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Alfred I Geller - One of the best experts on this subject based on the ideXlab platform.

  • overexpression of either lysine specific demethylase 1 or clock but not co rest improves long term expression from a modified neurofilament promoter in a Helper Virus free hsv 1 vector system
    Brain Research, 2012
    Co-Authors: Guorong Zhang, Hua Zhao, Haiyan Cao, Alfred I Geller
    Abstract:

    Long-term expression from Helper Virus-free Herpes Simplex Virus (HSV-1) vectors is required for many specific neural gene therapies and studies on neuronal physiology. We previously developed a promoter that supports long-term, neuron-specific expression by fusing the chicken s-globin insulator (INS), followed by an upstream enhancer from the rat tyrosine hydroxylase (TH) promoter, to a neurofilament heavy gene (NFH) promoter. Here, we examined the capability of specific transcription factors to further improve long-term expression from this promoter. Following a HSV-1 Virus infection, the Virus genome is localized to promyelocytic leukemia protein (PML) nuclear bodies (NB). At these sites, specific cellular transcription factors interact with HSV-1 encoded transcription factors, and together regulate HSV-1 gene expression. Importantly, lysine-specific demethylase-1 (LSD1), CLOCK, and Co-Rest each activate HSV-1 gene expression. However, gene expression from HSV-1 vectors differs in a number of important aspects from the Virus, including no HSV-1 genes are expressed. Nonetheless, these observations raise the possibility that specific transcription factors may improve long-term expression from specific promoters in HSV-1 vectors. Here, we show that overexpression of either LSD1 or CLOCK improves long-term expression from the INS-TH-NFH promoter, but overexpression of Co-Rest supports levels of long-term expression similar to those supported by a control vector. Further, overexpression of LSD1 is compatible with neuron-specific expression. Thus, overexpressing specific transcription factors can improve long-term expression from specific cellular promoters in HSV-1 vectors, and the chromatin structure of the vector has an important role in enabling expression.

  • a 16 bp upstream sequence from the rat tyrosine hydroxylase promoter supports long term expression from a neurofilament promoter in a Helper Virus free hsv 1 vector system
    Brain Research, 2011
    Co-Authors: Guorong Zhang, Hua Zhao, Soumya Awasthi, Alfred I Geller
    Abstract:

    Helper Virus-free Herpes Simplex Virus vector-mediated gene transfer has supported studies on neuronal physiology, and may support specific gene therapies. Long-term, neuron-specific expression is required for many of these applications. A neurofilament heavy gene (NFH) promoter does not support long-term expression. We previously developed a promoter that supports long-term expression by fusing 6.3 kb of upstream sequences from the rat tyrosine hydroxylase (TH) promoter to a NFH promoter, and this promoter has supported physiological studies. The TH promoter fragment contains an enhancer, as it has activity in both orientations and at a distance from the basal promoter. Identifying this enhancer may support further improvements in long-term expression. A previous deletion analysis identified two ~100 bp fragments that each support long-term expression, and are contained within an ~320 bp fragment located ~3 kb from the TH promoter transcription start site. As this analysis used overlapping fragments, the two ~100 bp fragments contained 44 or 23 bp of unique sequence. Here, we used mutagenesis to identify a short sequence that supports long-term expression. We studied a 42 bp sequence, centered on the 23 bp unique sequence. Analysis of the wt sequence, and five mutations containing clustered changes that spanned the sequence, identified two adjacent mutations that do not support long-term expression, which together defined a 16 bp maximum essential sequence. This 16 bp sequence contains a putative E2F-1/DP-1 transcription factor binding site, and this transcription factor is expressed in many brain areas.

  • a Helper Virus free hsv 1 vector containing the vesicular glutamate transporter 1 promoter supports expression preferentially in vglut1 containing glutamatergic neurons
    Brain Research, 2010
    Co-Authors: Guorong Zhang, Alfred I Geller
    Abstract:

    Abstract Multiple potential uses of direct gene transfer into neurons require restricting expression to specific classes of glutamatergic neurons. Thus, it is desirable to develop vectors containing glutamatergic class-specific promoters. The three vesicular glutamate transporters (VGLUTs) are expressed in distinct populations of neurons, and VGLUT1 is the predominant VGLUT in the neocortex, hippocampus, and cerebellar cortex. We previously reported a plasmid (amplicon) Herpes Simplex Virus (HSV-1) vector that placed the Lac Z gene under the regulation of the VGLUT1 promoter (pVGLUT1lac). Using Helper Virus-free vector stocks, we showed that this vector supported ∼ 90% glutamatergic neuron-specific expression in postrhinal (POR) cortex, in rats sacrificed at either 4 days or 2 months after gene transfer. We now show that pVGLUT1lac supports expression preferentially in VGLUT1-containing glutamatergic neurons. pVGLUT1lac vector stock was injected into either POR cortex, which contains primarily VGLUT1-containing glutamatergic neurons, or into the ventral medial hypothalamus (VMH), which contains predominantly VGLUT2-containing glutamatergic neurons. Rats were sacrificed at 4 days after gene transfer, and the types of cells expressing s-galactosidase were determined by immunofluorescent costaining. Cell counts showed that pVGLUT1lac supported expression in ∼ 10-fold more cells in POR cortex than in the VMH, whereas a control vector supported expression in similar numbers of cells in these two areas. Further, in POR cortex, pVGLUT1lac supported expression predominately in VGLUT1-containing neurons, and, in the VMH, pVGLUT1lac showed an ∼ 10-fold preference for the rare VGLUT1-containing neurons. VGLUT1-specific expression may benefit specific experiments on learning or specific gene therapy approaches, particularly in the neocortex.

  • Improved Long-term Expression from Helper Virus-free HSV-1 Vectors Packaged Using Combinations of Mutated HSV-1 Proteins that Include the UL13 Protein Kinase and Specific Components of the VP16 Transcriptional Complex
    BMC Molecular Biology, 2009
    Co-Authors: Xiaodan Wang, Alfred I Geller
    Abstract:

    Background Herpes Simplex Virus (HSV-1) gene expression is thought to shut off recombinant gene expression from HSV-1 vectors; however, in a Helper Virus-free HSV-1 vector system, a number of promoters support only short-term expression. These results raise the paradox that recombinant gene expression remains short-term even in the absence of almost all (~99%) of the HSV-1 genome, HSV-1 genes, and HSV-1 gene expression. To resolve this paradox, we hypothesized that specific proteins in the HSV-1 Virus particle shut off recombinant gene expression. In two earlier studies, we examined the effects on recombinant gene expression of packaging vectors using specific mutated HSV-1 proteins. We found that vectors packaged using mutated UL13 (a protein kinase), or VP16, or UL46 and/or UL47 (components of the VP16 transcriptional complex) supported improved long-term expression, and vectors packaged using mutated UL46 and/or UL47 also supported improved gene transfer (numbers of cells at 4 days). These results suggested the hypothesis that specific proteins in the HSV-1 particle act by multiple pathways to reduce recombinant gene expression. To test this hypothesis, we examined combinations of mutated proteins that included both UL13 and specific components of the VP16 transcriptional complex.

  • improved long term expression from Helper Virus free hsv 1 vectors packaged using combinations of mutated hsv 1 proteins that include the ul13 protein kinase and specific components of the vp16 transcriptional complex
    BMC Molecular Biology, 2009
    Co-Authors: Meng Liu, Xiaodan Wang, Alfred I Geller
    Abstract:

    Herpes Simplex Virus (HSV-1) gene expression is thought to shut off recombinant gene expression from HSV-1 vectors; however, in a Helper Virus-free HSV-1 vector system, a number of promoters support only short-term expression. These results raise the paradox that recombinant gene expression remains short-term even in the absence of almost all (~99%) of the HSV-1 genome, HSV-1 genes, and HSV-1 gene expression. To resolve this paradox, we hypothesized that specific proteins in the HSV-1 Virus particle shut off recombinant gene expression. In two earlier studies, we examined the effects on recombinant gene expression of packaging vectors using specific mutated HSV-1 proteins. We found that vectors packaged using mutated UL13 (a protein kinase), or VP16, or UL46 and/or UL47 (components of the VP16 transcriptional complex) supported improved long-term expression, and vectors packaged using mutated UL46 and/or UL47 also supported improved gene transfer (numbers of cells at 4 days). These results suggested the hypothesis that specific proteins in the HSV-1 particle act by multiple pathways to reduce recombinant gene expression. To test this hypothesis, we examined combinations of mutated proteins that included both UL13 and specific components of the VP16 transcriptional complex. A HSV-1 vector containing a neuronal-specific promoter was packaged using specific combinations of mutated proteins, and the resulting vector stocks were tested in the rat striatum. For supporting long-term expression, the preferred combination of mutated HSV-1 proteins was mutated UL13, UL46, and UL47. Vectors packaged using this combination of mutated proteins supported a higher efficiency of gene transfer and high levels expression for 3 months, the longest time examined. Vector particles containing this combination of mutated HSV-1 proteins improve recombinant gene expression. Implications of these results for strategies to further improve long-term expression are discussed. Moreover, long-term expression will benefit specific gene therapy applications.

Cornel Fraefel - One of the best experts on this subject based on the ideXlab platform.

  • herpes simplex Virus type 1 co infection leads to the formation of rolling circle amplification like adeno associated Virus dna replication products
    bioRxiv, 2020
    Co-Authors: Anita F Meier, Kurt Tobler, Remo Leisi, Anouk Lkharrazi, Carlos Ros, Cornel Fraefel
    Abstract:

    Adeno-associated Virus (AAV) genome replication only occurs in the presence of a co-infecting Helper Virus such as adenoVirus type 5 (AdV5) or herpes simplex Virus type 1 (HSV-1). AdV5-supported replication of the AAV genome has been described to occur in a strand-displacement rolling hairpin mechanism initiated at the AAV 3-prime inverted terminal repeat (ITR) end. It has been assumed that the same mechanism applies to HSV-1-supported AAV genome replication. We demonstrate the formation of double-stranded head-to-tail concatemers of AAV genomes in presence of HSV-1, and thus provide evidence for an unequivocal rolling circle amplification (RCA) mechanism. This study reveals the ability of AAV to modify the canonical rolling hairpin replication mechanism and to mimic the replication strategy of a co-infecting herpesVirus. This stands in contrast to the textbook model of AAV genome replication when HSV-1 is the Helper Virus. Furthermore, we introduce nanopore sequencing as a novel, high-throughput approach to study viral genome replication in unprecedented detail.

  • Definition of herpes simplex Virus type 1 Helper activities for adeno-associated Virus early replication events.
    PLoS Pathogens, 2009
    Co-Authors: Nathalie Alazard-dany, Cornel Fraefel, Armel Nicolas, Aurélie Ploquin, Regina Strasser, Anna Greco, Alberto L Epstein, Anna Salvetti
    Abstract:

    The human parvoVirus Adeno-Associated Virus (AAV) type 2 can only replicate in cells co-infected with a Helper Virus, such as AdenoVirus or Herpes Simplex Virus type 1 (HSV-1); whereas, in the absence of a Helper Virus, it establishes a latent infection. Previous studies demonstrated that the ternary HSV-1 helicase/primase (HP) complex (UL5/8/52) and the single-stranded DNA-Binding Protein (ICP8) were sufficient to induce AAV-2 replication in transfected cells. We independently showed that, in the context of a latent AAV-2 infection, the HSV-1 ICP0 protein was able to activate rep gene expression. The present study was conducted to integrate these observations and to further explore the requirement of other HSV-1 proteins during early AAV replication steps, i.e. rep gene expression and AAV DNA replication. Using a cellular model that mimics AAV latency and composite constructs coding for various sets of HSV-1 genes, we first confirmed the role of ICP0 for rep gene expression and demonstrated a synergistic effect of ICP4 and, to a lesser extent, ICP22. Conversely, ICP27 displayed an inhibitory effect. Second, our analyses showed that the effect of ICP0, ICP4, and ICP22 on rep gene expression was essential for the onset of AAV DNA replication in conjunction with the HP complex and ICP8. Third, and most importantly, we demonstrated that the HSV-1 DNA polymerase complex (UL30/UL42) was critical to enhance AAV DNA replication to a significant level in transfected cells and that its catalytic activity was involved in this process. Altogether, this work represents the first comprehensive study recapitulating the series of early events taking place during HSV-1-induced AAV replication.

  • improved Helper Virus free packaging system for hsv amplicon vectors using an icp27 deleted oversized hsv 1 dna in a bacterial artificial chromosome
    Molecular Therapy, 2001
    Co-Authors: Cornel Fraefel, Yoshinaga Saeki, Xandra O Breakefield, Tomotsugu Ichikawa, Antonio E Chiocca
    Abstract:

    Herpes simplex Virus type 1 (HSV-1) amplicons are prokaryotic plasmids containing one or more transcriptional units and two cis-acting HSV-1 sequences: a viral origin of DNA replication and a viral DNA cleavage/packaging signal. In the presence of HSV-1 "Helper" functions, amplicons are replicated and packaged into HSV-1 virions. Despite recent improvements in packaging methods, stocks of amplicon vectors are still contaminated with replication-competent Helper Virus at a frequency of 10(-4)-10(-6). To overcome this problem, we report that: (i) genetic modifications of HSV-1 genomes can be routinely achieved in Escherichia coli, either by homologous or site-specific recombination, (ii) a novel HSV-1 bacterial artificial chromosome (fHSVDeltapacDelta27 0+), which has a deletion in the essential gene encoding ICP27 and an addition of ICP0 "stuffer" sequences to increase its size to 178 kb, supports the replication and packaging of cotransfected amplicon DNA without generating replication-competent Helper Virus (<1 Helper Virus per 10(8) TU amplicon vectors), and (iii) the resulting amplicon stocks have titers of up to 3-10 x 10(8) TU/ml after concentration. Elimination of replication-competent Helper Virus from HSV-1 amplicon vector stocks further improves safety in gene transfer applications.

  • improved Helper Virus free packaging system for hsv amplicon vectors using an icp27 deleted oversized hsv 1 dna in a bacterial artificial chromosome
    Molecular Therapy, 2001
    Co-Authors: Cornel Fraefel, Yoshinaga Saeki, Xandra O Breakefield, Tomotsugu Ichikawa, Antonio E Chiocca
    Abstract:

    Abstract Herpes simplex Virus type 1 (HSV-1) amplicons are prokaryotic plasmids containing one or more transcriptional units and two cis -acting HSV-1 sequences: a viral origin of DNA replication and a viral DNA cleavage/packaging signal. In the presence of HSV-1 "Helper" functions, amplicons are replicated and packaged into HSV-1 virions. Despite recent improvements in packaging methods, stocks of amplicon vectors are still contaminated with replication-competent Helper Virus at a frequency of 10 −4 –10 −6 . To overcome this problem, we report that: (i) genetic modifications of HSV-1 genomes can be routinely achieved in Escherichia coli , either by homologous or site-specific recombination, (ii) a novel HSV-1 bacterial artificial chromosome (fHSVΔpacΔ27 0+), which has a deletion in the essential gene encoding ICP27 and an addition of ICP0 "stuffer" sequences to increase its size to 178 kb, supports the replication and packaging of cotransfected amplicon DNA without generating replication-competent Helper Virus ( 8 TU amplicon vectors), and (iii) the resulting amplicon stocks have titers of up to 3–10 × 10 8 TU/ml after concentration. Elimination of replication-competent Helper Virus from HSV-1 amplicon vector stocks further improves safety in gene transfer applications.

  • herpes simplex Virus type 1 dna amplified as bacterial artificial chromosome in escherichia coli rescue of replication competent Virus progeny and packaging of amplicon vectors
    Human Gene Therapy, 1998
    Co-Authors: Yoshinaga Saeki, Cornel Fraefel, Xandra O Breakefield, Antonio E Chiocca, Tomotsugu Ichikawa, Kurt Tobler, Akane Saeki, Mathias Ackermann
    Abstract:

    Herpes simplex Virus type 1 (HSV-1)-based amplicon vectors contain only approximately 1% of the 152-kb HSV-1 genome, and consequently, replication and packaging into virions depends on Helper functions. These Helper functions have been provided conventionally by a Helper Virus, usually a replication-defective mutant of HSV-1, or more recently, by a set of five cosmids that overlap and represent the genome of HSV-1 deleted for DNA cleavage/packaging signals (pac). In the absence of pac signals, potential HSV-1 genomes that are reconstituted from the cosmids via homologous recombination are not packageable. The resulting amplicon stocks are, therefore, virtually free of contaminating Helper Virus. To simplify this packing system, the HSV-1 genome was cloned and maintained stably as a single-copy, F plasmid-based bacterial artificial chromosome in E. coli. Such a plasmid containing the HSV-1 genome deleted for the pac signals (fHSV delta pac) did not generate replication-competent progeny Virus on transfection into mammalian cells, but rather, it was able to support the packaging of cotransfected amplicon DNA that contained a functional pac signal. The resulting amplicon vector stocks had titers of up to 10(7) transducing units per milliliter of culture medium and efficiently transduced neural cells in the rat brain, as well as hepatocytes in the rat. The capacity of generating infectious and replication-competent HSV-1 progeny following transfection into mammalian cells was restored after insertion of a pac signal into fHSV delta pac.

Xiaodan Wang - One of the best experts on this subject based on the ideXlab platform.

  • Improved Long-term Expression from Helper Virus-free HSV-1 Vectors Packaged Using Combinations of Mutated HSV-1 Proteins that Include the UL13 Protein Kinase and Specific Components of the VP16 Transcriptional Complex
    BMC Molecular Biology, 2009
    Co-Authors: Xiaodan Wang, Alfred I Geller
    Abstract:

    Background Herpes Simplex Virus (HSV-1) gene expression is thought to shut off recombinant gene expression from HSV-1 vectors; however, in a Helper Virus-free HSV-1 vector system, a number of promoters support only short-term expression. These results raise the paradox that recombinant gene expression remains short-term even in the absence of almost all (~99%) of the HSV-1 genome, HSV-1 genes, and HSV-1 gene expression. To resolve this paradox, we hypothesized that specific proteins in the HSV-1 Virus particle shut off recombinant gene expression. In two earlier studies, we examined the effects on recombinant gene expression of packaging vectors using specific mutated HSV-1 proteins. We found that vectors packaged using mutated UL13 (a protein kinase), or VP16, or UL46 and/or UL47 (components of the VP16 transcriptional complex) supported improved long-term expression, and vectors packaged using mutated UL46 and/or UL47 also supported improved gene transfer (numbers of cells at 4 days). These results suggested the hypothesis that specific proteins in the HSV-1 particle act by multiple pathways to reduce recombinant gene expression. To test this hypothesis, we examined combinations of mutated proteins that included both UL13 and specific components of the VP16 transcriptional complex.

  • improved long term expression from Helper Virus free hsv 1 vectors packaged using combinations of mutated hsv 1 proteins that include the ul13 protein kinase and specific components of the vp16 transcriptional complex
    BMC Molecular Biology, 2009
    Co-Authors: Meng Liu, Xiaodan Wang, Alfred I Geller
    Abstract:

    Herpes Simplex Virus (HSV-1) gene expression is thought to shut off recombinant gene expression from HSV-1 vectors; however, in a Helper Virus-free HSV-1 vector system, a number of promoters support only short-term expression. These results raise the paradox that recombinant gene expression remains short-term even in the absence of almost all (~99%) of the HSV-1 genome, HSV-1 genes, and HSV-1 gene expression. To resolve this paradox, we hypothesized that specific proteins in the HSV-1 Virus particle shut off recombinant gene expression. In two earlier studies, we examined the effects on recombinant gene expression of packaging vectors using specific mutated HSV-1 proteins. We found that vectors packaged using mutated UL13 (a protein kinase), or VP16, or UL46 and/or UL47 (components of the VP16 transcriptional complex) supported improved long-term expression, and vectors packaged using mutated UL46 and/or UL47 also supported improved gene transfer (numbers of cells at 4 days). These results suggested the hypothesis that specific proteins in the HSV-1 particle act by multiple pathways to reduce recombinant gene expression. To test this hypothesis, we examined combinations of mutated proteins that included both UL13 and specific components of the VP16 transcriptional complex. A HSV-1 vector containing a neuronal-specific promoter was packaged using specific combinations of mutated proteins, and the resulting vector stocks were tested in the rat striatum. For supporting long-term expression, the preferred combination of mutated HSV-1 proteins was mutated UL13, UL46, and UL47. Vectors packaged using this combination of mutated proteins supported a higher efficiency of gene transfer and high levels expression for 3 months, the longest time examined. Vector particles containing this combination of mutated HSV-1 proteins improve recombinant gene expression. Implications of these results for strategies to further improve long-term expression are discussed. Moreover, long-term expression will benefit specific gene therapy applications.

  • glutamatergic or gabaergic neuron specific long term expression in neocortical neurons from Helper Virus free hsv 1 vectors containing the phosphate activated glutaminase vesicular glutamate transporter 1 or glutamic acid decarboxylase promoter
    Brain Research, 2007
    Co-Authors: Morten Arendt Rasmussen, Xiaodan Wang, Lingxin Kong, Guorong Zhang, Meng Liu, Gabor Szabo, Norman P Curthoys, Alfred I Geller
    Abstract:

    Many potential uses of direct gene transfer into neurons require restricting expression to one of the two major types of forebrain neurons, glutamatergic or GABAergic neurons. Thus, it is desirable to develop Virus vectors that contain either a glutamatergic or GABAergic neuron-specific promoter. The brain/kidney phosphate-activated glutaminase (PAG), the product of the GLS1 gene, produces the majority of the glutamate for release as neurotransmitter, and is a marker for glutamatergic neurons. A PAG promoter was partially characterized using a cultured kidney cell line. The three vesicular glutamate transporters (VGLUTs) are expressed in distinct populations of neurons, and VGLUT1 is the predominant VGLUT in the neocortex, hippocampus, and cerebellar cortex. Glutamic acid decarboxylase (GAD) produces GABA; the two molecular forms of the enzyme, GAD65 and GAD67, are expressed in distinct, but largely overlapping, groups of neurons, and GAD67 is the predominant form in the neocortex. In transgenic mice, an approximately 9 kb fragment of the GAD67 promoter supports expression in most classes of GABAergic neurons. Here, we constructed plasmid (amplicon) Herpes Simplex Virus (HSV-1) vectors that placed the Lac Z gene under the regulation of putative PAG, VGLUT1, or GAD67 promoters. Helper Virus-free vector stocks were delivered into postrhinal cortex, and the rats were sacrificed 4 days or 2 months later. The PAG or VGLUT1 promoters supported approximately 90% glutamatergic neuron-specific expression. The GAD67 promoter supported approximately 90% GABAergic neuron-specific expression. Long-term expression was observed using each promoter. Principles for obtaining long-term expression from HSV-1 vectors, based on these and other results, are discussed. Long-term glutamatergic or GABAergic neuron-specific expression may benefit specific experiments on learning or specific gene therapy approaches. Of note, promoter analyses might identify regulatory elements that determine a glutamatergic or GABAergic neuron.

  • isolation of an enhancer from the rat tyrosine hydroxylase promoter that supports long term neuronal specific expression from a neurofilament promoter in a Helper Virus free hsv 1 vector system
    Brain Research, 2007
    Co-Authors: Qingshen Gao, Xiaodan Wang, Mei Sun, Alfred I Geller
    Abstract:

    Direct gene transfer into neurons, using a Virus vector, has been used to study neuronal physiology and learning, and has potential for supporting gene therapy treatments for specific neurological diseases. Many of these applications require high-level, long-term recombinant gene expression, in forebrain neurons. We previously showed that addition of upstream sequences from the rat tyrosine hydroxylase (TH) promoter to a neurofilament heavy gene (NF-H) promoter supports long-term expression in forebrain neurons, from Helper Virus-free Herpes Simplex Virus (HSV-1) vectors. This element in the TH promoter satisfied the definition of an enhancer; it displayed activity at a distance from the basal promoter, and in both orientations. This enhancer supported physiological studies that required long-term expression; a modified neurofilament promoter, containing an insulator upstream of the TH-NFH promoter, supported expression in ∼11,400 striatal neurons at 6 months after gene transfer, and expression for 7, 8, or 14 months, the longest times tested. In contrast, the NF-H promoter alone does not support long-term expression, indicating that the critical sequences are in the 6.3 kb fragment of the TH promoter. In this study, we performed a deletion analysis to identify the critical sequences in the TH promoter that support long-term expression. We localized these critical sequences to an ∼320 bp fragment, and two subfragments of ∼100 bp each. Vectors that contained each of these small fragments supported levels of long-term, neuronal-specific expression that were similar to the levels supported by a vector that contained the initial 6.3 kb fragment of the TH promoter. These small fragments of the TH promoter may benefit construction of vectors for physiological studies, and may support studies on the mechanism by which this enhancer supports long-term expression.

  • enhanced long term expression from Helper Virus free hsv 1 vectors packaged in the presence of deletions in genes that modulate the function of vp16 ul46 and ul47
    Journal of Neuroscience Methods, 2005
    Co-Authors: Meng Liu, Xiaodan Wang, Ju Tang, Tianzhong Yang, Alfred I Geller
    Abstract:

    Herpes simplex Virus (HSV-1) gene expression is hypothesized to shut off recombinant gene expression from HSV-1 vectors, but in a Helper Virus-free HSV-1 vector system, a number of promoters support only short-term expression. Thus paradoxically, recombinant gene expression remains short-term in the absence of almost all (approximately 99%) of the HSV-1 genome. To resolve this paradox, we hypothesize that specific HSV-1 proteins that affect the virion can shut off recombinant gene expression. In an earlier study, we examined the effects on recombinant gene expression of five different proteins that affect the HSV-1 virion. We found that vectors packaged in the presence of mutated vhs or U S 11 exhibited minimal changes in gene expression, vectors packaged in the presence of a mutated U S 3 supported improved gene transfer (numbers of cells at 4 days), and vectors packaged in the presence of mutated U L 13 or VP16 supported improved long-term expression. The capability of the VP16 transcriptional complex to reduce gene expression deserves additional study because VP16 is a powerful enhancer that interacts with a number of cellular and viral proteins. In particular, U L 46 and U L 47 are known to modulate the effects of VP16 on immediate early promoters. In this study, we examined expression from a HSV-1 vector that contains a neuronal-specific promoter and was packaged in the presence of deletions in U L 46, or U L 47, or both U L 46 and U L 47. In the rat striatum, each of these vector stocks supported both improved gene transfer (numbers of cells at 4 days) and improved long-term expression (2 months). Vectors packaged in the presence of a deletion in both U L 46 and U L 47 supported larger improvements in gene expression compared to vectors packaged in the presence of deletions in either gene alone. The implications of these results for strategies to improve long-term expression are discussed.

Guorong Zhang - One of the best experts on this subject based on the ideXlab platform.

  • overexpression of either lysine specific demethylase 1 or clock but not co rest improves long term expression from a modified neurofilament promoter in a Helper Virus free hsv 1 vector system
    Brain Research, 2012
    Co-Authors: Guorong Zhang, Hua Zhao, Haiyan Cao, Alfred I Geller
    Abstract:

    Long-term expression from Helper Virus-free Herpes Simplex Virus (HSV-1) vectors is required for many specific neural gene therapies and studies on neuronal physiology. We previously developed a promoter that supports long-term, neuron-specific expression by fusing the chicken s-globin insulator (INS), followed by an upstream enhancer from the rat tyrosine hydroxylase (TH) promoter, to a neurofilament heavy gene (NFH) promoter. Here, we examined the capability of specific transcription factors to further improve long-term expression from this promoter. Following a HSV-1 Virus infection, the Virus genome is localized to promyelocytic leukemia protein (PML) nuclear bodies (NB). At these sites, specific cellular transcription factors interact with HSV-1 encoded transcription factors, and together regulate HSV-1 gene expression. Importantly, lysine-specific demethylase-1 (LSD1), CLOCK, and Co-Rest each activate HSV-1 gene expression. However, gene expression from HSV-1 vectors differs in a number of important aspects from the Virus, including no HSV-1 genes are expressed. Nonetheless, these observations raise the possibility that specific transcription factors may improve long-term expression from specific promoters in HSV-1 vectors. Here, we show that overexpression of either LSD1 or CLOCK improves long-term expression from the INS-TH-NFH promoter, but overexpression of Co-Rest supports levels of long-term expression similar to those supported by a control vector. Further, overexpression of LSD1 is compatible with neuron-specific expression. Thus, overexpressing specific transcription factors can improve long-term expression from specific cellular promoters in HSV-1 vectors, and the chromatin structure of the vector has an important role in enabling expression.

  • a 16 bp upstream sequence from the rat tyrosine hydroxylase promoter supports long term expression from a neurofilament promoter in a Helper Virus free hsv 1 vector system
    Brain Research, 2011
    Co-Authors: Guorong Zhang, Hua Zhao, Soumya Awasthi, Alfred I Geller
    Abstract:

    Helper Virus-free Herpes Simplex Virus vector-mediated gene transfer has supported studies on neuronal physiology, and may support specific gene therapies. Long-term, neuron-specific expression is required for many of these applications. A neurofilament heavy gene (NFH) promoter does not support long-term expression. We previously developed a promoter that supports long-term expression by fusing 6.3 kb of upstream sequences from the rat tyrosine hydroxylase (TH) promoter to a NFH promoter, and this promoter has supported physiological studies. The TH promoter fragment contains an enhancer, as it has activity in both orientations and at a distance from the basal promoter. Identifying this enhancer may support further improvements in long-term expression. A previous deletion analysis identified two ~100 bp fragments that each support long-term expression, and are contained within an ~320 bp fragment located ~3 kb from the TH promoter transcription start site. As this analysis used overlapping fragments, the two ~100 bp fragments contained 44 or 23 bp of unique sequence. Here, we used mutagenesis to identify a short sequence that supports long-term expression. We studied a 42 bp sequence, centered on the 23 bp unique sequence. Analysis of the wt sequence, and five mutations containing clustered changes that spanned the sequence, identified two adjacent mutations that do not support long-term expression, which together defined a 16 bp maximum essential sequence. This 16 bp sequence contains a putative E2F-1/DP-1 transcription factor binding site, and this transcription factor is expressed in many brain areas.

  • a Helper Virus free hsv 1 vector containing the vesicular glutamate transporter 1 promoter supports expression preferentially in vglut1 containing glutamatergic neurons
    Brain Research, 2010
    Co-Authors: Guorong Zhang, Alfred I Geller
    Abstract:

    Abstract Multiple potential uses of direct gene transfer into neurons require restricting expression to specific classes of glutamatergic neurons. Thus, it is desirable to develop vectors containing glutamatergic class-specific promoters. The three vesicular glutamate transporters (VGLUTs) are expressed in distinct populations of neurons, and VGLUT1 is the predominant VGLUT in the neocortex, hippocampus, and cerebellar cortex. We previously reported a plasmid (amplicon) Herpes Simplex Virus (HSV-1) vector that placed the Lac Z gene under the regulation of the VGLUT1 promoter (pVGLUT1lac). Using Helper Virus-free vector stocks, we showed that this vector supported ∼ 90% glutamatergic neuron-specific expression in postrhinal (POR) cortex, in rats sacrificed at either 4 days or 2 months after gene transfer. We now show that pVGLUT1lac supports expression preferentially in VGLUT1-containing glutamatergic neurons. pVGLUT1lac vector stock was injected into either POR cortex, which contains primarily VGLUT1-containing glutamatergic neurons, or into the ventral medial hypothalamus (VMH), which contains predominantly VGLUT2-containing glutamatergic neurons. Rats were sacrificed at 4 days after gene transfer, and the types of cells expressing s-galactosidase were determined by immunofluorescent costaining. Cell counts showed that pVGLUT1lac supported expression in ∼ 10-fold more cells in POR cortex than in the VMH, whereas a control vector supported expression in similar numbers of cells in these two areas. Further, in POR cortex, pVGLUT1lac supported expression predominately in VGLUT1-containing neurons, and, in the VMH, pVGLUT1lac showed an ∼ 10-fold preference for the rare VGLUT1-containing neurons. VGLUT1-specific expression may benefit specific experiments on learning or specific gene therapy approaches, particularly in the neocortex.

  • glutamatergic or gabaergic neuron specific long term expression in neocortical neurons from Helper Virus free hsv 1 vectors containing the phosphate activated glutaminase vesicular glutamate transporter 1 or glutamic acid decarboxylase promoter
    Brain Research, 2007
    Co-Authors: Morten Arendt Rasmussen, Xiaodan Wang, Lingxin Kong, Guorong Zhang, Meng Liu, Gabor Szabo, Norman P Curthoys, Alfred I Geller
    Abstract:

    Many potential uses of direct gene transfer into neurons require restricting expression to one of the two major types of forebrain neurons, glutamatergic or GABAergic neurons. Thus, it is desirable to develop Virus vectors that contain either a glutamatergic or GABAergic neuron-specific promoter. The brain/kidney phosphate-activated glutaminase (PAG), the product of the GLS1 gene, produces the majority of the glutamate for release as neurotransmitter, and is a marker for glutamatergic neurons. A PAG promoter was partially characterized using a cultured kidney cell line. The three vesicular glutamate transporters (VGLUTs) are expressed in distinct populations of neurons, and VGLUT1 is the predominant VGLUT in the neocortex, hippocampus, and cerebellar cortex. Glutamic acid decarboxylase (GAD) produces GABA; the two molecular forms of the enzyme, GAD65 and GAD67, are expressed in distinct, but largely overlapping, groups of neurons, and GAD67 is the predominant form in the neocortex. In transgenic mice, an approximately 9 kb fragment of the GAD67 promoter supports expression in most classes of GABAergic neurons. Here, we constructed plasmid (amplicon) Herpes Simplex Virus (HSV-1) vectors that placed the Lac Z gene under the regulation of putative PAG, VGLUT1, or GAD67 promoters. Helper Virus-free vector stocks were delivered into postrhinal cortex, and the rats were sacrificed 4 days or 2 months later. The PAG or VGLUT1 promoters supported approximately 90% glutamatergic neuron-specific expression. The GAD67 promoter supported approximately 90% GABAergic neuron-specific expression. Long-term expression was observed using each promoter. Principles for obtaining long-term expression from HSV-1 vectors, based on these and other results, are discussed. Long-term glutamatergic or GABAergic neuron-specific expression may benefit specific experiments on learning or specific gene therapy approaches. Of note, promoter analyses might identify regulatory elements that determine a glutamatergic or GABAergic neuron.

  • coexpression of tyrosine hydroxylase gtp cyclohydrolase i aromatic amino acid decarboxylase and vesicular monoamine transporter 2 from a Helper Virus free herpes simplex Virus type 1 vector supports high level long term biochemical and behavioral cor
    Human Gene Therapy, 2004
    Co-Authors: Mei Sun, Xiaodan Wang, Lingxin Kong, Courtney Holmes, Qingsheng Gao, Guorong Zhang, Josef Pfeilschifter, David S Goldstein, Alfred I Geller
    Abstract:

    Parkinson's disease is due to the selective loss of nigrostriatal dopaminergic neurons. Consequently, many therapeutic strategies have focused on restoring striatal dopamine levels, including direct gene transfer to striatal cells, using viral vectors that express specific dopamine biosynthetic enzymes. The central hypothesis of this study is that coexpression of four dopamine biosynthetic and transporter genes in striatal neurons can support the efficient production and regulated, vesicular release of dopamine: tyrosine hydroxylase (TH) converts tyrosine to L-3,4-dihydroxyphenylalanine (L-DOPA), GTP cyclohydrolase I (GTP CH I) is the rate-limiting enzyme in the biosynthesis of the cofactor for TH, aromatic amino acid decarboxylase (AADC) converts L-DOPA to dopamine, and a vesicular monoamine transporter (VMAT-2) transports dopamine into synaptic vesicles, thereby supporting regulated, vesicular release of dopamine and relieving feedback inhibition of TH by dopamine. Helper Virus-free herpes simplex Virus...

Xandra O Breakefield - One of the best experts on this subject based on the ideXlab platform.

  • improved Helper Virus free packaging system for hsv amplicon vectors using an icp27 deleted oversized hsv 1 dna in a bacterial artificial chromosome
    Molecular Therapy, 2001
    Co-Authors: Cornel Fraefel, Yoshinaga Saeki, Xandra O Breakefield, Tomotsugu Ichikawa, Antonio E Chiocca
    Abstract:

    Herpes simplex Virus type 1 (HSV-1) amplicons are prokaryotic plasmids containing one or more transcriptional units and two cis-acting HSV-1 sequences: a viral origin of DNA replication and a viral DNA cleavage/packaging signal. In the presence of HSV-1 "Helper" functions, amplicons are replicated and packaged into HSV-1 virions. Despite recent improvements in packaging methods, stocks of amplicon vectors are still contaminated with replication-competent Helper Virus at a frequency of 10(-4)-10(-6). To overcome this problem, we report that: (i) genetic modifications of HSV-1 genomes can be routinely achieved in Escherichia coli, either by homologous or site-specific recombination, (ii) a novel HSV-1 bacterial artificial chromosome (fHSVDeltapacDelta27 0+), which has a deletion in the essential gene encoding ICP27 and an addition of ICP0 "stuffer" sequences to increase its size to 178 kb, supports the replication and packaging of cotransfected amplicon DNA without generating replication-competent Helper Virus (<1 Helper Virus per 10(8) TU amplicon vectors), and (iii) the resulting amplicon stocks have titers of up to 3-10 x 10(8) TU/ml after concentration. Elimination of replication-competent Helper Virus from HSV-1 amplicon vector stocks further improves safety in gene transfer applications.

  • improved Helper Virus free packaging system for hsv amplicon vectors using an icp27 deleted oversized hsv 1 dna in a bacterial artificial chromosome
    Molecular Therapy, 2001
    Co-Authors: Cornel Fraefel, Yoshinaga Saeki, Xandra O Breakefield, Tomotsugu Ichikawa, Antonio E Chiocca
    Abstract:

    Abstract Herpes simplex Virus type 1 (HSV-1) amplicons are prokaryotic plasmids containing one or more transcriptional units and two cis -acting HSV-1 sequences: a viral origin of DNA replication and a viral DNA cleavage/packaging signal. In the presence of HSV-1 "Helper" functions, amplicons are replicated and packaged into HSV-1 virions. Despite recent improvements in packaging methods, stocks of amplicon vectors are still contaminated with replication-competent Helper Virus at a frequency of 10 −4 –10 −6 . To overcome this problem, we report that: (i) genetic modifications of HSV-1 genomes can be routinely achieved in Escherichia coli , either by homologous or site-specific recombination, (ii) a novel HSV-1 bacterial artificial chromosome (fHSVΔpacΔ27 0+), which has a deletion in the essential gene encoding ICP27 and an addition of ICP0 "stuffer" sequences to increase its size to 178 kb, supports the replication and packaging of cotransfected amplicon DNA without generating replication-competent Helper Virus ( 8 TU amplicon vectors), and (iii) the resulting amplicon stocks have titers of up to 3–10 × 10 8 TU/ml after concentration. Elimination of replication-competent Helper Virus from HSV-1 amplicon vector stocks further improves safety in gene transfer applications.

  • herpes simplex Virus type 1 dna amplified as bacterial artificial chromosome in escherichia coli rescue of replication competent Virus progeny and packaging of amplicon vectors
    Human Gene Therapy, 1998
    Co-Authors: Yoshinaga Saeki, Cornel Fraefel, Xandra O Breakefield, Antonio E Chiocca, Tomotsugu Ichikawa, Kurt Tobler, Akane Saeki, Mathias Ackermann
    Abstract:

    Herpes simplex Virus type 1 (HSV-1)-based amplicon vectors contain only approximately 1% of the 152-kb HSV-1 genome, and consequently, replication and packaging into virions depends on Helper functions. These Helper functions have been provided conventionally by a Helper Virus, usually a replication-defective mutant of HSV-1, or more recently, by a set of five cosmids that overlap and represent the genome of HSV-1 deleted for DNA cleavage/packaging signals (pac). In the absence of pac signals, potential HSV-1 genomes that are reconstituted from the cosmids via homologous recombination are not packageable. The resulting amplicon stocks are, therefore, virtually free of contaminating Helper Virus. To simplify this packing system, the HSV-1 genome was cloned and maintained stably as a single-copy, F plasmid-based bacterial artificial chromosome in E. coli. Such a plasmid containing the HSV-1 genome deleted for the pac signals (fHSV delta pac) did not generate replication-competent progeny Virus on transfection into mammalian cells, but rather, it was able to support the packaging of cotransfected amplicon DNA that contained a functional pac signal. The resulting amplicon vector stocks had titers of up to 10(7) transducing units per milliliter of culture medium and efficiently transduced neural cells in the rat brain, as well as hepatocytes in the rat. The capacity of generating infectious and replication-competent HSV-1 progeny following transfection into mammalian cells was restored after insertion of a pac signal into fHSV delta pac.

  • gene transfer into hepatocytes mediated by Helper Virus free hsv aav hybrid vectors
    Molecular Medicine, 1997
    Co-Authors: Cornel Fraefel, Xandra O Breakefield, David R Jacoby, Christopher R Lage, Harold Hilderbrand, Janice Y Chou, Joseph A Majzoub
    Abstract:

    Background Vectors based on herpes simplex Virus type 1 (HSV-1) can efficiently transduce hepatocytes in the mouse liver, and vector genomes can persist for at least 2 months. However, 24 hr after gene transfer, the number of cells that express the transgene decreases rapidly and no transduced cells are detectable after 7 days. In this study, we examined the capability of a Helper Virus-free HSV/AAV hybrid amplicon vector to extend transgene expression in hepatocytes in vivo.

  • green fluorescent protein as a reporter for retroVirus and Helper Virus free hsv 1 amplicon vector mediated gene transfer into neural cells in culture and in vivo
    Neuroreport, 1997
    Co-Authors: K S Aboodyguterman, Peter J Wild, Peter Pechan, Nikolai G Rainov, Miguel Senaesteves, Alice K Jacobs, Evan Y Snyder, Elisabeth M Schraner, Kurt Tobler, Xandra O Breakefield
    Abstract:

    GREEN fluorescent protein (GFP) is an effective marker for retroVirus and herpes Virus vector-mediated gene transfer into various central nervous system-derived cells, both proliferative and non-proliferative, in culture and in vivo. RetroVirus vectors were used to stably transduce several rat and human glioma lines, and a multi-potent mouse neural progenitor line in culture. Implantation of selected pools of transduced glioma cells into rodent brain allowed clear visualization of the tumor and the invading tumor edge. Helper Virus-free HSV-1 amplicon vectors successfully transferred gfp into non-dividing primary neural cells in culture and in the rat brain. This study describes the versatility of GFP for: (i) labelling of glioma cells in experimental brain tumor models and neural progenitor cells by retroVirus vectors, and (ii) efficient, non-toxic delivery of genes to post mitotic cells of the nervous system using Helper-Virus free HSV-1 amplicon vectors.