Joseph C. Glorioso, Ph.D. - US grants

The role of three viral glycoproteins, gB, gC and gD in inducing humoral and cell-mediated immune responses in mice will be analyzed genetically and immunologically with wild-type HSV-1 (KOS) and mutants for the three glycoproteins. Two types of mutants will be sought: (1) Mutants that are deficient in the expression of one or a combination of the three glycoproteins, induced by in vitro mutagenesis of cloned restriction enzyme fragments; and (2) Mutants that express functional glycoproteins altered in primary structure, selected as resistant to neutralization by monoclonal antibodies against the glycoproteins, monoclonal antibody resistant mutants, mar mutants. Mutants will be mapped by genetic recombination and marker rescue techniques and characterized biochemically and immunologically. Immune responses will be induced in mice by infection with wild-type virus and by immunization with wild-type and a battery of mutant infected cells expressing the various subsets of three glycoproteins. A selected number of immune reactions will be monitored. For the humoral response, these will include virus neutralization, complement-mediated immune cytolysis, total antibody production by ELISA assay and quantitation of antibody forming cells. In testing for the cell-mediated response, assays of cytotoxic T lymphocytes and T cell proliferation will be used. The battery of mutant infected cells will be used as reactants in the in vitro assays to identify which of the glycoproteins are most active in inducing particular immune responses, that is, which are immunodominant. Evidence for interactions between the immunogens in inducing specific immune responses may emerge. In addition, genetic and immunological analyses of mar mutants with libraries of monoclonal antibodies will be used to (1) identify the predominant antibody specificities generated in the immune response to HSV-1, (2) estimate the number of antigenic determinants in a glycoprotein and (3) genetically map mutations affecting determinant sites and the immunogenicity of the glycoprotein. Finally, syngeneic mouse cells, transformed with the structural genes for single glycoproteins will be sought and used as unique material for immunization.

Herpes simplex virus (HSV) is a ubiquitous human pathogen capable of causing a variety of clinically important diseases. The role of the immune response in controlling primary and recurrent HSV infection is under investigation. HSV glycoprotein C (gC) is highly immunogenic for both humoral and cellular immune responses and consequently plays a major role in the immunobiology of infection. The central aims of this competing renewal application are (i) to develop a more complete understanding at the molecular level of the antigenic structure of gC as recognized by both antibodies and cytotoxic T lymphocytes (CTL), with an emphasis on a comparative analysis of the organization of antigenic domains and component epitopes between gC of the two serotypes and (ii) to explore the potential usefulness of gC as a subunit vaccine against virus-induced encephalitis and zosteriform lesions using the mouse as a model host. The central hypothesis to be tested is that the antigenic domains of gC-1 and gC-2 are colinear and that the predominant type-specificity exhibited by these domains is due to the presence of dissimilar amino acids within the component epitopes. The experimental strategy for defining type-specific epitope structure and organization will be to compare the reactivity of large panels of gC-1 and gC-2 specific mAbs with (i) a series of genetically engineered gC-1:gC-2 chimeric gene products and (ii) panels of wild-type and mutant synthetic peptides. The peptides will also be used to develop antibody reagents to discover new antigenic determinants and for a more detailed analysis of known antigenic sites. Correlation of the epitope mapping studies with antigenic variation among fresh clinical isolates will be sought in order to assess the stability of epitope structure and to detect genetically invariant domains. Studies of the CTL responses to HSV gC will be continued using short-term and long-term CTL clones to (i) confirm that gC of both serotypes is the major inducer and target antigen for CTLs (ii), define CTL-specific epitopes at a molecular level, and (iii) determine the extent to which the B and T cell repertoires overlap in their recognition of gC. Finally, purified gC derived with a baculovirus expression vector system in combination with synthetic peptides will be used in immunization protocols to induce protective immunity against HSV-induced neurological and peripheral lesions with emphasis on prevention of latent infection of ganglion neurons.

Herpes simplex virus infects cells through a multi-step process involving virus attachment to the cell plasma membrane followed by fusion of the virus envelope with the membrane resulting in penetration of the nucleocapsid into the cytoplasm. Two HSV envelope glycoproteins, gB and gD, are required for penetration. Both glycoproteins also play a role in syncytia formation, an aberrant form of virus-mediated cell-cell membrane fusion. The overall goal of this proposal is to fine map mutations in these glycoprotein genes which affect virus penetration and syncytia formation and to identify critical molecular structures which mediate these functions. The genetics of virus penetration and syncytia formation will be explored to determine whether these two processes are different manifestations of the same biological activity. A combination of deletion, linker insertion and bisulfite mutagenesis strategies will be used to saturate biologically important domains in these molecules with mutations. Mutant glycoprotein genes will be introduced into a specialized recipient viral genome using the P1 cre-lox site specific recombination system in a shuttle vector. Lethal and nonlethal mutations will be studied during replication on complementing and noncomplementing cell lines. Studies of gB will define functionally important structures within the external, transmembrane and cytoplasmic domains. Phenotypes to be explored include penetration minus, altered dimer formation, complementation inhibition, altered rate of entry, mab resistance, temperature sensitivity and syncytial plaque formation. Cooperativity among syn gene products will be examined using transient assays. For gD, linker insertion mutagenesis will be initiated across the whole gene to search for functionally important domains. Emphasis will be on mutations which result in the penetration minus phenotype. Virus neutralizing gD-specific mabs block virus penetration. Thus, neutralizing determinants on gD will be mapped by studies of the reactivity patterns of mAbs with a panel of gD truncated polypeptides, linker insertion mutants and by DNA sequencing of mar mutants. Finally, structures within gD that block virus penetration when the glycoprotein is expressed in transformed cell membranes will e defined, using a panel of cell lines transformed with a novel retrovirus vector carrying mutant forms of gD. Together, these studies of gB and gD should genetically map and uncover essential structures that contribute to virus penetration and syncytia formation.

The overall aim of this research is to use aneurovirulent mutant forms of Herpes simplex virus (HSV) as direct gene transfer vectors to study the pathogenesis of Alzheimer's disease (AD). We will test the hypothesis that altered production or aberrant processing of amyloid precursor protein (APP) is directly responsible for the development of Alzheimer-like pathology in vivo. Genetically engineered recombinant HSV vectors will be used to introduce specific forms of the APP gene into the mature rat brain in a manner in which these genes are exclusively expressed from an otherwise quiescent viral genome during latency. We will also test a related hypothesis that chronic production of nerve growth factor (NGF) can rescue basal forebrain cholinergic neurons from age-related cell death using a similar vector. Finally, we will determine whether there exists an interrelationship between APP and NGF gene expression in brain using a combination of virus vectors. Three specific aims are outlined: (i) We will first complete development of HSV as a gene transfer latency vector for the rat CNS. The virus will be engineered to establish life-long latent infections in specific brain regions following direct stereotactic injection. Lac-z gene cassettes will be introduced into the virus vector at the U(s)3 locus using a Pl phage cre-lox recombination system and B-galactosidase expression from the latent HSV genomes will be induced in hippocampal cell populations using either the latency-specific (LAT) promoter or the neuron-specific neurofilament (NF) promoter. We will confirm that there is no virus -induced neuropathology created by the recombinant vector alone. (ii) The genes encoding for the full length APP, with and without the protease inhibitor insert, and the gene for the A4 fragment of APP, will be similarly recombined into the HSV latency vector and introduced into the hippocampus. We will determine whether any of these recombinants cause Alzheimer-like pathology in the rat brain, and whether these gene products effect the level of NGF expression. (iii) The gene for NGF will be expressed in hippocampus using an HSV::NGF recombinant latency vector in order to determine whether chronic NGF production can reverse the age-related loss of basal forebrain cholinergic neurons. We will also use expression from this vector in brain to determine whether chronic overproduction of NGF effects endogenous brain expression, and whether NGF delivered in this manner plays any role in modulating specific CNS pathology.___

Herpes simplex virus (HSV) is a ubiquitous human pathogen capable of causing a variety of clinically important diseases. The role of the immune response in controlling primary and recurrent HSV infection is under investigation. HSV glycoprotein C (gC) is highly immunogenic for both humoral and cellular immune responses and consequently plays a major role in the immunobiology of infection. The central aims of this competing renewal application are (i) to develop a more complete understanding at the molecular level of the antigenic structure of gC as recognized by both antibodies and cytotoxic T lymphocytes (CTL), with an emphasis on a comparative analysis of the organization of antigenic domains and component epitopes between gC of the two serotypes and (ii) to explore the potential usefulness of gC as a subunit vaccine against virus-induced encephalitis and zosteriform lesions using the mouse as a model host. The central hypothesis to be tested is that the antigenic domains of gC-1 and gC-2 are colinear and that the predominant type-specificity exhibited by these domains is due to the presence of dissimilar amino acids within the component epitopes. The experimental strategy for defining type-specific epitope structure and organization will be to compare the reactivity of large panels of gC-1 and gC-2 specific mAbs with (i) a series of genetically engineered gC-1:gC-2 chimeric gene products and (ii) panels of wild-type and mutant synthetic peptides. The peptides will also be used to develop antibody reagents to discover new antigenic determinants and for a more detailed analysis of known antigenic sites. Correlation of the epitope mapping studies with antigenic variation among fresh clinical isolates will be sought in order to assess the stability of epitope structure and to detect genetically invariant domains. Studies of the CTL responses to HSV gC will be continued using short-term and long-term CTL clones to (i) confirm that gC of both serotypes is the major inducer and target antigen for CTLs (ii), define CTL-specific epitopes at a molecular level, and (iii) determine the extent to which the B and T cell repertoires overlap in their recognition of gC. Finally, purified gC derived with a baculovirus expression vector system in combination with synthetic peptides will be used in immunization protocols to induce protective immunity against HSV-induced neurological and peripheral lesions with emphasis on prevention of latent infection of ganglion neurons.

The treatment of disease by genetic manipulation is emerging as a practical tool in medicine. The goal of this program project is to contribute to the development of gene therapy as a therapeutic modality through the study of model systems. The focus of the project is to use model systems to reveal obstacles and provide solutions to the impediments to gene therapy. Cellular and animal models will be employed to study transfer of potentially therapeutic genes, promote their expression, and measure the effects on a system that is predictive of similar responses in man. While clinical trials are not foreseen as within the scope of the proposal for all four projects the studies proposed in the program are intended to provide substantial information upon which clinical trials will be based. The program seeks through its four interactive projects and four cores to contribute data relevant to the general approach of gene therapy. While diverse in the organ systems studied in the program, the central purpose of the project is single and focused on the methods of gene transfer that may be needed to target and express human genes in different tissues. This goal will be accomplished by a thorough analysis of the vectors developed to transfer a variety of genes and by consideration of the problems of transduction and expression that are peculiar to each model system.

Although the ability of herpes simplex virus (HSV) to establish long term latency is sensory ganglion neurons is well documented, the virus-host interactions involved in transcriptional control of viral genes during latency remain largely unknown. Two latency active promoters have been discovered in the LAT region of the genome. LAP1 contains a TATA box while LAP2 is TATA-less. Reports indicate that LAP1 expresses a large unstable polyA+ 8.7kb transcript from which a small stable 2.0kb polyA- LAT RNA is derived by splicing. Moreover, numerous less abundant LATs of unknown origin have been described. Recent evidence, however, suggests that the 2kb LAT is a primary transcript and associated with polyribosomes. Our laboratory has evidence that LAP2 functions to express reporter genes in both the LAT and an ectopic locus. Experiments are described in the following three interrelated aims to carefully (i) determine whether one or both LAPs express LATs and under what circumstances, (ii) define the cis and trans elements which determine LAP activity and (iii) examine the potential role of chromatin structure in governing latency gene expression. (1) The question of whether LAP1 or LAP2 drive expression of either the major or minor LATs during lytic infection in vitro and during latent infection of the mouse trigeminal ganglion will be addressed through careful examination of transcript termini and through the use of mutants altered in LAP regulatory elements and putative LAT splice donor/acceptor sites. (2) Depending on the outcome of experiments in Aim 1, genetic studies involving recombinant viruses carrying linker-scanner and site-directed mutations in one of both LAPs will be conducted to determine the cis-acting signals that positively and/or negatively regulate LAP function in vitro and in vivo. Biochemical assays are also planned to study the binding of nuclear proteins to specific promoter sequences which contribute to the ability of LAP to function during latency, thereby establishing a connection between LAP functional elements and transcriptional activator or repressor proteins. (3) Studies re outlined to evaluate the role of chromatin structure in governing latency gene expression. The distribution of nucleosomes on latent HSV DNA will be determined by nuclease digestion and correlated with the pattern of gene expression during latency. Additional assays using cell lines carrying chromosomally integrated lytic and latency genes will be employed to evaluate differences between transcriptionally active and silent promoters.

This proposal requests funds to enable young investigators in training to attend the 18th International Herpesvirus Workshop to be held in Pittsburgh, Pennsylvania July 25-30, 1993. This workshop is the most important meeting for investigators in the herpesvirus field. It is the forum where the most recent advances in the field are first presented. This meeting also provides an opportunity for new graduate students and postdocs to obtain an appreciation for the scope, complexity and current state of knowledge of the Herpesvirus field. The strength of this workshop results from the high level of participation by investigators in all areas of herpesvirus research. The meeting is attended by researchers from all over the world with both basic and clinical interests. The broad based interests of the participants results in the cross-fertilization of individual research programs and also provides an atmosphere that encourages the exchange of new information. Herpes viruses cause a wide range of diseases in humans. Herpes simplex viruses types 1 and 2 (HSV-1, HSV-2) cause skin, eye and genital ulcerations. Varicella-zoster virus (VZV) causes chicken pox and shingles, while Epstein-Barr virus (EBV) is responsible for infectious mononucleosis. Congenital infection with HSV-2 and cytomegalovirus (CMV) are significant causes of mental retardation, while HSV encephalitis can be fatal. HHV 6 has been associated with a febrile illness in infancy resembling exanthem subitem. HHV 7 has recently been isolated from healthy individuals. Neoplasias such as nasopharyngeal carcinoma, B-cell lymphoma, and Hodgkins disease are all associated with EBV. Several members of the herpesvirus have been identified as possible cofactors in the growth and pathogenesis of human immunodeficiency virus. The hallmark of herpesviruses is the ability to establish a latent infection, persist in the host for years and subsequently reactivate, causing disease. Due to this property, herpes viruses are opportunistic infectious agents contributing to morbidity and mortality in the immuno- compromised. Organ transplant recipients and AIDS patients are susceptible to EBV associated systemic, central nervous system and oral infection, CMV retinitis and pneumonitis, and disseminated CMV, VZV and HSV infections. An emerging area that takes advantage of this aspect of herpesviruses is the development of modified herpesviruses for gene transfer purposes. Animal herpesviruses have significant economical importance to the poultry (Marek's Disease Virus), swine (pseudorabies virus), dairy (bovine rhinotracheitis virus) and equine herpesvirus) industries. Sessions will be convened to discuss the following topics: gene regulation, DNA replication, genome structure, enzymes and non-structural proteins, glycoproteins and structural proteins, receptors and virus entry, transformation and neoplasia, immunology, viral pathogenesis, viral vectors, anti-virals and resistance, vaccines and therapy.

The over goal of this research is to engineer a herpes simplex virus type l (HSV-1) replication defective mutant capable of stable transfer and expression of the murine beta-glucuronidase gene in the brains of a mouse model of the lysosomal storage disease mucopolysaccharidosis type VH (Sly syndrome). High level enzyme expression in brain should be therapeutic since enzyme should be released and taken up by neighboring brain cells in an active form thus obviating the need for vector infection of all brain cells. The design of the HSV vector will take advantage of and improve on the large capacity of HSV for foreign DNA and the latency specific expressiOn system that naturally evolved in the HSV life cycle. Four related aims are proposed to achieve this goal: (i) Recombinant promoters will be constructed using the HSV latency promoter/neuronal specific elements in combination with strong proximal and/or basal control elements in order to achieve vigorous foreign gene expression during latency. The design of these promoters will be determined by experiments to elucidate the identity of neuronal specific elements and how these elements functionally interact or cooperate with basal and/or general transcription factors. (ii) Constitutive (e.g. GAL4:VPI6 fusion gene ) and drug-inducible autogene (VPI6:GAL4:HBD fusion gene) systems will be developed to amplify or regulate gene expression from promoters carrying the GAL4 DNA binding elements in cis, such that sustained expression from the vector can be achieved or induced by a drug capable of crossing the blood-brain barrier, RU486. (iii) Vectors with greatly packaging capacity extending up to 40kb will be constructed. This will enable the transduction of large genomic genes as well as multiple cDNAs. These vectors will be defective in two essential genes and therefore be completely defective. In addition, the system has been engineered to eliminate the generation of wild-type recombinants when the vector stock is being prepared on transformed laboratory cell lines, and will also eliminate the potential for the generation of replication proficient virus carrying the transducted gene, which could potentially arise by recombination with latent wild-type virus in vivo. (iv) The best features of these vectors will be combined to express '3- glucuronidase in a murine model of mucopolysacchridosis type VII. This will involve the transduction of both the cDNA and genomic gene encoding beta- glucuronidase and the subsequent analysis for appropriate and therapeutic levels of gene expression.

Herpes simplex virus infects cells through a multi-step process involving virus attachment to the cell plasma membrane followed by fusion of the virus envelope with the membrane resulting in penetration of the nucleocapsid into the cytoplasm. Two HSV envelope glycoproteins, gB and gD, are required for penetration. Both glycoproteins also play a role in syncytia formation, an aberrant form of virus-mediated cell-cell membrane fusion. The overall goal of this proposal is to fine map mutations in these glycoprotein genes which affect virus penetration and syncytia formation and to identify critical molecular structures which mediate these functions. The genetics of virus penetration and syncytia formation will be explored to determine whether these two processes are different manifestations of the same biological activity. A combination of deletion, linker insertion and bisulfite mutagenesis strategies will be used to saturate biologically important domains in these molecules with mutations. Mutant glycoprotein genes will be introduced into a specialized recipient viral genome using the P1 cre-lox site specific recombination system in a shuttle vector. Lethal and nonlethal mutations will be studied during replication on complementing and noncomplementing cell lines. Studies of gB will define functionally important structures within the external, transmembrane and cytoplasmic domains. Phenotypes to be explored include penetration minus, altered dimer formation, complementation inhibition, altered rate of entry, mab resistance, temperature sensitivity and syncytial plaque formation. Cooperativity among syn gene products will be examined using transient assays. For gD, linker insertion mutagenesis will be initiated across the whole gene to search for functionally important domains. Emphasis will be on mutations which result in the penetration minus phenotype. Virus neutralizing gD-specific mabs block virus penetration. Thus, neutralizing determinants on gD will be mapped by studies of the reactivity patterns of mAbs with a panel of gD truncated polypeptides, linker insertion mutants and by DNA sequencing of mar mutants. Finally, structures within gD that block virus penetration when the glycoprotein is expressed in transformed cell membranes will e defined, using a panel of cell lines transformed with a novel retrovirus vector carrying mutant forms of gD. Together, these studies of gB and gD should genetically map and uncover essential structures that contribute to virus penetration and syncytia formation.

DESCRIPTION: (Adapted from the applicant's abstract) - Rheumatoid arthritis is a chronic inflammatory disease affecting an estimated 10 million individuals in the United States alone. Currently, no effective long-term treatment other than joint replacement surgery is available. The overall aim of this proposal is to develop an in vivo gene therapy protocol for the treatment of arthritis. The first-generation gene therapy protocol using retroviral-mediated gene transduction demonstrated that antagonists of two cytokines, interleukin-l (IL-l) and tumor necrosis factor alpha (TNFa), can provide a significant therapeutic outcome in animal models of arthritis. These results are encouraging and provide the foundation of these proposed studies; however, it will never be practical to treat the millions of Americans who suffer from arthritis using an ex vivo strategy. Ex vivo treatments require sophisticated laboratories and protocols for transduction and selection of explanted cells. The goal of these studies is to develop an in vivo protocol in which gene transduction requires nothing more than an injection into the afflicted joint and thus, could become widely available in settings as simple as a physician's office. In vivo gene delivery will be accomplished with the injection of engineered Herpes simplex virus 1 (HSV1) vectors. HSV-l vectors have the advantages of high infectivity and the potential to express multiple transgenes. The major disadvantage of HSV vectors, their cytotoxicity, has largely been overcome by the deletion of the cytotoxic genes, ICP4, ICP22, ICP27, and UL41. These multiple deletion mutants are capable of replication only in complementing cell lines. They show greatly reduced cytotoxicity both in cell culture and in rabbit synovium with the acquired capacity for durable transgene expression in vivo. By using these replication-defective vectors, the principal investigator will be able to transduce synoviocytes in vivo to express antagonists of IL-l and TNFa, setting the stage for a simple and effective gene therapy treatment for arthritis. Four specific aims are proposed: 1) To construct HSV-l gene therapy vectors with further reduced cytotoxicity by deletion of the ICP0 gene; 2) to engineer HSV-1 vectors for coordinated expression of therapeutic genes; 3) to assess the effect of prior immunization with HSV on vector persistence and expression and to determine the effect of "antigenic stealthing" genes; 4) to assess the efficacy of HSV-l vectors for the direct in vivo gene therapy of the antigen-induced rabbit model of arthritis.

DESCRIPTION: The mechanism of viral gene silencing during HSV latency is not understood. While transcription of the viral IE genes is silenced, an RNA referred to as latency associated transcript (LAT) is transcribed. LAT is considered a stable intron. Viral protein associated with the LAT transcription unit has not been identified. This application describes the use of HSV mutants to investigate HSV gene expression during latency. Mutant viruses with as many as three IE genes have been deleted. These IE genes are ICPO, 4, 22, and 27. The specific aims of this proposal are as follows: (i) To determine the genetic basis of IE gene promoter silencing and its role in the establishment of latency. (ii) To characterize cis-acting elements associated with the ICPO promoter during latency or reactivation. (iii) To determine the role of the LAT intron in silencing ICPO expression (iv) To characterize cis-acting elements associated with two LAT promoters designated LAP1 and LAP2. (v) To determine the role of chromatin and DNA methylation in promoter silencing during latency. During infection of neurons with IE triple mutants of HSV, the viral ICPO promoter remains active for 2 to 4 weeks in contrast to wild type HSV. Since ICP4 deleted mutants still silence ICPO transcription, the applicant proposes that the ICP27 and ICP22 viral gene products have a role in silencing ICPO promoter activity. To further investigate these observations, promoter switching experiments whereby the ICPO promoter is substituted by either the ICP4, 27, or 47 promoters is proposed. The mutant viruses will be inoculated into the hippocampus of rat brain and the activity of the promoters relative to the number of viral genome equivalents will be determined by RT-PCR. Mutant viruses with the ICPO gene substituted by a reported gene (lacZ) will be analyzed to determine if the ICPO gene product autoregulates its own promoter. The effects of the level of ICP4, 27, and 22 gene expression on ICPO gene expression will be determined by using a regulation system for gene expression consisting of a Gal4 DNA binding domain-VP16-RU486 fusion described by the O'Malley lab that is induced by the hormone progesterone. In the second specific aim, the cis-acting elements in the ICPO promoter that are required for escape from silencing and reactivation from latency will be investigated. Site-directed mutagenesis of potential cis-acting elements and repeat sequences, 5' and 3' truncations and internal deletions will be used to analyze the promoter. The putative cis-acting elements in the ICPO intron will also be analyzed. Latency will be established by the corneal scarification method using mice and the number of latent genomes in the trigeminal ganglia will be determined by quantitative PCR. The applicant proposes that the transcription factor E2F may have a role in ICPO promoter activity. In the third specific aim, the role of the LAT RNA in regulating the level of ICPO RNA will be analyzed. Since the two viral RNAs have complementary sequences, the LAT RNA may target the ICPO RNA for degradation. Mutant viruses that fail to express the 2 kb LAT RNA may fail to downregulate ICPO RNA levels whereas over expression of the 2 kb LAT RNA may efficiently downregulate. To extend this line of research, the applicant proposes to use a yeast system with selectable markers and reporter genes. The yeast system could be used to determine the regions of the LAT RNA required for hybridization to ICPO RNA and, subsequent destruction of ICPO RNA. In the fourth specific aim, the functional elements of the two latency active promoters, LAP1 and LAP2, will be subjected to site-directed mutagenesis. Both common cis-acting sites and brain specific cis-acting sites will be mutagenized. An interesting approach will be to use transcription factor Brn knock-out mice to determine the effects of the brain specific transcription factors and cis-acting elements. In the fifth specific aim, the viral chromatin and the methylation state of the viral DNA will be analyzed with an emphasis on the ICPO and LAP regions. Chromatin arrangement and/or DNA methylation may have a role in silencing IE gene transcription. The differences in the chromatin profile or methylation profiles between wild type virus and IE deletion virus will be determined. Differences in nucleosome arrangement will be analyzed by a nuclease sensitivity assay. Differences in the CpG sequence methylation will be analyzed.

Neuropathy is a common and debilitating complication of diabetes. Several lines of evidence suggest that deficiencies in the tropic factor nerve growth factor (NGF) play a role in the pathogenic sequence leading from insulin deficiency to neuropathy, and studies have shown that treatment with NGF can reverse specific aspects of neuropathy in animal models in the short term. Over the past 5 years, we have made substantial progress in engineering gene transfer vectors from recombinant herpes simplex type 1 (HSV-1), a neurotropic virus which naturally establishes a permanent latent state within neurons. We have also demonstrated that specific viral latency associated promoter sequences (LAP2) are effective in producing prolonged transgene expression from replciation-defective HSV vector genomes persisting in neuron sensory ganglia in a manner similar to latency. This proposal outlines a series of studies in four Specific Aims to test the hypothesis that delivery of NGF by HSV-mediated gene transfer is an effective means of preventing the progression of diabetic neuropathy. Aim 1: To establish the parameters of vector-mediated transgene production following both footpad and intestinal inoculation. Aim 2: To examine the effect of prior infection with HSV on HSV-mediated transgene production. Aim 3: To examine the possibility that HGF expression can be controlled by a drug-inducible transcriptional transactivator produced by the persistent vector in sensory neurons. Aim 4: To compare the therapeutic effect of local(intraneuronal) and systemic (following intestinal inoculation) transgene-produced NGF in prevent diabetic neuropathy in STZ diabetic rats using detailed neurophysiologic, morphologic, and behavioral measures of peripheral nerve structure and function. The studies described will provide insight into the mechanism of action of the NGF in the diabetic neuropathy model. Success in achieving these aims could lead directly to the development of a novel therapeutic modality for the treatment and preention of diabetic neuropathy.

Despite the dramatic advances in prevention, diagnosis, and treatment made during the last half of the 20th century cardiovascular disease remains the number-one cause of morbidity and death in the United States. The success of angioplasty, vascular surgery procedures, and even heart transplantation (all approaches to treat vaso-occlusive disease) is limited by intimal hyperplasia. The success of angioplasty, vascular surgery procedures, and even heart transplantation (all approaches to treat vaso-occlusive constant infusions of inotropes or with left ventricular assist devices. However, in many instances these treatments only transiently postpone the inevitable death or transplantation. Clearly more effective and sustainable therapies are needed. Gene therapy offers perhaps the greatest opportunity to make the next major advance in preventing or treating cardiovascular disease. While pharmacologic methods typically require frequent closing, a single gene therapy application may be adequate to prevent, attenuate, or reverse even chronic disease. including useful vectors, methods of delivery and the molecular basis of many cardiovascular diseases in humans is the natural progression of ongoing research in cardiovascular gene therapy at the University of Pittsburgh. Two independent research programs, one aimed at developing gene therapy approaches to treat heart failure and the investigators in the Pittsburgh Human Gene Therapy Center (PHGTC). We now propose to further link these established research programs with the extensive resources of the PHGTC and other key resources in the Cores cardiovascular therapy programs from the bench to the bedside. Second, through our proposed preclinical projects, we will acquire the essential data needed to determine if other promising genes, gene targets and vectors projects, we will develop more effective vectors to target cardiovascular tissues. Fourth, through an organized and comprehensive training program, we will prepare clinician scientists for careers in gene therapy for cardiovascular disease. Fifth, through our coordinating data management core, we will provide communication mechanisms and data organization for our consortium centers.

In vivo gene therapy applications will be enhanced if viral vectors can be targeted to cells where transgene expression is desired. Herpes simplex virus type 1 (HSV-1) has significant potential utility has a powerful gene vector system, but its broad host range limits general clinical applicability. Accordingly, the overall goal of this research is to develop suitable methods for targeting infection by directing virus attachment to novel receptors and contribute to a fundamental understanding of viral attachment and entry. To this end,, we will attempt to (i) alter the natural binding functions of glycoproteins C, B, and D with N-terminal addition of novel receptor-binding ligands, and (ii) explore further the utility of endosomal release of vector using internalizing receptors in combination with chloroquine. Specifically we propose to: (I) target infection via gC by evaluating the ability of an HS/HveA (HVEM) binding deficient (ridl) gC:EPO (erythropoietin) recombinant virus to infect CHO cells bearing the EPO receptor alone or in combination with either chloroquine or the gD-specific HveC receptor; (II) target infection via gB by evaluating the ability of an HS/HveA binding deficient virus carrying a g:B bungarotoxin (BTX) chimeric glycoprotein to infect CHO cells expressing the alpha7 subunit of the acetylcholine receptor either alone or in combination with HveC; (III) target infection via gD by constructing HveA-binding deficient viruses in which (i) the BTX sequence replaces N-terminal residues 6-24 (created in an infectious HSV-BAC, bacterial artificial chromosome) and selecting from the library of progeny viruses produced by transfection those that are infectious for refractory CHO cells; and (IV) target infection via gD defective for recognition of HveA and HveC by constructing gD mutants which can mediate infection via HveA but not HveC using two strategies; (i) saturation mutagenesis of residues 216-234 or 28-70 using the HSV:BAC system and selection of recombinants on CHO-HveA cells following neutralization with soluble HveC receptor, and (ii) replacement of residues 28-70 with random peptide sequences and selection of mutant which rescue entry into CHO- HveA but not CHO-HveC cells. HveA binding will then be disrupted by replacing N-terminal residues 6-24 with BTX or novel CHO binding ligands identified in Aim III and recombinant virus mutants selected for infection of CHO-alpha7 or CHO cells; and (V) study the mechanism of infection using VSV-G and chloroquine by evaluating the infectivity of gD deficient VSV-G recombination vectors carrying other altered or deleted HSV glycoproteins.

DESCRIPTION: (provided by applicant) A variety of diseases affect the viability and function of motor neurons located within the spinal cord and projecting to muscle throughout the body. In order to develop an effective gene therapy application to treat MN disease it will be necessary to efficiently transduce MNs in vivo. Herpes simplex virus type 1 (HSV-1) has been engineered to be a versatile gene vector for transduction of sensory nerves in vivo, the natural site for wild-type virus latency. While high multiplicity infection of spinal cord will result in virus transduction of motor neurons (MNs), replication defective HSV vectors fail to transduce motor neurons in adult rodent models following skin or muscle inoculation. A similar result is likely to occur in human gene therapy applications since HSV is not found in MNs in natural infections. Thus treatment of MN diseases will require the enhancement of HSV vector infection of MNs with the longer-range goal of limiting the virus host-range to MN receptors. Because little is known about the effects of introducing new receptor binding ligands into the HSV-1 envelope, the applicants will explore strategies to enhance and eventually retarget HSV infection to MNs through modification of the receptor binding activities of three fundamentally different envelope components, glycoproteins B, C, and D. Because tetanus toxin receptors (TTR) are specifically found on MN synaptic junctions, we will focus our efforts on using nontoxic TTR binding components of the tetanus toxin engineered to be presented on the virus envelope as recombinant glycoproteins capable of initiating virus attachment and entry into MNs both in vitro and in vivo. Aim 1 is designed to target HSV to MNs by substitution of the native heparan sulfate attachment determinants presented by HSV glycoproteins B and C (gB and gC) with a tetanus toxin (TTx) heavy chain C-terminal fragment (HcC). Aim 2 is intended to restrict virus infection via HveB binding and evaluate re-targeting of gD via HcC. Aim 3 is designed to test soluble HveC in combination with HcC ligand bearing gC and gB or HveC-HcC soluble fusion proteins as effectors of MN targeting. The soluble HveC adapter will be expected to block gD binding to cellular HveC while directly or indirectly re-targeting the virus to TTx receptors.

Survival of patients with malignant glioma remains poor despite the availability of surgical debulking, radiation therapy, and chemotherapeutic regimens. Progress in applying gene therapy to the treatment of cancer provides an additional strategy which may prove effective in combination with more standard therapies. NUREL-C2 is a completely inactivated herpes simplex virus (HSV)- based gene transfer vehicle that expresses the four novel therapeutic proteins ICP0, thymidine kinase, connexin-43 and TNFalpha which work in concert to kill tumor cells when used in combination with intravenous administration of the anti-cancer drug ganciclovir (GCV) and radiosurgery. Animal experiments using this combination of gene and conventional therapies to treat intracerebral implants of radiosensitive human glioblastoma cells have resulted in excellent tumor control and improved survival. To establish the maximum the maximum potential of this approach, additional preclinical studies are proposed to optimize the contributions of each component to the combined treatment and to evaluate efficacy in models of radioresistant human glioblastoma (Aim 1). The vector and combined therapy will be systematically tested for safety and dose-limiting toxicity in normal mice and rhesus monkeys to expand our current results (Aim 2). A Phase I clinical trial is proposed with two consecutive components involving a) pre- and post-surgical intracranial NUREL-C2 inoculation followed by GCT treatment, and b) stereotactic NUREL-C2 delivery into the tumor with maintenance on GCV and gamma knife radiosurgery two days later. Using a battery of molecular, serological, imaging and clinical tests, patients will be evaluated for adverse effects of viral vector implantation, vector toxicity prior to, during, and after GCV treatment, short-term vector distribution and transgene expression in the tumor, metabolic activity of the tumor, and imaging responses to therapy. Safe vector dose will be determined in the first aim of the trial by dose escalation between consecutive groups of 3 patients. In the second arm, potential changes in toxicity profile and safe dose due to the combination with radiosurgery will be identified. Concurrent manifestations of efficacy will be recorded (Aim 3). In the final Aim, the therapeutic potential of HSV vectors expressing radiosensitizing genes or novel genes from Projects 1 and 2 will be tested for effectiveness in glioma models. Effective genes will be incorporated into NUREL-C2 and the new derivatives tested for improved cytocidal qualities in vitro and efficacy in vivo to arrive at an optimally effective gene transfer agent for the treatment of malignant glioma (Aim 4).

Muscular dystrophy is a common genetic disease that affects 1 in 3500 male births annually. The disease is characterized by early muscle hypertrophy followed by muscle degeneration and early death in adolescence resulting from failure of heart and diaphragm muscle. The disease results from mutations that affect expression or function of dystrophin, an important structural component of the subplasma membrane. Currently no treatment is available. The overall goal of the proposed research is to develop gene and cell therapeutic methods for treatment of muscular dystrophy. Clinical, pre-clinical and basic muscle cell development studies are described in which experts in gene transfer, muscle cell biology, animal models of muscular dystrophy and clinical applications are brought together in a manner to achieve the highest level of data sharing, synergy and creative solution finding will be possible. Project 1 (J Mendell) will define clinical end-points and identify cohorts of patient that would participate in a phase I dose escalation safety clinical trial using an AAV gene vector carrying the functional dystrophin minigene delivered to a single skeletal muscle and continue gene therapy clinical trials for limb girdle MD. In Project 2 (X Xiao and J Kornegay), will explore methods for improved AAV-dys gene delivery using the dog model. In Project 3 (J Huard), experiments using muscle stem cells will be carried out using dystrophic mouse models in attempts to achieve muscle delivery of normal muscle derived stem cells to engraft into diseased heart. In Project 4 (J Glorioso), a novel functional genomics approach to identify genes that participate in differentiation of mouse embryonic stem cells toward muscle cell lineages is proposed using HSV gene vector cDNA libraries obtained from muscle derived stem cells. The core programs are designed to directly support the projects in the form of Administration (Core A: J Glorioso and P Robbins), Clinical Vector Production (Core B: J Barranger), a muscular dystrophy dog colony (Core C: J Kornegey) and Imaging (Core D: S Watkins) to provide information on the results of gene transfer in animals and patients. Finally, our center includes a training program for residents interested in gene therapy for muscle disease. We believe this to be a timely and highly innovative proposal which is likely to provide new armroaches to the treatment of muscular dvstrophv.

Heart transplantation is the preferred therapy for patients with a variety of end-stage heart diseases. Over the last two decades, developments in immunosuppressive medications, technical innovations, and improvements in postoperative care have significantly improved outcomes following heart transplantation. Cardiac allograft ischemia-reperfusion (I-R) injury, however, remains a major source of morbidity and mortality leading to both early allograft dysfunction as well as long-term morbidity. Considerable evidence supports an important role for heme oxygenase (HO-1) in protection against I-R. This enzyme catabolizes heme into biliverdin, free iron and carbon monoxide (CO), which act to reduce inflammation and cell death. The goal of this proposal is to develop suitable HSV-HO-1 gene vectors that specifically target infection of heart blood vessel endothelium and express HO in sufficient levels and duration to ameliorate this type of ischemic disease. In four specific aims, a retargeted HSV vector (HSV-HOT) will be engineered in which the endothelial-specific receptors VEGF-R2 (vascular endothelial growth factor receptor) [and/or Tie-2 (angiopoietin receptor)] will be used for virus attachment and penetration in lieu of the natural HSV receptors HveA and HveC. If needed, we will also explore an alternative retargeting strategy that utilize a targeting soluble adapter (Aim 1). A highly engineered, replication defective vector backbone will be used for gene delivery. This vector is noncytotoxic, highly stable, capable of vigorous transgene expression and suitable for high titer manufacture and purification using cell lines that are engineered to complement the defective viral functions in trans. Preclinical studies will be carried out in vitro using human endothelial cells (Aim 2) followed by in vivo studies in rodent (Aim 3), pig and primate (Aim 4) heart transplantation models. Vector application studies will include the development of procedures for vector administration to heart blood vessels, analyses of vector dosing and distribution, characterization of HO-1 expression and byproduct synthesis (e.g. CO), and evaluation of protection from I-R. It is our long-term goal to develop the preclinical efficacy and safety data needed to perform early phase I trials in heart transplant recipients.

Glioblastoma multiforme(GBM) is a devastating disease that almost invariably leads to patient death despite best efforts using standard therapies that include surgery, radiation and chemotherapy. New therapeutic interventionsare needed which may be used in combination with standard medical practice. Among these treatments, gene therapy potentially holds promise for treatment of GBM however impediments to effective gene delivery remain. Highly attenuated replication competent HSV-1 vectors providea powerful opportunityto provide effective gene delivery in additionto the natural lytic features (oncolysis) and early phase human trials support the safety of this approach. In this proposal, experiments are outlinedto explore methodsto enhancethe potency of HSV oncolyticvectors through improvedvector distribution within the tumor mass, the testing of additional mutant vector backbones whose performance may be improved by enhanced and more specific intra-tumoralreplicationand through the use of additionaltransgenes that may be more effective in destruction of the tumor mass including locally infiltrating tumor cells into normal brain tissue. Throughout this investigationvector performanceand tumor killing will be evaluated in combinationwith radiosurgery. We will use the performance of G207 as a benchmark with which to compare any vector improvements. In four related specific aims we will: (i) Exploit the use of collagenases to enhance intra-tumoral vector distribution as visualized by advanced vital microscopy; (ii) Examine new genetic alterations in the HSV genome in a search for more active mutant oncolytic vectors that have the same or better safety profiles as vectors currently used in early phasepatient studies (e.g. G207); (iii) Develop retargeting strategies to enable tumor-specific HSV infection through recognition of tumor-cell receptors; and (iv) Introduce novel anti-tumor transgenes into the vector backbone that include (a) purine nucleoside phosphorylase (PNP) in combination with 6-methylpurine(MeP) treatment, (b) chlorotoxin (CltX), a peptide that inhibits tumor cell migration and may induce tumor cell apoptosis (c) tumor necrosis factor (TNFoc)that acts to sensitize tumor cells and the tumor vasculature to radiosurgical methods. The outcome of these studies are intended to discover new vectors, more effective transgenes and delivery strategies which together may provide gene therapy as an effective approach to at least prolong the survival of patients with recurrent GBM over currently available treatment methods.

The vanilloid receptor (TRPV1; formerly known as VR1) plays an important role in primary hyperalgesia, a[unreadable] component of chronic pain. TRPV1 is functionally up-regulated in patho-physiologic states such as painful[unreadable] diabetic neuropathy and cystopathy. Activation of TRPV1 occurs through a number of convergent signaling[unreadable] pathways and emerging evidence suggests that TRPV1 function is also negatively regulated by numerous[unreadable] mechanisms. The discovery of novel cellular inhibitors or negative modulators of TRPV1 function and their[unreadable] mode of action should provide additional insights into the role of TRPV1 in peripheral pain signaling and by[unreadable] extension, suggest novel approaches for their application to the control of primary hyperalgesia. We propose[unreadable] to test the hypotheses that (i) novel cellular products play an important role in the inhibition or negative[unreadable] modulation of TRPV1 function and (ii) that vector-based expression of these molecules can control TRPV1[unreadable] signaling in vivo. To test these hypotheses, we have devised a combination of HSV vector-based genetic[unreadable] strategies to identify these potential regulatory molecules and biochemical methods to characterize their role[unreadable] in the negative control of TRPV1 function. Specifically, we will attempt to identify products that (a) inhibit[unreadable] TRPV1 activation by capsaicin (CAP) and resiniferatoxin (RTX) and (b) interfere with TRPV1 potentiation by[unreadable] protein kinase C epsilon (PKCe) activated by Phorbol 12-myristate 13-acetate (PMA). TRPV1 inhibitory[unreadable] genes that either have been engineered or naturally occur, will be studied in some detail to determine the[unreadable] molecular mechanism underlying their ability to impede TRPV1-mediated calcium influx. In four specific aims[unreadable] we intend to (i) create model systems in which HSV TRPV1 expression vectors can be used to examine[unreadable] potential inhibitors of TRPV1 function or calcium overload (ii) create a library of vectors expressing cDNAs[unreadable] derived from rat dorsal root ganglia, (iii) characterize mechanisms by which the selected neuronal gene[unreadable] products inhibit or negatively modulate TRPV1 function and (iv) evaluate the analgesic effects of the[unreadable] engineered and novel TRPV1 inhibitors in vivo using rat models of pain where TRPV1 antagonism has been[unreadable] shown to reduce pain signaling. Both the model inhibitor genes and novel genes obtained by the HSV[unreadable] screening methods will be provided to Projects 1 and 2 for evaluation of their potential analgesic effects in[unreadable] diabetes-related neuropathic pain and models of bladder pain.

[unreadable] In the previous period of funding we developed gene transfer vectors based on the herpes simplex virus[unreadable] (HSV) to deliver genes with high efficiency to peripheral sensory neurons. We have exploited this property[unreadable] to develop non-replicating HSV vectors that provide an analgesic effect in models of somatic pain[unreadable] (inflammatory pain, neuropathic pain, and pain resulting from cancer) and visceral pain (bladder[unreadable] inflammation). In this renewal we focus our efforts to further develop HSV vectors for treatment of acute and[unreadable] chronic pain related to diabetes and painful bladder syndromes. Project 1 will explore the use of HSV-mediated[unreadable] transfer of genes that produce inhibitory neurotransmitters (glutamic acid decaraboxylase and[unreadable] proenkephalin) and anti-inflammatory peptides (IL4 and TNFDSR) in models of painful diabetic neuropathy.[unreadable] Project 2 will explore the analgesic effect of these vectors in acute and chronic rodent models of bladder[unreadable] pain. Project 3 will use a vector-based functional genomics approach to identify and characterize novel[unreadable] cellular gene products that inhibit or negatively modulate the activity of the vanilloid receptor TRPV1.[unreadable] Projects 1 and 2 are designed to provide preclinical evidence for vectors that may be developed for novel[unreadable] treatments of human disease, while Project 3 will lead to the identification and evaluation of novel gene[unreadable] products that would then be tested in a similar fashion. Administration (Core A), Preclinical Vector Production[unreadable] (Core B) and Gene Transfer Assays and Imaging (Core C) cores directly support the activities of all three[unreadable] projects. We believe this to be a timely and highly innovative proposal which is likely to provide new[unreadable] approaches to treatment of chronic pain related to diabetes and inflammation.

21+ years old; A Mouse; Abscission; Adult; Aliquot; Appearance; Articulation; Assay; BACs (Chromosomes); Bacteria; Bacterial Artificial Chromosomes; Bioassay; Biochemical; Biologic Assays; Biological Assay; Biological Models; Blood Serum; Body Tissues; CDF; Candidate Disease Gene; Candidate Gene; Cell Communication and Signaling; Cell Differentiation; Cell Differentiation process; Cell Line; Cell Lineage; Cell Lines, Strains; Cell Signaling; Cell division; CellLine; Cells; Cellular Expansion; Cellular Growth; Cholinergic Differentiation Factor; Cloning; Cloning Vectors; Code; Coding System; Commit; Complement; Complement Proteins; Complementary DNA; Complex; Condition; D-Factor; DNA Binding; DNA Binding Interaction; DNA Library; DNA Recombination; DNA bank; DNA recombination (naturally occurring); DNA, Complementary; Data; Data Banks; Data Bases; Databank, Electronic; Databanks; Database, Electronic; Databases; Defective Hybrids; Defective Interfering Particles; Defective Interfering Viruses; Defective Viruses; Detection; Development; ES Cell Line; ES cell; Early Promoters; Eating; Elements; Embryo; Embryonic; Embryonic Stem Cell Line; Engineering; Engineerings; Enhancers; Essential Genes; Event; Excision; Expression Library; Extirpation; FP593; Figs; Figs - dietary; Fluorescence; Food Intake; GFAC; Gene Expression; Gene Products, RNA; Gene Targeting; Generalized Growth; Genes; Genes, Essential; Genes, Immediate-Early; Genes, Reporter; Genetic Recombination; Genome; Goals; Growth; Growth Agents; Growth Factor; Growth Factors, Proteins; Growth Substances; HHV-1; HILDA; HSV; HSV vector; HSV-1; HSV1; Herpes Simplex Virus; Herpes Simplex Virus 1; Herpes Simplex Virus Type 1; Herpes Simplex Virus Vector; Herpes labialis Virus; Herpesvirus 1 (alpha), Human; Herpesvirus 1, Human; Herpesvirus hominis; Human Interleukin in DA Cells; Human herpes simplex virus type 1; Human herpesvirus 1; Human herpesvirus type 1; Human, Adult; ICP47; IGF-1; IGF-I; IGF-I-SmC; IGF1; IGF1 gene; IGFI; Image; Immediate-Early Genes; In Vitro; Incomplete Viruses; Infection; Insulin-Like Growth Factor 1; Insulin-Like Growth Factor I; Insulin-Like Somatomedin Peptide I; Intracellular Communication and Signaling; Investigators; Joints; Kinetic; Kinetics; LIF; LIF gene; Left; Libraries; Life; MLPLI; Mammals, Mice; Melanoma-Derived LPL Inhibitor; Methods; Mice; Model System; Modeling; Models, Biologic; Mother Cells; Murine; Mus; Muscle; Muscle Cells; Muscle Cells, Mature; Muscle Development; Muscle Tissue; Muscle satellite cell; Muscular Development; Myocytes; Nature; PCR; PRR protein; Pathway interactions; Phenotype; Plasmid Cloning Vector; Plasmid Vector; Plasmids; Play; Polymerase Chain Reaction; Population; Process; Production; Progenitor Cells; Promoter; Promoters (Genetics); Promoters, Early; Promotor; Promotor (Genetics); Proteins; RNA; RNA, Non-Polyadenylated; Range; Receptor Protein; Recombination; Recombination, Genetic; Removal; Reporter Genes; Research Personnel; Researchers; Ribonucleic Acid; Role; Serum; Signal Transduction; Signal Transduction Systems; Signaling; Simplexvirus; Somatomedin C; Spinal Column; Spine; Staging; Standards; Standards of Weights and Measures; Stem cells; Surgical Removal; System; System, LOINC Axis 4; TK Gene; Targetings, Gene; Technology; Testing; Thymidine Kinase Gene; Time; Tissue Growth; Tissues; Trans-Acting Factors; Trans-Activators; Transactivators; Transcription Regulation; Transcriptional Control; Transcriptional Regulation; Transplantation; VP 16; VP 16 (drug); VP16; Vero Cells; Vertebral column; Viral; Viral Gene Products; Viral Gene Proteins; Viral Proteins; Virion; Virus; Virus Particle; Virus Replication; Viruses, General; adult human (21+); backbone; base; biological signal transduction; cDNA; cDNA Expression; cDNA Library; cell engineering; cell growth; cellular engineering; clinical data repository; clinical data warehouse; cultured cell line; cytotoxic; data repository; day; drFP583; ds red protein; dsFP593; embryonic stem cell; engineering design; experience; experiment; experimental research; experimental study; functional genomics; gene product; herpes simplex i; herpes virus 1, human; herpesvirus; human alphaherpesvirus 1; human herpesvirus 1 group; imaging; insight; interest; leukemia inhibitory factor; muscle stem cell; mutant; myogenesis; nectin; novel; ontogeny; particle; pathway; progenitor; protein expression; receptor; red fluorescent protein; relational database; research study; resection; satellite cell; social role; stem; stem cell of embryonic origin; trans acting factor (genetic); transgene expression; transplant; vector; virus multiplication; virus protein

Glioblastoma mutliforme (GBM) is the most common form of primary brain cancer. Despite aggressive therapies including surgery, radiotherapy, and chemotherapy, recurrent disease is nearly always fatal. Oncolytic HSV vectors (e.g. G207) have shown some promise in the treatment of GBM however there have been few complete responses, a disappointing outcome most likely related to inadequate vector infection and growth, particularly among tumor cells that migrate from the tumor mass and invade normal brain tissue. Thus a central goal of Project 3 is to improve oncolytic vector delivery, replication and spread while maintaining safety and tumor specificity. Because changes in the tumor microenvironment greatly influence virus growth, we propose further to arm these oncolytic vectors with genes that improve vector distribution, overcome local anti-viral responses and enhance susceptibility to apoptotic mechanisms. Specifically, we propose to: (i) to explore the growth, spread and anti-tumor potential of a highly active HSV -1 strain KOS Oncolytic Vector (KOV) deleted for the non-essential immediate early (I.E.) genes ICPO, ICP22 and ICP47, (ii) to employ a recombinant KOV vector expressing a secreted matrix metalloproteinase protease (ADAMTS-8) with strong anti-angiogenic activity in an effort to increase initial vector distribution and to facilitate vector spread during replication, (iii) examine the use of a recombinant KOV capable of expressing VH1, binl and a dominant negative IKB (kBaM) as inhibitors of the interferon gamma (IFNy) and indoleamine 2,3-dioxygenase (IDO) antiviral and cytokine induction pathways and (iv) to evaluate the ability of recombinant KOV expressing (a) a novel dominant negative PKCe (DNP) that blocks its anti-apoptotic function, (b) caspase 8a to launch the apoptotic cascade and (c) an optimized recombinant soluble TRAIL (orsTRAIL) to induce tumor cell apoptosis. Ultimately, it is our intention to create a powerful oncolytic vector that exploits these combined growth-facilitating, anti-tumor functions that will set a new standard for this form of glioma therapy. This new vector will be compared to G207 to demonstrate improved anti-tumor responses. The highly engineered vector will also provide opportunities to better understand glioma cell biology, greatly improve the use of anti-cancer drugs in collaboration with Project 1 and assist the induction of tumor-specific immunity in collaboration with Project 2. Together our replication competent gene vectors should be useful in the development of an effective multi-modal therapy, an important overall goal of our program project grant.

DESCRIPTION (provided by applicant): Chronic pain is a major health concern affecting 80 million Americans at some time in their lives. Current pharmacotherapies are not effective long-term, which has led to the development and testing of gene therapy approaches. We and others have demonstrated that herpes simplex virus type 1 (HSV) based vectors can deliver highly effective pain-modulating transgenes to sensory neurons in vivo following inoculation of peripheral tissue. One major advantage of this approach is that painful tissue can be specifically targeted by local vector delivery. We believe that this advantage can be further extended by targeting specific neuron types responsible for chronic pain, thus enabling gene transfer to be tailored to specific types of pain, such as inflammatory or neuropathic pain, while simultaneously reducing deleterious side effects. We have recently shown that HSV-mediated transfer and long-term expression of the glycine receptor 11 subunit (GlyR11) can be used to control the timing and duration of afferent silencing with exogenous administration of glycine. Based on these findings and our recent success in the creation of highly efficient, fully retargeted HSV vectors, we hypothesize that we can selectively silence distinct subpopulations of primary afferents responsible for neuropathic and inflammatory pain, therefore providing injury specific pain relief. These studies will enable us to determine whether the same or different afferent populations underlie inflammatory and neuropathic pain and provide the rational basis for the future development of HSV-based gene transfer vectors designed to restrict analgesia to the relevant primary afferents. We anticipate that these studies will (i) provide a novel platform technology that will allow us to selectively express transgenes designed to modulate the function of sensory afferent subpopulations, a strategy that can be readily extended to other types of sensory nerve disorders; (ii) develop initial functional and physical maps of sensory afferent subtypes that upon silencing will block different persistent pain conditions, thereby providing essential information needed for targeted pain control; (iii) identify afferents that have been functionally altered to respond to painful stimuli providing further information on nerve fiber plasticity; and, (iv) identify potential risks associated with silencing of an inappropriate population of sensory neurons (e.g. altered proprioception). To achieve these goals, we have proposed a series of interrelated experiments described in 3 Specific Aims. In Aim 1, the infectivity of HSV vectors will be retargeted to selectively transduce (a) A2 fibers, (b) peptidergic and (c) nonpeptidergic C fibers. In Aim 2, we will complement transductional retargeting with transcriptional targeting using transgene promoters that will, when combined with transductional targeting, fine tune silencing specificity. The combination strategy is intended to create initial fine maps of subpopulations of sensory fibers within the larger transductionally targeted groups representing critical afferents for the response to different painful stimuli. In Aim 3, the retargeted HSV vectors will be introduced into the DRG by peripheral inoculation of animal models of inflammatory and neuropathic pain and the analgesic efficacy and side effect profiles will be established following glycine-induced GlyR11-mediated silencing.

The goal of this proposal is to develop improved oncolytic herpesviral vectors (oHSV) for the treatment of glioblastomas (GBM), a deadly cancer of the nervous system for which adequate therapy remains elusive Oncolytic viruses derive their anti-cancer activity from their ability to lyse cancer cells and spread within the tumor while sparing normal cells. Their selectivity for cancer cells is generally based on natural or engineered defects in the viral genome that inactivate key replication functions in normal cells but are complemented by the abnormal environment within cancer cells. HSV is an attractive vector for oncolytic therapy for tumors of the nervous system because of its natural neurotropism, high lytic activity, well-defined life cycle controlled by two essential genes, and capacity to deliver multiple exogenous anti-cancer protein products to enhance their efficacy. Clinical trials of current oncolytic HSV (oHSV) have demonstrated safety but limited efficacy, due in part to barriers examined by each of the projects of this program grant. Our study seeks to take advantage of new vector engineering technologies to achieve greater replication efficiency and oHSV spread within the tumor without compromising safety. We hypothesize that restricting oHSV infection to tumor cells by engineering steps referred to as retargeting, combined with measures to block virus replication in normal cells based on signature differences in microRNA expression profiles between normal and cancer cells, will provide a new generation of oHSVs that will prove both highly effective and safe. In Aim 1, we will redirect oHSV infectivity to glioblastoma cells by viral envelope engineering and in collaboration with Project 2-3, test, the effect of HDAC inhibition and virus-mediated ectopic chondroitinase expression on oHSV distribution and spread. In Aim 2, we will install genetic elements in the oHSV that will allow replication exclusively in glioblastoma cells as a function of the unique microRNA signatures of these cells. In Aim 3, animal experiments will be performed to test the safety and efficacy of selected oHSVs that combine key features from the first two aims. With potential further improvements based on feedback from all other projects of this program, we expect that we will engineer a promising new generation of oHSV for evaluation in clinical trials.

? DESCRIPTION (provided by applicant): There are a number of diseases that affect the bladder that do not have adequate treatments. For example, interstitial cystitis (IC) and overactive active bladder (OAB) are chronic urological disorders characterized by increased micturition frequency and urgency; IC is distinct from OAB in that patients additionally suffer from pelvic/suprapubic pain. Likewise, inability to empty the bladder is a common debilitating problem (often associated with severe pain) among spinal cord injury patients and for some, is the major contributor to decreased quality of life. In all of these examples, it is not completely clear how distinct subpopulations of bladder afferents differentially drive these symptoms and thus, there are basic science issues that need to be addressed before mechanism-based approaches can be explored. Our laboratory (the Davis lab) has been using genetic mouse lines that express channelrhodopsin (ChR2) or halorhodopsin (NpHR) in sensory neurons. Preliminary data presented in this application demonstrate that these light sensitive channels can be used to regulate the visceromotor reflex (a surrogate for bladder pain) as well as control the micturition reflex. Unfortunately, our previous studies rely on transgenic mouse models that have significant limitations inherent in a genetic technology that produces permanent gene changes that are activated during embyogenesis and lack temporal and spatial control. New tools are needed to determine if our observations can be extended to other relevant animal models, as well as developed into effective treatments for human. This application is in response to RFA-RM-15-002 that strives to develop tools that will be tailored to the specific use case/mechanism under study and whose end deliverable is a tool or technology, NOT a biological discovery (from Common Fund pdf provided to potential applicants). The co-PI on this application (Dr. Glorioso) is a pioneer in the use of sensory neuron-specific viruses that can be used to activate or silence sensory neurons. In particular, he has developed a novel, druggable chloride channel (ivermectin) construct that is effective in blocking somatic pain. In addition, the Glorioso lab has produced other novel viral constructs that have passed phase I human trials, allowing the proposed studies to meet the criteria of translatability that is require by this RFA. This proof of concept data while exciting, will only be applicable to human bladder (as well as other animal models) if appropriate strategies are developed that will allow expression of these molecules in humans. The proposed research program will combine the efforts of these two laboratories; one studying pain and regulation of visceral organ function and a second that has a long track-record in designing sensory neuron-specific viruses that can be used to target expression of novel genes to primary afferents. We will produce and test 9 different viral constructs (expressing three novel genes, under three different promoters), targeted to different sensory neuron populations and determine their effectiveness for control micturition and the visceromotor reflex (a surrogate for bladder pain).

DESCRIPTION (provided by applicant): Glioblastoma multiforme (GBM) is a deadly brain tumor without effective therapy. Oncolytic herpes simplex viral vectors (OVs) provide an attractive treatment alternative based on preclinical studies however clinical trials have thus far been disappointing showing only anecdotal evidence for complete responses. We propose vector design strategies to overcome impediments to robust vector performance. Important vector limitations include (i) inadequate intra-tumoral virus growth due to the introduction of attenuating mutations (ii) reduced vector distribution due to poor initial penetration of the tumor mass and limited spread of new virions, and (iii) tumor cell migration away from the tumor margin, limiting their accessibility to infectious virus. To meet these challenges, we propose to create an innovative oHSV that retains the full complement of HSV replication functions that will ensure optimal vector replication and lytic activity. Vector safety will be ensured by vector targeting to tumor-related receptors including tumor stem cell markers and the expression of essential virus genes will be controlled by naturally occurring micro-RNAs (miRs) that are differentially expressed in normal brain and tumor cells. In addition, the vector will be armed wit human matrix metalloproteases (MMPs) that impair formation of the extracellular matrix (ECM), a natural barrier to virus spread, and with an antagonist of c-Met activation, an essential functio for tumor cell growth and migration. Together these transgenes are expected to enhance intra-tumoral vector spread, increase tumor access of infectious virus and impede tumor cell migration. The safety of these vectors will be evaluated in toxicology studies using immunocompetent mice, and treatment efficacy will be examined in orthotopic mouse models involving intracranial implantation of primary human glioblastoma initiating cells (GICs) that represent the four classes of stage IV GBM. The xenografts exhibit characteristics of human glioblastoma including tumor cell migration and provide a means to evaluate the utility of this next generation of oHSV as an effective tumor therapy. We anticipate that this combination of tumor targeting, robust vector replication and enhanced vector access to tumor cells will provide a powerful technology to eradicate tumors with the potential for translation to patients.

Oncolytic HSV vectors (oHSV) offer considerable promise in the treatment of Glioblastoma Multiforme (GBM). In previous studies we designed a new base vector (KGN-4:T124) that is blocked for replication in normal brain tissue by brain-specific cellular miR-124 targeting of ICP4 expression. This base vector was further retargeted (KGNE-4:T124) to achieve selective infection of GBM cells expressing EGFR/EGFRvIII. Arming of this vector with a matrix metalloproteinase (KGNE-4:T124-MMP9) enhanced vector spread and oncolysis in xenogeneic animal models. However, virolysis alone is unlikely to achieve complete elimination of tumor cells that can cause recurrence. Effective elimination of all tumor cells will require the induction of innate and adaptive anti-tumor immunity. Unfortunately, GBM down-regulates the cellular machinery needed to sustain activation of immune cells including NK, macrophages and cytotoxic T cells. Therefore we propose to test the hypothesis that GBM treatment with our oHSV base vectors, KGN-4:T124 and KGNE-4:T124, can be enhanced through vector arming with immunomodulatory genes that inhibit tumor progression and induce innate and adaptive anti-tumor immune responses. To this end, we will initially exploit a tumor cell line (BAGL1) derived from a GBM tumor induced in BALB/c mice by Sleeping Beauty transposition of genes encoding luciferase, anti-p53 shRNA, N-RasV12 and human EGFRvIII. These experiments will be extended to a genetically-induced GBM model developed by Dr. Eric Holland that avoids tumor cell injection. Our aims will seek to counteract three aspects of immune evasion in GBM: (i) ADAM17-mediated shedding of NK cell activators and reduced NK function, (ii) expression of the immune checkpoint molecules that lead to NK and T cell exhaustion, and (iii) the presence of M2 macrophages that contribute to an immunosuppressive tumor microenvironment (TME). In Aim 1, we will test the hypothesis that our unarmed base vectors, collectively referred to as KGN(E)-4:T124, will provide an effective GBM treatment in immune competent mice. We will determine the impact of vector dosing on intratumoral vector distribution, TME composition and animal survival, and assess the value of EGFR targeting. We will also examine the effect of prior HSV immunization on treatment outcomes. In Aim 2, we will test the hypothesis that KGN(E)- 4:T124 derivatives armed with TIMP-3 (ADAM17 inhibitor/VEGF-R2 antagonist) will improve therapy. We will evaluate the impact of TIMP-3 expression on NK cell activation, tumor spread and animal survival. In Aim 3, we will test the hypothesis that vectors armed with single chain antibody (scFv) checkpoint inhibitors (anti-PD-L1 or anti-CTLA4), alone or in combination with depletion of the immunosuppressive (M2) TAM population using BLZ945-mediated inhibition of colony-stimulating factor-1 receptor (CSF-1R), will produce effective anti-tumor immunity, particularly in HSV immune mice. Anti-tumor immunity will be established using a bi-lateral tumor model and confirmed by selective antibody-mediated T cell depletion.

PROJECT SUMMARY ? PROJECT 1 Oncolytic herpes simplex virus (oHSV) vectors offer considerable promise in the treatment of Glioblastoma Multiforme (GBM). In the current funding period we designed a new series of HSV vectors capable of safe but efficient tumor destruction through (i) full vector retargeting to prevalent human GBM receptors (EGFR/EGFRvIII) (vector KGNE), (ii) cellular miR-124 blockade of vector replication in normal brain (KGN-4:T124: our base vector), or (iii) a combination of these features (KGNE-4:T124). Further arming of the combination vector with a matrix metalloproteinase (KGNE-4:T124-MMP9) enhanced vector spread and oncolysis in animal models. However, vector replication in tumors can still be limited by Natural Killer (NK) cells that normally protect the host from HSV infection. Projects 2 and 4 have recently demonstrated that interference with NK cell activation and killing of virus-infected cells enhances GBM treatment by allowing more robust and broader oncolytic virus spread. We propose to validate and extend these findings by comparing the impact of vector transgene-mediated NK blockade on the oncolytic activities of the two base vectors of this PPG, our KGN-4:T124 (strain KOS) and vector rQNestin34.5 (strain F) from Project 2, in three distinct syngeneic mouse models of GBM. Because these base vectors have different genetic backgrounds and are controlled by different safety mechanisms, their comparison in these experiments will be important for choosing the most effective backbone and arming genes for future trials. We will exploit two HSV-permissive mouse glioma cell lines, CT2A and GL261N4, to generate brain tumors in immunocompetent mice by tumor cell injection. In addition, we have an established collaboration with Eric Holland (U. of Washington) to verify key results in his genetically induced mouse models of GBM based on the RCAS/tv-a system. In Aim 1, we will establish base-line tumor treatment efficacies for KGN-4:T124 versus rQNestin34.5. We will perform tumor-treatment dose-response analyses, and characterize and compare the effects of the two vectors on the composition of the tumor microenvironment (TME). In Aim 2, we will take advantage of these base-line data to evaluate the effect of NK cell antagonism on tumor therapy. In collaboration with Projects 2 and 4, we will determine the effects of vector arming with genes encoding soluble forms of HCMV UL141 and/or PRV glycoprotein D that reduce the expression of glioma cell ligands recognized by NK cells. In Aim 3, we will express the Fc?-binding ectodomains of two other HCMV-derived proteins, gp34 and gp68, that are known to inhibit NK cell activation. In collaborative experiments with Project 4, we will test the hypothesis that sgp34 will inhibit classical ADCC (antibody-dependent cellular cytotoxicity), enhance vector replication and tumor virolysis while sgp34 and/or sgp68 will interfere with Fc bridging cellular cytotoxicity (FcBCC). We will also provide TIMP-3 expression vectors to Project 3 to inhibit Notch signaling among macrophages and thereby reduce macrophage recognition of infected tumor cells and prolong oHSV persistence.