The effect of herpes simplex virus vector-mediated gene therapy of protein phosphatase 1α on bladder overactivity and nociception.

AIMS: We studied the effect of herpes simplex virus (HSV) vectors-based gene transfer of protein phosphatase 1α (PP1α) on bladder hypersensitivity in rats. METHODS: Using adult female Sprague-Dawley rats, non-replicating HSV vectors carrying PP1α or green fluorescent protein (GFP) were injected into the bladder wall. At one week after vector inoculation, cystometry and Western blot assay were performed, whereas the other experiments were performed at 2 weeks after vector inoculation. RESULTS: GFP-expressing cells were identified in the bladder as well as in L6/S1 dorsal root ganglia at 14 days. In cystometry, intercontraction intervals (ICI) after resiniferatoxin (RTx; TRPV1 agonist) irrigation was significantly reduced in the PP1α group in comparison with the GFP group. Moreover, RTx-induced freezing behavior events were observed significantly more frequently in the PP1α group than the GFP group. The number of c-Fos positive cells in the L6 spinal dorsal horn was significantly less in the PP1α group than in the GFP group. Western blot assay revealed lower levels of phosphorylated inositol 1, 4, 5-triphosphate receptor (p-IP3 R), and phosphorylated TRPV1 in the PP1α compared with the GFP group. CONCLUSIONS: HSV vectors-mediated PP1α gene therapy may be an alternative treatment modality for cystitis-related hypersensitive bladder condition at least in part via modulation of the IP3 R signaling pathway.

Use of miRNA response sequences to block off-target replication and increase the safety of an unattenuated, glioblastoma-targeted oncolytic HSV.

Glioblastoma multiforme (GBM) is an aggressive brain cancer for which there is no effective treatment. Oncolytic HSV vectors (oHSVs) are attenuated lytic viruses that have shown promise in the treatment of human GBM models in animals, but their efficacy in early phase patient trials has been limited. Instead of attenuating the virus with mutations in virulence genes, we engineered four copies of the recognition sequence for miR-124 into the 3'UTR of the essential ICP4 gene to protect healthy tissue against lytic virus replication; miR-124 is expressed in neurons but not in glioblastoma cells. Following intracranial inoculation into nude mice, the miR-124-sensitive vector failed to replicate or show overt signs of pathogenesis. To address the concern that this safety feature may reduce oncolytic activity, we inserted the miR-124 response elements into an unattenuated, human receptor (EGFR/EGFRvIII)-specific HSV vector. We found that miR-124 sensitivity did not cause a loss of treatment efficiency in an orthotopic model of primary human GBM in nude mice. These results demonstrate that engineered miR-124 responsiveness can eliminate off-target replication by unattenuated oHSV without compromising oncolytic activity, thereby providing increased safety.

Novel mutations in gB and gH circumvent the requirement for known gD Receptors in herpes simplex virus 1 entry and cell-to-cell spread.

Both entry and cell-to-cell spread of herpes simplex virus (HSV) involve a cascade of cooperative interactions among the essential glycoproteins D, B, and H/L (gD, gB, and gH/gL, respectively) initiated by the binding of gD to a cognate HSV entry receptor. We previously reported that a variant (D285N/A549T) of glycoprotein B (gB:NT) enabled primary virus entry into cells that were devoid of typical HSV entry receptors. Here, we compared the activities of the gB:NT variant with those of a newly selected variant of glycoprotein H (gH:KV) and a frequently coselected gB variant (gB:S668N). In combination, gH:KV and gB:S668N enabled primary virus entry into cells that lacked established HSV entry receptors as efficiently as did gB:NT, but separately, each variant enabled only limited entry. Remarkably, gH:KV uniquely facilitated secondary virus spread between cells that lacked canonical entry receptors. Transient expression of the four essential entry glycoproteins revealed that gH:KV, but not gB:NT, induced fusion between cells lacking the standard receptors. Because the involvement of gD remained essential for virus spread and cell fusion, we propose that gH:KV mimics a transition state of gH that responds efficiently to weak signals from gD to reach the active state. Computational modeling of the structures of wild-type gH and gH:KV revealed relatively subtle differences that may have accounted for our experimental findings. Our study shows that (i) the dependence of HSV-1 entry and spread on specific gD receptors can be reduced by sequence changes in the downstream effectors gB and gH, and (ii) the relative roles of gB and gH are different in entry and spread.

Specific differentially methylated domain sequences direct the maintenance of methylation at imprinted genes.

Landmark features of imprinted genes are differentially methylated domains (DMDs), in which one parental allele is methylated on CpG dinucleotides and the opposite allele is unmethylated. Genetic experiments in the mouse have shown that DMDs are required for the parent-specific expression of linked clusters of imprinted genes. To understand the mechanism whereby the differential methylation is established and maintained, we analyzed a series of transgenes containing DMD sequences and showed that imperfect tandem repeats from DMDs associated with the Snurf/Snrpn, Kcnq1, and Igf2r gene clusters govern transgene imprinting. For the Igf2r DMD the minimal imprinting signal is two unit copies of the tandem repeat. This imprinted transgene behaves identically to endogenous imprinted genes in Dnmt1o and Dnmt3L mutant mouse backgrounds. The primary function of the imprinting signal within the transgene DMD is to maintain, during embryogenesis and a critical period of genomic reprogramming, parent-specific DNA methylation states established in the germ line. This work advances our understanding of the imprinting mechanism by defining a genomic signal that dependably perpetuates an epigenetic state during postzygotic development.

Preimplantation expression of the somatic form of Dnmt1 suggests a role in the inheritance of genomic imprints.

BACKGROUND: Identical DNA methylation differences between maternal and paternal alleles in gametes and adults suggest that the inheritance of genomic imprints is strictly due to the embryonic maintenance of DNA methylation. Such maintenance would occur in association with every cycle of DNA replication, including those of preimplantation embryos. RESULTS: The expression of the somatic form of the Dnmt1 cytosine methyltransferase (Dnmt1s) was examined in cleavage-stage preimplantation mouse embryos. Low concentrations of Dnmt1s are found in 1-, 2-, 4-, and 8-cell embryos, as well as in morulae and blastocysts. Dnmt1s is present in the cytoplasm at all stages, and in the nuclei of all stages except the 1-cell, pronuclear-stage embryo. The related oocyte-derived Dnmt1o protein is also present in nuclei of 8-cell embryos, along with embryo-synthesized Dnmt1s. Dnmt1s protein expressed in 1-cell and 2-cell embryos is derived from the oocyte, whereas the embryo synthesizes its own Dnmt1s from the 2-cell stage onward. CONCLUSION: These observations suggest that Dnmt1s provides maintenance methyltransferase activity for the inheritance of methylation imprints in the early mouse embryo. Moreover, the ability of Dnmt1o and Dnmt1s proteins synthesized at the same time to substitute for one another's maintenance function, but the lack of functional interchange between oocyte- and embryo-synthesized Dnmt1 proteins, suggests that the developmental source is the critical determinant of Dnmt1 function during preimplantation development.

Profound phenotypic variation among mice deficient in the maintenance of genomic imprints.

BACKGROUND: An alteration in the mechanism that maintains the monoallelic, imprinted expression of genes can result in their biallelic expression and lead to disruptions in fetal development. Here, we examined the consequences of a loss of maintenance methylation at one specific stage of preimplantation, induced by a deficiency of the oocyte-derived Dnmt1o protein and known to produce biallelic expression of imprinted genes. METHODS: Phenotypes of mid-gestation Dnmt1o-deficient mouse embryos were assessed by a scoring system based on the developmental stage of 17 anatomical features and by magnetic resonance microscopy. RESULTS: Many mid-gestation embryos developing without Dnmt1o protein exhibited significant developmental delays of multiple organ systems (P < 0.05) and a wide variety of morphologic anomalies compared with wild-type embryos. Most of the remaining mid-gestation Dnmt1o-deficient embryos appeared normal. CONCLUSIONS: These findings indicate that a profound range of gestational phenotypes can be induced by the loss of a single protein at a specific preimplantation developmental stage. This is best explained by the formation of epigenetic mosaic early embryos, composed of somatic cells with different spectra of normal intact genomic imprints. These findings have important implications for understanding the types of embryonic phenotypes related to the disruption of inherited imprints, and thus may provide a model of altered imprinting in humans. In particular, because Dnmt1o functions in the preimplantation embryo, a complete or partial loss of Dnmt1o function may play a role in epigenetic abnormalities seen in assisted reproduction technology births.

Conserved features of imprinted differentially methylated domains.

Genomic imprinting is a conserved epigenetic phenomenon in eutherian mammals, with regards both to the genes that are imprinted and the mechanism underlying the expression of just one of the parental alleles. Epigenetic modifications of alleles of imprinted genes are established during oogenesis and spermatogenesis, and these modifications are then inherited. Differentially methylated domains (DMDs) of imprinted genes are the genomic sites of these inherited epigenetic imprints. We previously showed that CpG-rich imperfect tandem direct repeats within three different mouse DMDs (Snurf/Snrpn, Kcnq1 and Igf2r), each with a unique sequence, play a central role in maintaining the differential methylation. This finding implicates repeat-related DNA structure, not sequence, in the imprinting mechanism. To better define the important features of this signal, we compared sequences of these three DMD tandem repeats among mammalian species. All DMD repeats contain short indirect repeats, many of which are organized into larger unit repeats. Even though the larger repeat units undergo deletion and addition during evolution (most likely through unequal crossovers during meiosis), the size of DMD tandem repeated regions has remained remarkably stable during mammalian evolution. Moreover, all three DMD tandem repeats have a high-CpG content, an ordered arrangement of CpG dinucleotides, and similar predicted secondary structures. These observations suggest that a structural feature or features of these DMD tandem repeats is the conserved DMD imprinting signal.

Shared role for differentially methylated domains of imprinted genes.

For most imprinted genes, a difference in expression between the maternal and paternal alleles is associated with a corresponding difference in DNA methylation that is localized to a differentially methylated domain (DMD). Removal of a gene's DMD leads to a loss of imprinting. These observations suggest that DMDs have a determinative role in genomic imprinting. To examine this possibility, we introduced sequences from the DMDs of the imprinted Igf2r, H19, and Snrpn genes into a nonimprinted derivative of the normally imprinted RSVIgmyc transgene, created by excising its own DMD. Hybrid transgenes with sequences from the Igf2r DMD2 were consistently imprinted, with the maternal allele being more methylated than the paternal allele. Only the repeated sequences within DMD2 were required for imprinting these transgenes. Hybrid transgenes containing H19 and Snrpn DMD sequences and ones containing sequences from the long terminal repeat of a murine intracisternal A particle retrotransposon were not imprinted. The Igf2r hybrid transgenes are comprised entirely of mouse genomic DNA and behave as endogenous imprinted genes in inbred wild-type and mutant mouse strains. These types of hybrid transgenes can be used to elucidate the functions of DMD sequences in genomic imprinting.

Deletion of the Virion Host Shut-off Gene Enhances Neuronal-Selective Transgene Expression from an HSV Vector Lacking Functional IE Genes.

The ability of herpes simplex virus (HSV) to establish lifelong latency in neurons suggests that HSV-derived vectors hold promise for gene delivery to the nervous system. However, vector toxicity and transgene silencing have created significant barriers to vector applications to the brain. Recently, we described a vector defective for all immediate-early gene expression and deleted for the joint region between the two unique genome segments that proved capable of extended transgene expression in non-neuronal cells. Sustained expression required the proximity of boundary elements from the latency locus. As confirmed here, we have also found that a transgene cassette introduced into the ICP4 locus is highly active in neurons but silent in primary fibroblasts. Remarkably, we observed that removal of the virion host shutoff (vhs) gene further improved transgene expression in neurons without inducing expression of viral genes. In rat hippocampus, the vhs-deleted vector showed robust transgene expression exclusively in neurons for at least 1 month without evidence of toxicity or inflammation. This HSV vector design holds promise for gene delivery to the brain, including durable expression of large or complex transgene cassettes.

Genomic imprinting: cis-acting sequences and regional control.

This review explores the features of imprinted loci that have been uncovered by genetic experiments in the mouse. Imprinted genes are expressed from one parental allele and often contain parent-specific differences in DNA methylation within genomic regions known as differentially methylated domains (DMDs). The precise erasure, establishment, and propagation of methylation on the alleles of imprinted genes during development suggest that parental differences in methylation at DMD sequences are a fundamental distinguishing feature of imprinted loci. Furthermore, targeted mutations of many DMDs have shown that they are essential for the imprinting of single genes or large gene clusters. An essential role of DNA methylation in genomic imprinting is also shown by studies of methyltransferase-deficient embryos. Many of the DMDs known to be required for imprinting contain imprinted promoters, tandem repeats, and CpG-rich regions that may be important for regulating parent-specific gene expression.

An HSV-based library screen identifies PP1α as a negative TRPV1 regulator with analgesic activity in models of pain.

Transient receptor potential vanilloid 1 (TRPV1) is a pronociceptive cation channel involved in persistent inflammatory and neuropathic pain. Herpes simplex virus (HSV) vector expression of TRPV1 causes cell death in the presence of capsaicin, thereby completely blocking virus replication. Here we describe a selection system for negative regulators of TRPV1 based on rescue of virus replication. HSV-based coexpression of TRPV1 and a PC12 cell-derived cDNA library identified protein phosphatase 1α (PP1α) as a negative regulator of TRPV1, mimicking the activity of "poreless" (PL), a dominant-negative mutant of TRPV1. Vectors expressing PP1α or PL reduced thermal sensitivity following virus injection into rat footpads, but failed to reduce the nocifensive responses to menthol/icilin-activated cold pain or formalin, demonstrating that the activity identified in vitro is functional in vivo with a degree of specificity. This system should prove powerful for identifying other cellular factors that can inhibit ion channel activity.

Inhibition of Indoleamine-2,3-dioxygenase (IDO) in Glioblastoma Cells by Oncolytic Herpes Simplex Virus.

Successful oncolytic virus treatment of malignant glioblastoma multiforme depends on widespread tumor-specific lytic virus replication and escape from mitigating innate immune responses to infection. Here we characterize a new HSV vector, JD0G, that is deleted for ICP0 and the joint sequences separating the unique long and short elements of the viral genome. We observed that JD0G replication was enhanced in certain glioblastoma cell lines compared to HEL cells, suggesting that a vector backbone deleted for ICP0 may be useful for treatment of glioblastoma. The innate immune response to virus infection can potentially impede oncolytic vector replication in human tumors. Indoleamine-2,3-dioxygenase (IDO) is expressed in response to interferon γ (IFNγ) and has been linked to both antiviral functions and to the immune escape of tumor cells. We observed that IFNγ treatment of human glioblastoma cells induced the expression of IDO and that this expression was quelled by infection with both wild-type and JD0G viruses. The role of IDO in inhibiting virus replication and the connection of this protein to the escape of tumor cells from immune surveillance suggest that IDO downregulation by HSV infection may enhance the oncolytic activity of vectors such as JD0G.

Design and application of oncolytic HSV vectors for glioblastoma therapy.

Glioblastoma multiforme is one of the most common human brain tumors. The tumor is generally highly infiltrative, making it extremely difficult to treat by surgical resection or radiotherapy. This feature contributes to recurrence and a very poor prognosis. Few anticancer drugs have been shown to alter rapid tumor growth and none are ultimately effective. Oncolytic vectors have been employed as a treatment alternative based on the ability to tailor virus replication to tumor cells. The human neurotropic herpes simplex virus (HSV) is especially attractive for development of oncolytic vectors (oHSV) because this virus is highly infectious, replicates rapidly and can be readily modified to achieve vector attenuation in normal brain tissue. Tumor specificity can be achieved by deleting viral genes that are only required for virus replication in normal cells and permit mutant virus replication selectively in tumor cells. The anti-tumor activity of oHSV can be enhanced by arming the vector with genes that either activate chemotherapeutic drugs within the tumor tissue or promote anti-tumor immunity. In this review, we describe current designs of oHSV and the experience thus far with their potential utility for glioblastoma therapy. In addition, we discuss the impediments to vector effectiveness and describe our view of future developments in vector improvement.

A developmental window of opportunity for imprinted gene silencing mediated by DNA methylation and the Kcnq1ot1 noncoding RNA.

The Kcnq1 imprinted domain encodes a paternally expressed noncoding RNA Kcnq1ot1 and several paternally repressed protein-coding genes. Transcriptional regulation is controlled by the Kcnq1ot1 gene whose maternal germline methylation imprint overlaps with the Kcnq1ot1 promoter. The domain can be divided into two groups of genes. One group is imprinted in all lineages and is reliant on DNA methylation for its imprinting. The other group contains genes that are imprinted specifically in the placenta and retain their imprinting in the absence of Dnmt1, the primary DNA maintenance methylase. In the placenta paternal Kcnq1ot1 expression is associated with the acquisition of repressive histone modifications throughout the domain. Using the Dnmt1o knockout, we have analyzed the effect of removing DNA maintenance methylation at the eight-cell stage on the Kcnq1 imprinted domain. In the placenta the expression of the normally silent maternal Kcnq1ot1 allele leads to reduced expression of the surrounding maternally expressed genes. This repression is seen in both the placental-specific imprinted genes and the ubiquitously imprinted genes. Conversely, reduction of functional Dnmt1 results solely in reduced expression of the ubiquitously imprinted genes in the placenta. This suggests that Kcnq1ot1 expression can epigenetically silence placentally imprinted genes in the cluster only during a specific developmental window. This highlights the possibility that Kcnq1ot1-mediated repression is temporally regulated leading to epigenetic silencing of placental-specific genes. We show that allele-specific histone modifications are still present in the Dnmt1 ( -/- ) trophoblast at placental-specific imprinted loci and are likely responsible for maintaining the imprinting of these genes in the absence of DNA methylation.

Specific dynamic and noninvasive labeling of pancreatic beta cells in reporter mice.

Noninvasive detection of differentiated cells is increasingly demanded for accurate and reliable assessments of both in vitro and in vivo experimental systems. Here we present an efficient, innovative approach for imaging the beta cells of the pancreatic islets of Langerhans. The main physiologic function of beta cells is glucose-stimulated insulin secretion. This function is facilitated through the synthesis and storage of insulin in secretory vesicles of beta cells, which then release their contents when beta cells are exposed to hyperglycemic conditions. To visualize beta cells in vivo in the mouse, we used targeted mutagenesis techniques to construct a modified insulin II (InsII) gene allele, InsII(EGFP), that expresses a proinsulin-EGFP (enhanced green fluorescent protein) fusion peptide. The EGFP portion of this fusion is entirely within the C-peptide portion of the proinsulin peptide. This fusion protein is processed in beta cells to insulin and EGFP-tagged C peptide, which are stored together in cytoplasmic secretory vesicles. The large amount of vesicular EGFP-tagged C peptide is evident as a characteristic robust and specific fluorescence pattern in the beta cells of InsII(EGFP) mice. This innovative method of visualizing beta cells will be a useful tool in the study of both beta cell physiology and the development of the endocrine cells of the pancreas.