VSV and other NNS viruses has made it generation of high-titer vectors driving high level GALV envelope possible to engineer the genome of these viruses for the expression resulting in extensive intratumoral cell to cell fusion co~struction. of novel vectors that have potential uses as gene and limited local intratumoral spread of the vector genome. dehve1?' vehicles. :me major limitation ofVSV as a gene delivery The GALV.fus cDNA was PCR cloned into the Sindbis vector vector 1s that cells mfected with VSV begin exhibiting cytopathic pSinrep5. Vector RN As both for Sinrep/GALV and SinrepBgal effects as soon as 4 hours post-infection and are usually killed within 18 to 24 hours. were produced by in vitro transcription and co-transfected with Here we describe a noncytopathic variant ofVSV that is being DH-BB helper RNA into BHK-1 cells. The viral supernatants were developed as a gene delivery vector. The vector (GTx-v200) is titrated on DF-1 cells. To determine the infectivity of replication and assembly competent, but completely lacks all of the Sinrep/GALV on different tumor cell lines, the viral supernatant cytopathic activities of the lab-adapted, wild-type strain ofVSV was used to infect a set of human cancer cell lines. Different size (Indiana ). The mutations responsible for the of fusions were observed in those cell lines. Cytotoxicity was also nonc)'.':opathic phenotype reside entirely within the matrix gene. observed in nonfusing cells, presumably mediated by the Sindbis Insertion ofheterologous genes into GTx-v200 results in long• nonstructural proteins. The Sinrep/GALV was also trasfected into term, high-level expression in a variety of cell types in vitro. By BHK-1 cell without the helper DH-BB. The transfected BHK-1 repl~cmg the VSV-G gene with that of a targeting complex cells were sonicated and supernatant was used to infect DF-1. cons1stmg of a membrane-anchored single-chain variable fragment Infectious particles were released in this way and could be scored and a membrane fusion protein, we have modified the tropism of as multinucleated syncytia on the infected DF-1 monolayer. An in GTx-v200 such that heterologous gene expression can be limited vivo study was then performed to assess the therapeutic potential to a specific subset of cells carrying the appropriate scFv surface of the Sinrep/GAL V vector. Athymic mice bearing subcutaneous receptor. Preclinical studies to examine the safety ofGTx-v200- U87 xenografts received Sinrep/GAL V or Sinrep/Bgal viral based vectors are currently in progress. vectors or DMEM intratumorally, Tumors completely regressed in the majority of Sinrep/GAL V treated mice but continued to progress in the control mice. Further in vivo studies are underway.

Gene Therapy