Fragile X mental retardation protein replacement restores hippocampal synaptic function in a mouse model of fragile X syndrome.

Fragile X syndrome (FXS) is caused by a mutation that silences the fragile X mental retardation gene (FMR1), which encodes the fragile X mental retardation protein (FMRP). To determine whether FMRP replacement can rescue phenotypic deficits in a fmr1-knockout (KO) mouse model of FXS, we constructed an adeno-associated virus-based viral vector that expresses the major central nervous system (CNS) isoform of FMRP. Using this vector, we tested whether FMRP replacement could rescue the fmr1-KO phenotype of enhanced long-term depression (LTD), a form of synaptic plasticity that may be linked to cognitive impairments associated with FXS. Extracellular excitatory postsynaptic field potentials were recorded from CA3-CA1 synaptic contacts in hippocampal slices from wild-type (WT) and fmr1-KO mice in the presence of AP-5 and anisomycin. Paired-pulse low-frequency stimulation (PP-LFS)-induced LTD is enhanced in slices obtained from fmr1 KO compared with WT mice. Analyses of hippocampal synaptic function in fmr1-KO mice that received hippocampal injections of vector showed that the PP-LFS-induced LTD was restored to WT levels. These results indicate that expression of the major CNS isoform of FMRP alone is sufficient to rescue this phenotype and suggest that post-developmental protein replacement may have the potential to improve cognitive function in FXS.

Different tropism of adenoviruses and adeno-associated viruses to corneal cells: implications for corneal gene therapy.

PURPOSE: Diseased corneas are potential targets for viral-based gene therapy to normalize (stimulate or inhibit) the expression of specific proteins. The choice of viral vectors is important to achieve optimal effect. The purpose of this study was to compare the tropism to different corneal cells of recombinant adenovirus (rAV) and recombinant adeno-associated virus (rAAV) constructs using live rabbit and organ-cultured human corneas. METHODS: rAV constructs harbored the green fluorescent protein (GFP) gene under the control of major immediate early cytomegalovirus (CMV) promoter. rAAV constructs from virus serotypes 1, 2 5, 7, and 8 had GFP under the chicken beta-actin promoter and CMV enhancer. For organ culture, 16 healthy and diabetic postmortem human corneas were used. Five or fifteen microl rAV at 10(7) plaque forming units per 1 microl were added for 2 days to culture medium of uninjured corneas that were further cultured for 5-32 days. rAAV were added at 1.2-7.8x10(10) vector genomes per cornea for 3 days to each cornea; the culture then continued for another 14-23 days. Corneal cryostat sections were examined by immunohistochemistry. Live rabbit corneas were used following excimer laser ablation of the corneal epithelium with preservation of the basal cell layer. Equal numbers of rAAV particles (2x10(11) vector genomes) were applied to the cornea for 10 min. After seven days to allow for corneal healing and gene expression the animals were euthanized, the corneas were excised, and sections analyzed by immunohistochemistry. RESULTS: By direct fluorescence microscopy of live organ-cultured human corneas GFP signal after rAV transduction was strong in the epithelium with dose-dependent intensity. On corneal sections, GFP was seen in all epithelial layers and some endothelial cells but most keratocytes were negative. In rAAV-transduced organ-cultured human corneas GFP signal could only be detected with anti-GFP antibody immunohistochemistry. GFP was observed in the epithelium, keratocytes, and endothelium, with more pronounced basal epithelial cell staining with rAAV1 than with other serotypes. No difference in the GFP expression patterns or levels between normal and diabetic corneas was noted. The rabbit corneas showed very similar patterns of GFP distribution to human corneas. With all rAAV serotype vectors, GFP staining in the epithelium was significantly (p=0.007) higher than the background staining in non-transduced corneas, with a trend for rAAV1 and rAAV8 to produce higher staining intensities than for rAAV2, rAAV5 (p=0.03; rAAV5 versus rAAV1), and rAAV7. rAAV serotype vectors also transduced stromal and endothelial cells in rabbit corneas to a different extent. CONCLUSIONS: rAAV appears to reach many more corneal cells than rAV, especially keratocytes, although GFP expression levels were lower compared to rAV. rAV may be more useful than rAAV for gene therapy applications requiring high protein expression levels, but rAAV may be superior for keratocyte targeting.

Novel nuclear signaling pathway mediates activation of fibroblast growth factor-2 gene by type 1 and type 2 angiotensin II receptors.

In bovine adrenal medullary cells synergistically acting type 1 and type 2 angiotensin II (AII) receptors activate the fibroblast growth factor-2 (FGF-2) gene through a unique AII-responsive promoter element. Both the type 1 and type 2 AII receptors and the downstream cyclic adenosine 1',3'-monophosphate- and protein kinase C-dependent signaling pathways activate the FGF-2 promoter through a novel signal-transducing mechanism. This mechanism, which we have named integrative nuclear FGF receptor-1 signaling, involves the nuclear translocation of FGF receptor-1 and its subsequent transactivation of the AII-responsive element in the FGF-2 promoter.

The role of intratumour therapy with electroporation and bleomycin in the management of advanced squamous cell carcinoma of the head and neck.

OBJECTIVES: To determine the safety and efficacy of electroporation with bleomycin in patients with advanced squamous cell carcinoma of the head and neck. METHODS: Two open-label, multicenter, single-arm Phase II studies of intratumour electroporation therapy. Sixty-two patients with 86 squamous cell carcinoma tumours of the head and neck were enrolled. Twenty-five patients were treated with bleomycin alone. Fifty-four patients (17 initially treated with bleomycin alone) were treated with electroporation and bleomycin therapy. Local tumour response was measured. RESULTS: In the bleomycin alone group, one tumour showed a partial response and 36 tumours showed no response to treatment. In the bleomycin with electroporation groups, 17 tumours showed complete response, 22 tumours showed partial response and 30 failed to achieve more than a 50% reduction in tumour size (no response). Bleomycin with electroporation had a significantly (p<0.001) greater number of patients showing a partial or complete response to the therapy when compared to bleomycin alone. Thirteen adverse events were reported which included five episodes of local bleeding, six local infections, one local tongue swelling and one cardiac arrhythmia. CONCLUSIONS: Fifty-seven percent of squamous cell carcinomas of the head and neck demonstrated a partial or complete response to intratumour electroporation with bleomycin suggesting that further work investigating its use as a treatment for local control of these lesions should be pursued.

In vivo gene transfer to the brain cortex using a single injection of HSV-1 vector into the medial septum.

This study shows that an ICP4-replication-deficient herpes simplex virus containing the Moloney murine leukaemia virus LTR fused with the coding sequence for the beta-galactosidase gene can be used as a very effective vector for delivering the beta-galactosidase reporter gene into the rat brain septum. F344 rats received bilateral stereotaxic injections into the nucleus of the diagonal band and into the medial septum. The X-gal stain was used to detect the activity of the expressed beta-galactosidase enzyme. The delivered reporter gene was expressed successfully not only in the neuronal cells of the injected areas but also in cells that project to the injection area such as cortex cells about 6 mm away from the injection sites. Expression was visible at 1, 3 and 9 weeks following injection. We conclude that this vector can effectively deliver genes into different regions of the mature mammalian brain and also to areas distant from the injection site.

A revised HindIII map and sequence analysis of a large 'left-hand' non-essential region of the rabbit poxvirus genome.

Sequence analysis of a 21.5 kb region of the left-hand non-essential region of rabbit poxvirus (RPV) was performed to analyze the structure and gene organization of this region. Initial mapping and cloning studies revealed that the early published HindIII maps are incorrect in that the location of the terminal HindIII B and C fragments, as previously published, are reversed. The sequence of this region was compared to similar regions of several strains of vaccinia virus. The results indicate that this region of RPV has a high degree of homology with a similar region of vaccinia. Analysis of the translated open reading frames (ORFs) has facilitated the detection of several previously unreported ORFs related to human complement proteins. In addition, two defective copies of a homologue of the 77 kDa cowpox virus (CPV) host range gene which allows CPV to grow on Chinese hamster ovary cells have been detected, suggesting a gene duplication has occurred. However, one copy of this gene is segmented into three separate ORFs. Analysis has also identified a large number of indirect repeat elements. A possible role of these repeats in the generation of deletions and gene duplications is suggested.

Long-term expression of a reporter gene from latent herpes simplex virus in the rat hippocampus.

A problem in utilizing herpes simplex virus (HSV) as a vector for expression of foreign genes in CNS neurons has been the inability to facilitate long-term expression of the engineered genes. Previously, we showed that the murine moloney leukemia virus LTR would drive beta-galactosidase (beta-gal) transcription for extended periods from the latent viral genome in sensory, but not motor neurons. In this communication we further evaluate the utility of the LTR promoter for use in long-term expression vectors. Following stereotactic injection of 8117/43 (an ICP4 minus, non-replicating virus with the LTR driving the beta-gal gene, or KD6 (an ICP4 minus non-replicating virus not expressing beta-gal) into the hippocampus of rats, polymerase chain reaction (PCR) analysis of viral DNA after 2 months indicated that latent infections were established. Assaying by both x-gal staining and reverse transcriptase PCR we demonstrate that (1) beta-gal can be detected for at least 6 months in hippocampal neurons, and (2) although the number of beta-gal transcripts in these cells drops considerably by 2 weeks, they can be detected during the period studied. These studies indicate that the LTR promoter is active and affords long-term expression in the CNS, albeit at comparatively low levels compared to those observed at acute times.

HSV Vectors for Gene Therapy.

A number of aspects of the natural biology of herpes simplex virus (HSV) make it an attractive candidate for a vector to express foreign genes within the nervous system. Some of the advantages of an HSV vector are 1 Establishment of a life-long latent infection within peripheral and central nervous system neurons (for a review, see ref,1); 2 Latent HSV genomes exist as multiple episomal copies/neuron and integration is not known to occur (2), and 3. Nonreplicating HSV recombinants can establish a latent infection efficiently (3).

Leiomyoma of the nasal septum.

Leiomyoma is a benign myogenic tumor that may develop wherever smooth muscle is present. It occurs commonly in the uterus, skin, and gastrointestinal tract and is rare within the nasal cavity. Only three of twenty-four reported cases of sinonasal leiomyoma have been found to originate from the nasal septum. Treatment of choice for these neoplasms is surgical excision. We present two cases of nasal septal leiomyoma. Unique features discussed include recurrence of one neoplasm and the technique used to endoscopically repair a cerebrospinal fluid leak resulting from resection of the neoplasm.

Gene transfer into the central nervous system using herpes simplex virus-1 vectors.

Manipulation of gene expression in developing or in mature central nervous systems (CNS) holds a promise for the resolution of many compelling neurobiological questions, including the feasibility of gene therapy to treat diseases of the brain. In this context, a number of viral vectors have been used in recent years to introduce and express genes into the CNS. This article discusses a gene transfer system based on the Herpes Simplex Virus-1 (HSV-1). We describe here the use of non-replicating, non-toxic HSV-1 vector, 8117/43, in a series of studies carried in our joint program. This vector proves further the utility of HSV-1 as a delivery vehicle to a number of distinct sites within the CNS.

HSV-1-mediated NGF delivery delays nociceptive deficits in a genetic model of diabetic neuropathy.

A previous phase III clinical trial failed to show significant therapeutic benefit of repeated subcutaneous nerve growth factor (NGF) administration in the treatment of diabetic neuropathy. Animal studies have since shown that site-specific viral-mediated expression of NGF in the lumbar dorsal root ganglia prevents peripheral nerve dysfunction associated with chemically induced neuropathy. Using a Herpes simplex virus expression vector, we have investigated the effect of localized NGF expression in a genetic mouse model of progressive diabetic neuropathy, the +/+ Leprdb mouse. We found that site-specific delivery of NGF initially delayed the appearance of hypoalgesia, assessed by the Hargreaves test, by 1 month and effectively attenuated this deficit for 2 months over the approximately 10 months normal life-span of these animals. Once the disease progressed into its more severe stages, NGF, although still capable of altering the electrophysiological profile of the sensory A- and C-fibers and influencing the expression of p75 and substance P in the dorsal root ganglia, could no longer maintain normal nociception. These data suggest that maximal therapeutic benefit in future NGF-based gene therapy trials will be gained from early applications of such viral-mediated neurotrophin delivery.

Experimental investigation of herpes simplex virus latency.

The clinical manifestations of herpes simplex virus infection generally involve a mild and localized primary infection followed by asymptomatic (latent) infection interrupted sporadically by periods of recrudescence (reactivation) where virus replication and associated cytopathologic findings are manifest at the site of initial infection. During the latent phase of infection, viral genomes, but not infectious virus itself, can be detected in sensory and autonomic neurons. The process of latent infection and reactivation has been subject to continuing investigation in animal models and, more recently, in cultured cells. The initiation and maintenance of latent infection in neurons are apparently passive phenomena in that no virus gene products need be expressed or are required. Despite this, a single latency-associated transcript (LAT) encoded by DNA encompassing about 6% of the viral genome is expressed during latent infection in a minority of neurons containing viral DNA. This transcript is spliced, and the intron derived from this splicing is stably maintained in the nucleus of neurons expressing it. Reactivation, which can be induced by stress and assayed in several animal models, is facilitated by the expression of LAT. Although the mechanism of action of LAT-mediated facilitation of reactivation is not clear, all available evidence argues against its involving the expression of a protein. Rather, the most consistent models of action involve LAT expression playing a cis-acting role in a very early stage of the reactivation process.

Generation and use of recombinant reporter viruses for study of herpes simplex virus infections in vivo.

It has become increasingly clear that the fate of herpes simplex virus (HSV) infections involves complex interactions between the virus and the specific cell types that comprise the tissues of the animal host. No reliable cell culture system for studying the establishment of latency and reactivation exists, and therefore these studies must be performed within animal models. One difficulty in elucidating the molecular regulation of these events is in determining the transcriptional activity of key viral genes during different stages of the infection in vivo. The heterogeneous cell types comprising infected tissues make PCR analysis of tissue homogenates difficult to interpret. The need to characterize expression of multiple transcriptional markers reliably and quantitatively at the level of individual cells is therefore key to determining the interplay between viral and cellular genes during latency and reactivation. Here we discuss the construction and evaluation of HSV reporter viruses that have been used in these analyses. HSV-1 recombinants have been engineered with representative viral promoters driving beta-galactosidase as a reporter. Methodology used to evaluate the levels of gene expression using (1) quantitative enzyme assays, (2) precipitatable substrate assays to localize the positive cells, and (3) immunohistochemistry and fluorescence assays to look at colocalization of markers during in vivo infection is presented. In addition to studying the molecular pathogenesis of HSV, the application of similar reporter viruses to evaluate promoters for targeting various differentiated tissues will be useful in developing these viruses as potential vectors for gene therapy.

Neuron-specific restriction of a herpes simplex virus recombinant maps to the UL5 gene.

We have previously shown that, when compared with either parent, a herpes simplex virus type 1/herpes simplex virus type 2 intertypic recombinant (R13-1) is attenuated by 10,000-fold with respect to neurovirulence in mice. Despite this, after intracranial inoculation, R13-1 replicated to titers of 10(5) PFU per brain. We present evidence that the restriction is specific for replication in neurons and have taken a three-step approach in determining the basis of the attenuation by (i) characterizing cellular tropism of the virus in both central and peripheral nervous systems, (ii) defining where in the viral replication cycle the restriction is manifest, and (iii) identifying the genetic basis of the restriction through marker rescue analysis. Following inoculation into the animal, R13-1 viral antigens predominate in nonneuronal cells, and the block to replication in neurons was found to be beyond the level of adsorption and penetration. Despite the restricted replication within neurons, the virus established a latent infection in spinal ganglia and could be reactivated by in vitro cocultivation of the ganglia. In studies carried out in cell culture, R13-1 was found to replicate normally in mouse embryo fibroblasts and primary mouse glial cells but was restricted by 1,000-fold in primary mouse neurons and PC12 cells. R13-1 appeared to produce normal levels of early RNA in these cells, but production of DNA and late RNA was less than that of the wild type. Marker rescue analysis localized the fragment responsible for restoring neurovirulence to UL5, a component of the origin-binding complex implicated in replication of the viral genome. Our results with this virus, with a cell-specific restriction, suggest that a neuron-specific component is involved in viral replication.

The nature of naturally occurring mutations in the hemagglutinin gene of vaccinia virus and the sequence of immediately adjacent genes.

The expression of the vaccinia IHD-J hemagglutinin (HA) gene is regulated by two promoters, an early/late and a second distinct late promoter. The first promoter results in transcripts that begin early and are synthesized throughout the infection. All transcripts from this promoter initiate at the same 5' site. A second promoter is only active late in infection and initiates transcription some distance upstream of the first promoter. We have previously shown that the transcriptional start site controlled by this second, "late only" promoter lies within the coding sequence of an upstream reading frame (p16-ORF), whereas the termination of early transcription of the HA gene utilizes a transcription termination signal (TTTTTNT) located just beyond the coding region of an immediately downstream reading frame (p17-ORF). In order to assist our understanding of HA gene expression, we report here the sequence of these two ORFs adjacent to the HA gene. The HA-ORF itself consists of 945 bp, whereas the upstream p16-ORF consists of 429 bp and the downstream p17-ORF of 453 bp, sufficient to encode polypeptides of 16 and 17 kD, respectively. While many strains of vaccinia are HA+, rabbitpox virus and the variant of vaccinia IHD-J, designated IHD-W, are HA-. We report and compare here the HA gene sequences of wild-type rabbit poxvirus, two spontaneous HA+ revertants of rabbit poxvirus, and the HA- vaccinia strain IHD-W to that of the previously sequenced (1) prototype HA+ IHD-J strain of vaccinia. All differences were found to occur within the HA open reading frame.(ABSTRACT TRUNCATED AT 250 WORDS)

LAT expression during an acute HSV infection in the mouse.

Previous studies using cell culture systems to evaluate LAT expression demonstrated that the LAT promoter expresses at much higher levels in neuroblastoma cell lines than fibroblast lines. The high level of LAT expression in neuronal-derived cell lines correlates with the high level of LAT accumulation observed in sensory ganglia neurons during a latent infection. We have found that using LAT promoters to express reporter genes from recombinant viruses in vivo produces high levels of LAT promoter activity in the epithelium of the mouse foot. An analysis of LAT promoter activity during an acute infection in the mouse clearly demonstrates that in contrast to studies performed with selected cell lines, the LAT promoter expresses similar levels of reporter gene product in peripheral cells and in neurons. In addition, the amount of reporter gene product is higher when the LAT promoter is located within the R(L) as compared to the U(L) region, and when expression is adjusted for copy number of the reporter construct, expression is roughly the same. These results suggest the activity of the LAT promoter varies greatly according to cell type and that high levels of expression is not limited solely to neurons, especially in the in vivo setting.

Herpes simplex virus type 1 DNA replication and gene expression during explant-induced reactivation of latently infected murine sensory ganglia.

Infectious virus assays and PCR amplification of DNA and RNA were used to investigate herpes simplex virus DNA replication and gene expression in two murine in vitro models for virus reactivation. We examined latent infections with wild-type (wt), precisely defined latency-associated transcript-negative (LAT-) mutants, and LAT+ rescuants of these mutants of the 17syn+ strain of virus in both murine trigeminal and lumbosacral ganglia and of the KOS(M) strain in the latter. In explants of ganglia latently infected with the LAT- mutant of strain 17syn+ virus, a reduction in number of cultures exhibiting cytopathic effects due to virus reactivation and measurable delays in virus recovery were observed compared with wt or the LAT+ rescuant. This LAT-specific effect was not seen in explants of lumbosacral ganglia latently infected with mutants derived from the KOS(M) strain of virus. Although there was appreciable variation between individual animals, no significant difference between LAT+ and LAT- virus in time of onset of viral DNA replication in explanted ganglia was seen with use of either virus strain. There was a slight decrease in the relative amount of viral DNA recovered compared with internal cellular controls in latently infected ganglia harboring the LAT- mutant of 17syn+ compared with the wt virus or the LAT+ rescuant. This reduced relative amount ranged from 0 to as much as 50% but averaged 20%. Such differences were not seen in infections with KOS(M)-derived mutants. In contrast, although expression of productive-cycle transcripts could be detected within 4 h following explant cultivation of latently infected ganglia, no differences between LAT+ and LAT- viruses could be seen. As discussed, these data place specific constraints on possible models for the role of LAT expression in in vitro reactivation systems.

Recruitment to the cytoplasm of a cellular lamin-like protein from the nucleus during a poxvirus infection.

Monoclonal antibodies (Mabs) directed against core proteins of rabbit poxvirus (RPV) have proven effective in the identification of host cell proteins such as RNA polymerase II (Pol II) that may play a role in the infectious process (D. K. Morrison and R. W. Moyer, 1986, Cell 44, 587-596). In this article we describe a Mab that has allowed the detection and characterization of a lamin-like protein derived from the nucleus of the infected cell, which like Pol II is recruited to the cytoplasm following RPV infection. A portion of the gene encoding this protein has been isolated through the screening of a lambda gt11 expression vector library. Sequence analysis of the gene shows it to be derived from a member of the HindIII 1.9-kb repetitive element, a family of mammalian repetitive sequences that are highly conserved. Immunoblot analysis and sequence analysis of the open reading frame show divergent relatedness to certain nuclear lamins. The protein is not, however, one of the three principal lamins characterized to date, but instead appears to be a perinuclear protein related to the highly conserved nuclear lamins that is recruited to the cytoplasm during the infectious process.