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J C Glorioso

Publications and source records attributed to J C Glorioso.

At least 73 records · Page 4Linked to original sources
PubMed1997

Viral gene delivery to skeletal muscle: insights on maturation-dependent loss of fiber infectivity for adenovirus and herpes simplex type 1 viral vectors.

The mechanisms causing age-dependent loss of muscle fiber infectivity observed in vivo for both adenoviral (Ad) and herpes simplex virus type 1 (HSV-1) gene delivery vectors remain poorly understood. Here we investigate the possible bases for this phenomenon using the novel application of enzymatically isolated, viable, single muscle fibers. We show that maturation-dependent loss of fiber infectivity is recapitulated in single fibers, and, thus, is not solely due to host immune response. Using localized irradiation of muscle in vivo, we show data suggesting that Ad infectivity of differentiated myofibers depends, at least in part, on myoblasts to mediate fiber transduction. On the other hand, infection of single fibers by HSV-1 is not affected by irradiation. Using confocal microscopy, we show that the basal lamina of myogenic cells efficiently infected by HSV-1 is structurally less organized than that of fibers resistant to infection by HSV-1. As well, we show that single myofibers isolated from adult, basal lamina-defective mice (merosin-deficient, dy/dy) are at least 10-fold more susceptible to infection by HSV-1 than are myofibers isolated from control mice. Together, these observations support the hypothesis that the basal lamina acts as a physical barrier to HSV-1 infection of mature muscle.

Adenoviridae↗
PubMed1997

LacZ gene transfer to skeletal muscle using a replication-defective herpes simplex virus type 1 mutant vector.

Herpes simplex virus type 1 (HSV-1) represents a promising new viral vector capable of efficient transduction of myofibers in vivo. Here we report on the use of a replication-defective HSV-1 mutant vector (DZ) deleted for the essential immediate early (IE) gene ICP4 for studies of reporter gene transfer and expression following direct inoculation of mouse skeletal muscle. The recombinant vector was engineered to contain the Escherichia coli lacZ gene under transcriptional control of the strong human cytomegalovirus (HCMV) IE promoter. The effect of vector cytotoxicity on the durability of transgene expression following infection of muscle cells in culture and myofibers in vivo revealed that this first-generation HSV vector was cytopathic, limiting the persistence of vector-transduced cells. UV irradiation of vector preparations reduced viral cytotoxicity for myoblasts in culture without reducing significantly beta-galactosidase production. Moreover, muscle cell viability and the durability of transgene expression was enhanced by several days following UV inactivated-vector infection in vivo. Nevertheless, the viral DNA was subsequently lost from vector-inoculated muscle tissue within 2 weeks. This observation indicated that vector toxicity alone did not account for the lack of persistent transgene expression. Longer-term vector transduction and transgene expression was observed, however, following inoculation of immunodeficient SCID mice, indicating that host immunocompetence played an important role in determining the duration of transgene expression in animals. To support this hypothesis, cells expressing CD4 and CD8 antigens have been found in the HSV-1 injected muscle of immunocompetent mice. These data demonstrated that both vector toxicity and vector-induced immunity are significant obstacles to the use of HSV-1 vectors for muscle gene transfer. These impediments must be overcome to further develop HSV vectors for muscle gene therapy applications.

Animals↗
PubMed1997

Development of an HSV-based vector for the treatment of Parkinson's disease.

The restricted pattern of neurodegeneration seen in Parkinson's disease, and the identification of trophic factors that prevent toxin-induced degeneration of dopaminergic neurons, has spurred research into potential gene therapy for this disease. Herpes simplex virus (HSV-1) is a neurotrophic virus which naturally establishes latency in neurons. HSV-based vectors have been demonstrated to transfer and transiently express transgenes in neurons in brain in vivo. Recent experiment have shown that deletion of multiple immediate-early HSV genes reduces the potential cytotoxicity of these vectors, and in addition results in altered patterns of transgene expression that may allow for long-term expression required for human gene therapy applications.

Gene Expression↗
PubMed1997

Gene transfer to muscle using herpes simplex virus-based vectors.

The main goal of gene therapy for Duchenne muscular dystrophy (DMD) is to restore dystrophin into as many muscle cells as necessary to be therapeutic. Herpes simplex virus type 1 (HSV-1) represents a promising new viral vector capable of efficient transduction of myofibers in vivo. The viral genome is large and can accommodate multiple or large non-viral genes including the full-length dystrophin. Here we report on the use of a replication defective HSV-1 mutant vector (DZ) deleted for the essential immediate early (IE) gene ICP4 for studies of reporter gene transfer and expression following direct inoculation of mouse skeletal muscle. Our initial experiments showed that HSV-1 can efficiently infect and express a foreign reporter gene in myoblasts and myotubes in vitro. Furthermore, the intramuscular inoculation of HSV-1 resulted in transduction of a significant number of muscle fibers in newborn mice and some muscle fibers in adult animals. We have attempted to exploit these features to develop new HSV mutant vectors for dystrophin gene delivery to DMD muscle, however two impediments to using this virus for muscle gene delivery have to be overcome: namely viral cytotoxicity and the differential transducibility with HSV-1 throughout the development of muscle fibers. To solve the first problem, virus mutants deleted for the immediate early (IE) genes (ICP4, ICP22, ICP27 and UL41) were constructed and the multiple deleted virus was greatly reduced in cytotoxicity relative to our first generation HSV vector strains. Current work is aimed at incorporating full-length dystrophin under muscle specific promoter (muscle creatine kinase MCK) into these new viral vectors. To address the second problem we have analysed by immunohistochemistry the spreading of the HSV-1 in newborn versus adult muscles to determine whether mature basal lamina which surrounds the adult muscle fibers blocks the HSV-1 entry into the mature muscle fibers.

Animals↗
PubMed1997

Engineering herpes simplex virus vectors for gene transfer to neurons.

HSV has a unique relationship with neurons which the virus naturally utilizes for efficient gene transfer and long-term gene expression. Progress in developing defective viral mutants with reduced cytotoxicity, and increasing insight into the state of the viral genome during latency and the functional elements of the latency promoter system, suggest that HSV vectors may be developed in which these natural features are exploited for effective transgene expression in brain.

Animals↗
PubMed1997

Rapid method for construction of recombinant HSV gene transfer vectors.

Herpes simplex virus type 1 (HSV-1) is a neurotrophic human pathogen that naturally persists in neurons in a latent state and carries a large number of viral functions which can be replaced by foreign genes to create a vector for gene therapy applications. In this report we describe a two-step method for insertion/deletion mutagenesis of HSV genes and the efficient insertion of transgenes into these locations in the viral genome. The first step is the insertion of a reporter gene (lacZ) cassette flanked by Pacl restriction enzyme sites not otherwise found in the viral genome, using standard marker transfer procedures to interrupt a portion of the target HSV gene. The second step is substitution of the reporter gene with other foreign cDNAs by digestion of the vector DNA with Pacl to remove the lacZ gene and subsequent repair of the vector genome by homologous recombination with a transgene expression plasmid. Potential recombinants identified by a 'clear plaque' phenotype after X-gal staining arose at high frequency (80-100%). Of these recombinants containing the transgene in place of the lacZ gene ranged from 19-65%. Insertion of the transgene expression construct into the viral genome eliminates the Pacl sites, allowing this method to be used repeatedly for the sequential deletion of multiple HSV genes while inserting multiple transgenes. This procedure was repeated in succession to produce a vector carrying two independent expression cassettes at distinct viral loci.

DNA, Viral↗
PubMed1997

Deletion of the S component inverted repeat sequence c' and the nonessential genes U(S)1 through U(S)5 from the herpes simplex virus type 1 genome substantially impairs productive viral infection in cell culture and pathogenesis in the rat central nervous system.

A distinctive feature of the genetic make-up of herpes simplex virus type 1 (HSV-1), a human neurotropic virus, is that approximately half of the 81 known viral genes are not absolutely required for productive infection in Vero cells, and most can be individually deleted without substantially impairing viral replication in cell culture. If large blocks of contiguous viral genes could be replaced with foreign DNA sequences, it would be possible to engineer highly attenuated recombinant HSV-1 gene transfer vectors capable of carrying large cellular genes or multiple genes having related functions. We report the isolation and characterization of an HSV-1 mutant, designated d311, containing a 12 kb deletion of viral DNA located between the L-S Junction a sequence and the U(S)6 gene, spanning the S component inverted repeat sequence c' and the nonessential genes U(S)1 through U(S)5. Replication of d311 was totally inhibited in rat B103 and mouse Neuro-2A neuroblastoma cell lines, and was reduced by over three orders of magnitude in human SK-N-SH neuroblastoma cells compared to wild-type (wt) HSV-1 KOS. This suggested that the deleted genes, while nonessential for replication in Vero cells, play an important role in HSV replication in neuronal cells, particularly those of rodent origin. Unlike wt KOS which replicated locally and spread to other regions of brain following stereotactic inoculation into rat hippocampus, d311 was unable to replicate and spread within the brain, and did not cause any apparent local neuronal cell damage. These results demonstrate that d311 is highly attenuated for the rat central nervous system. d311 and other mutants of HSV containing major deletions of the nonessential genes within U(S) have the potential to serve as useful tools for gene transfer applications to brain.

Animals↗
PubMed1997

Herpes simplex virus vectors for gene transfer to the nervous system.

Herpes simplex virus (HSV) represents a candidate gene transfer vector for the treatment of nervous system disease. It has many natural biological features which make it attractive for gene delivery to a variety of tissues. The virus naturally establishes a latency in sensory neurons of the peripheral nervous system, wherein the virus in maintained as an extrachromosomal DNA element in the absence of viral lytic gene expression without altering the metabolism of the host neuron. The virus possesses a neuronal latency-specific promoter system which remains active long-term, while other viral and cellular promoters are repressed. Replication defective virus recombinants have been engineered to delete multiple essential immediate early gene functions rendering these new mutants significantly less cytotoxic to neurons and other cells in culture. Further developments in regulating transgene expression and reducing virus toxicity will continue to aid the design and use of these vectors for therapeutic applications for the nervous system.

Animals↗
PubMed1996

Human immunodeficiency virus tat gene transfer to the murine central nervous system using a replication-defective herpes simplex virus vector stimulates transforming growth factor beta 1 gene expression.

The high incidence of neurological disorders in patients afflicted with acquired immunodeficiency syndrome (AIDS) may result from human immunodeficiency virus type 1 (HIV-1) induction of chemotactic signals and cytokines within the brain by virus-encoded gene products. Transforming growth factor beta1 (TGF-beta1) is an immunomodulator and potent chemotactic molecule present at elevated levels in HIV-1-infected patients, and its expression may thus be induced by viral trans-activating proteins such as Tat. In this report, a replication-defective herpes simplex virus (HSV)-1 tat gene transfer vector, dSTat, was used to transiently express HIV-1 Tat in glial cells in culture and following intracerebral inoculation in mouse brain in order to directly determine whether Tat can increase TGF-beta1 mRNA expression. dSTat infection of Vero cells transiently transfected by a panel of HIV-1 long terminal repeat deletion mutants linked to the bacterial chloramphenicol acetyltransferase reporter gene demonstrated that vector-expressed Tat activated the long terminal repeat in a trans-activation response element-dependent fashion independent of the HSV-mediated induction of the HIV-1 enhancer, or NF-kappaB domain. Northern blot analysis of human astrocytic glial U87-MG cells transfected by dSTat vector DNA resulted in a substantial increase in steady-state levels of TGF-beta1 mRNA. Furthermore, intracerebral inoculation of dSTat followed by Northern blot analysis of whole mouse brain RNA revealed an increase in levels of TGF-beta1 mRNA similar to that observed in cultured glial cells transfected by dSTat DNA. These results provided direct in vivo evidence for the involvement of HIV-1 Tat in activation of TGF-beta1 gene expression in brain. Tat-mediated stimulation of TGF-beta1 expression suggests a novel pathway by which HIV-1 may alter the expression of cytokines in the central nervous system, potentially contributing to the development of AIDS-associated neurological disease.

Animals↗
PubMed1996

The basal lamina is a physical barrier to herpes simplex virus-mediated gene delivery to mature muscle fibers.

A major impediment to successful implementation of gene therapy for treatment of muscular dystrophy is the restricted infectivity of mature muscle fibers with viral vectors. This phenomenon has been observed with adenovirus vectors and more recently with herpes simplex virus type 1 (HSV-1)-based vectors. Here we report findings of morphological studies designed to experimentally determine the mechanism underlying the rapid reduction in vector-mediated gene delivery concomitant with the maturation of muscle fibers. Using immunohistochemistry and confocal microscopy, we have colocalized HSV-1 and collagen IV, a major component of the basal lamina, in HSV-1-injected muscles and determined that the virus penetrates and expresses a transgene (lacZ) in muscle fibers of newborn animals but cannot efficiently penetrate adult myofibers. This was observed in normal as well as in immunocompromised animals, suggesting that the lack of adult myofiber transduction is not a result of an immune response and clearance of the viral vector. Since heparan sulfate proteoglycan, the initial attachment receptor for HSV-1, was shown to be preserved during the maturation of the myofibers by immunofluorescence assay and HSV-1 was able to infect isolated, viable myofibers in vitro, we suggest that the low-level HSV-1 transduction of mature myofibers is not a consequence of the loss of viral attachment sites on the surfaces of mature muscle fibers. Rather, our results indicate that the mature basal lamina acts as a physical barrier to HSV-1 infection of adult myofibers. This conclusion was further supported by the finding that HSV-1 displayed an intermediate level of transduction in mature dy/dy muscle which is defective for normal basal lamina formation. Together, these experiments suggest that efficient HSV vector transduction in mature skeletal muscle requires methods to permeabilize the basal lamina.

Animals↗
PubMed1996

Glycoprotein B of herpes simplex virus type 1 oligomerizes through the intermolecular interaction of a 28-amino-acid domain.

Herpes simplex virus type 1 glycoprotein B (gB) is an envelope component that plays an essential role in virus infection. The biologically active form of gB is an oligomer that contributes to the process of viral envelope fusion with the cell surface membrane, resulting in viral penetration and initiation of the replication cycle. In previous studies, two discontinuous sites for oligomer formation were identified: a nonessential upstream site located between residues 93 and 282 and an essential downstream site located between residues 596 and 711. In this study, in vitro-transcribed and -translated gB test molecules were used to characterize the more active essential membrane-proximal domain. A series of gB test polypeptides mutated in this downstream oligomerization domain were assayed for their abilities to form oligomers with a mutant gB capture polypeptide containing the analogous wild-type domain. Detection of oligomers was achieved by coimmunoprecipitation of two gB mutant molecules by using a monoclonal antibody specific for a hemagglutinin epitope tag introduced into the coding sequence of the capture polypeptide. Analysis of the immune-precipitated products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that the downstream oligomerization domain resided within residues 626 to 676. This region was further resolved into two segments, residues 626 to 653 and 653 to 675, each of which was independently sufficient to form oligomers. However, residues 626 to 653 provided for a stronger interaction between gB monomers. Moreover, this stretch of 28 amino acids was shown to form oligomers when introduced into the carboxy-terminal region of gB monomers lacking this domain at the normal site, thus indicating that this domain was functionally independent of its natural location within the gB molecule. Further analysis of the sequence within residues 596 to 653 by using mutant test polypeptides altered in individual amino acids revealed that cysteines 9 and 10 located at positions 596 and 633, respectively, were not required for oligomer formation but contributed to dimer formation and/or stabilization. The results of this study suggest that oligomerization of gB monomers is induced by interactions between contiguous residues localized within the ectodomain near the site of molecule insertion into the viral envelope membrane.

Amino Acid Sequence↗
PubMed1996

cis-acting elements involved in transcriptional regulation of the herpes simplex virus type 1 latency-associated promoter 1 (LAP1) in vitro and in vivo.

Latency-associated promoter 1 (LAP1) of herpes simplex virus type 1 is required to generate a series of latency-associated transcripts (LATs) in sensory neurons of latently infected animals. Sequence analysis and DNA binding studies have suggested the existence of several cis-acting elements within LAP1 that are potentially important for promoter function, although their role in LAT gene expression during latency is largely unexplored. In this report, we present evidence that the LAP1 TATA box is essential for transcription initiation in vitro. A reduction in LAT synthesis measured by in situ hybridization and reverse transcription-PCR (RT-PCR) of rat brain tissue latently infected with a LAP1 TATA substitution virus demonstrated that this sequence was required for full LAP1 activity in vivo. Analysis of additional site-directed and 5'-deletion mutants of LAP1 by in vitro transcription-primer extension assays showed that upstream elements including the USF and cyclic AMP response element (CRE) site specifically contributed to LAP1 function and that sequences beginning at position -620 relative to the transcription start site were essential for full promoter activity. The combination of deleting USF, CRE, and TATA completely abolished LAT expression in the brain, identifying these as essential elements for the neuron-specific functioning of LAP1 during latency. Mutation of the transcription start site did not abolish transcription, suggesting the absence of an initiator element. However, one of the most exciting findings from this study is that the region downstream of the TATA box appears to contain a true enhancer that is not only essential for transcription, but also functional when positioned 1.6 kb downstream of the start site of transcription. It was concluded that (i) the TATA box was essential for full transcriptional activity from LAP1 both in vitro and in vivo, (ii) the USF element and CRE contribute to LAP1 function during latency in combination with the TATA element, (iii) multiple trans-acting factors besides the USF- and CRE-binding proteins were required for full promoter activity in vitro, and (iv) sequences downstream of the TATA box enhanced promoter activity in vitro.

Animals↗
PubMed1996

Detection of herpes simplex virus type 1 latency-associated transcript expression in trigeminal ganglia by in situ reverse transcriptase PCR.

One of the defining characteristics of herpes simplex virus type 1 (HSV-1) infection is the ability of the virus to establish a lifelong latent state in neurons. We previously demonstrated (R. Ramakrishnan, A.J. Fink, G. Jiang, P. Desai, J. C. Glorioso, and M. Levine, J. Virol. 68:1864-1873, 1994) by in situ PCR that many more neurons contain viral genomes than are detected by in situ hybridization for HSV latency-associated transcripts (LATs). To determine whether all cells which contain genomes express LATs, we examined trigeminal ganglia for LATs 1 and 8 weeks after corneal scarification with ribonucleotide reductase-deficient HSV-1 by in situ reverse transcriptase PCR. The number of LAT-positive cells detected by in situ reverse transcriptase was substantially greater than the number of cells positive by in situ hybridization and appeared to be similar to the number of cells containing HSV genomes by in situ PCR and the number of ganglionic neurons that project to the cornea as detected by retrograde labeling with Fluorogold. These results demonstrate LAT expression in many neurons containing HSV-1 genomes.

Animals↗
PubMed1996

Involvement of a high-mobility-group protein in the transcriptional activity of herpes simplex virus latency-active promoter 2.

Latency-active promoter 2 (LAP 2) is a TATA-less promoter in herpes simplex virus type 1 (HSV-1) that can express genes during viral latency. Four regions of LAP2 are protected from DNase I digestion in vitro by either HeLa cell nuclear extracts or purified Sp1. Transient gene expression assays of LAP2 substitution mutants demonstrate that two of the regions protected by Sp1 and three other regions protected by nuclear extract are important for promoter function. The mutation causing the most significant reduction in expression alters a stretch of 23 thymidine residues (T23) that binds a protein with several properties common to high-mobility-group (HMG) proteins. The T23 binding activity is heat stable, can be inhibited by poly(dA-dT).poly(dA-dT), and is inhibited by minor-groove-binding drugs. Antiserum directed against HMG I(Y) blocked the formation of one of the DNA-protein complexes on the T23 oligonucleotide, suggesting that a protein antigenically related to HMG I(Y) binds to LAP2 in vitro. Direct evidence of HMG I(Y) involvement in LAP2 function is provided by the findings that recombinant HMG I(Y) protein facilitates Sp1 binding to LAP2 in mobility shift assays and that antisense HMG I(Y) RNA specifically inhibits LAP2 function in vivo. These results suggest that DNA structure may be an important determinant of the activity of a promoter that is capable of escaping the global shutoff of transcription that occurs during viral latency.

Animals↗