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The use of mutant strains of oncolytic herpes simplex virus (oHSV) in early-phase human clinical trials for the treatment of glioblastoma multiforme (GBM) has proven safe, but limited efficacy suggests that more potent vector designs are required for effective GBM therapy. Inadequate vector performance may derive from poor intratumoral vector replication and limited spread to uninfected cells. Vector replication may be impaired by mutagenesis strategies to achieve vector safety, and intratumoral virus spread may be hampered by vector entrapment in the tumor-specific extracellular matrix (ECM) that in GBM is composed primarily of type IV collagen. In this report, we armed our previously described epidermal growth factor receptor (EGFR)vIII-targeted, neuronal microRNA-sensitive oHSV with a matrix metalloproteinase (MMP9) to improve intratumoral vector distribution. We show that vector-expressed MMP9 enhanced therapeutic efficacy and long-term animal survival in a GBM xenograft model. Keywords: matrix metalloproteinase 9 = MMP9, extracellular matrix = ECM, Oncolytic vectors = OVs, Glioblastoma multiforme = GBM, OVs derived from herpes simplex virus = oHSV

To evaluate the expression of genes encoding cytokines, grow factors and cell cycle regulators in the proliferative endometrium of women with chronic endometritis (CE) compared to controls. We performed a case-control study on seven women with CE as diagnosed by hysteroscopy and histology (Cases) compared to six women without CE (Controls). All women underwent diagnostic hysteroscopy plus endometrial biopsy during the mid-proliferative phase of the menstrual cycle. Endometrial samples were divided into two different aliquots for histological and molecular analyses. The endometrial expression profile of 16 genes encoding proteins involved in the inflammatory process, proliferation and cell cycle regulation/apoptosis was assessed by using high-throughput qPCR. Study endpoints were between-group differences in the expression of VEGF A, VEGF B, VEGF C, EGF, TNF, TGF B1, IFNG, TP73, TP73L, BAXva, CDC2, CDC2va, CCND3, CCNB1, BAX and IL12. RESULTS: VEGF A, VEGF B, VEGF C, EGF, TNF, TGF B1, IFNG, TP73, TP73L, BAXva, CDC2, CDC2va, CCND3, CCNB1 were significantly overexpressed in women with CE compared to controls, while BAX and IL12 had similar expression between groups. In women with CE, we found an altered endometrial expression of genes involved in inflammatory, cell proliferation, and apoptosis processes. The dominance of proliferative and anti-apoptotic activity in CE may potentially promote the development of polyps and hyperplastic lesions.

International audience; G protein-coupled receptors ( GPCRs) are classically characterized as cell-surface receptors transmitting extracellular signals into cells. Here we show that central components of a GPCR signaling system comprised of the melatonin type 1 receptor (MT1), its associated G protein, and beta-arrestins are on and within neuronal mitochondria. We discovered that the ligand melatonin is exclusively synthesized in the mitochondrial matrix and released by the organelle activating the mitochondrial MT1 signal-transduction pathway inhibiting stress-mediated cytochrome c release and caspase activation. These findings coupled with our observation that mitochondrial MT1 overexpression reduces ischemic brain injury in mice delineate a mitochondrial GPCR mechanism contributing to the neuroprotective action of melatonin. We propose a new term, "automitocrine," analogous to "autocrine" when a similar phenomenon occurs at the cellular level, to describe this unexpected intracellular organelle ligand-receptor pathway that opens a new research avenue investigating mitochondrial GPCR biology.

FRET-based sensors for cAMP and PKA activity reveal that mitochondrial subcompartments host segregated cAMP cascades with distinct functional and kinetic signatures., Cyclic AMP (cAMP)-dependent phosphorylation has been reported to exert biological effects in both the mitochondrial matrix and outer mitochondrial membrane (OMM). However, the kinetics, targets, and effectors of the cAMP cascade in these organellar domains remain largely undefined. Here we used sensitive FRET-based sensors to monitor cAMP and protein kinase A (PKA) activity in different mitochondrial compartments in real time. We found that cytosolic cAMP did not enter the matrix, except during mitochondrial permeability transition. Bicarbonate treatment (expected to activate matrix-bound soluble adenylyl cyclase) increased intramitochondrial cAMP, but along with membrane-permeant cAMP analogues, failed to induce measureable matrix PKA activity. In contrast, the OMM proved to be a domain of exceptionally persistent cAMP-dependent PKA activity. Although cAMP signaling events measured on the OMM mirrored those of the cytosol, PKA phosphorylation at the OMM endured longer as a consequence of diminished control by local phosphatases. Our findings demonstrate that mitochondria host segregated cAMP cascades with distinct functional and kinetic signatures.

Gene therapy treatment strategies for Huntington's disease (HD) depend on a vector that (i) allows targeted delivery of large therapeutic transgenes to specific cell populations and (ii) is replication-defective and non-cytotoxic. Thus, our goal is to develop a replication-defective HSV-based gene therapy vector to deliver therapeutic transgenes to the cells most affected by expression of the mutant Huntington gene (Htt). Strategies for full retargeting of HSV require virus detargeting from its cognate receptors (HVEM and nectin1), recognized by the virus attachment/entry component glycoprotein D (gD), and introduction of a new ligand into gD that allows entry through recognition of the corresponding cellular receptors. To target an HSV vector for entry exclusively into cells expressing the receptor GFRα1 we employed the ligand GDNF. We replaced the signal peptide and HVEM binding domain of gD with pre-pro-(pp)GDNF to create a GFRα1 targeting protein, gD(Y38)_GDNF, that can still bind nectin1. Virus expressing gD(Y38)_GDNF was propagated on cells expressing nectin1 and purified virus was shown to enter nectin1-deficient J1.1-2 and B78H1 cells in a GFRα1-dependent manner. U2OS cells engineered to express GFRα1 demonstrated the most robust virus entry and spread, allowing us to create and propagate a fully retargeted virus that can no longer bind nectin1, gD(Δ38)_GDNF. Once the functionality of gD(Δ38)_GDNF was confirmed, we transferred this mutation to a replication-defective backbone lacking essential immediate early gene expression. Complementing cells were screened and those cells that best supported growth of the replication-defective backbone were selected. Virus stocks were propagated and tested for receptor specificity in vitro and experiments are currently underway to assess GFRα1-specific entry in vivo. We anticipate that the use of neuronal-specific targeting ligands, such as GFRα1, in replication-defective backbones will promote homing of non-toxic HSV gene therapy vectors to cells affected in HD.

Early phase human clinical trials using several versions of oncolytic herpes simplex virus type 1 (oHSV) have shown promise in the treatment of glioblastoma multiforme, but efficacy has been limited. Impediments to oHSV therapy include poor virus spread due in part to the tumor extracellular matrix, and insufficient replication in tumor cells as a result of attenuating mutations. Thus, our central goal was to improve oncolytic vector delivery, replication, and spread while maintaining safety and tumor specificity. We had already developed a new class of two stage tumor targeted oHSV combining (i) selective infection through tumor-specific receptors and (ii) selective replication based on differential expression of microRNAs (miRs) in tumor and normal cells. We further modified our vector by arming it with the matrix metalloproteinase 9 (MMP9) as a means to reduce vector trapping in the tumor extracellular matrix. MMP9 degrades collagen type IV, a major component of the extracellular matrix (ECM) and basement membranes of glioblastomas. Here we show that: (i) MMP9 expression improves vector spread in GBM neurospheres in vitro; (ii) MMP9 enhances the oncolytic therapeutic efficacy in animal model of human GBM without compromising vector safety.

A set of 67 novel LTR-retrotransposon has been identified by in silico analyses of the Culex quinquefasciatus genome using the LTR_STRUC program. The phylogenetic analysis shows that 29 novel and putatively functional LTR-retrotransposons detected belong to the Ty3/gypsy group. Our results demonstrate that, by considering only families containing potentially autonomous LTR-retrotransposons, they account for about 1% of the genome of C. quinquefasciatus. In previous studies it has been estimated that 29% of the genome of C. quinquefasciatus is occupied by mobile genetic elements. The potential role of retrotransposon insertions strictly associated with host genes is described and discussed along with the possible origin of a retrotransposon with peculiar Primer Binding Site region. Finally, we report the presence of a group of 38 retrotransposons, carrying tandem repeated sequences but lacking coding potential, and apparently lacking “master copy” elements from which they could have originated. The features of the repetitive sequences found in these non-autonomous LTR retrotransposons are described, and their possible role discussed. These results integrate the existing data on the genomics of an important virus-borne disease vector.

Insulators or chromatin boundary are DNA elements that organize the genome into discrete regulatory domains by limiting the actions of enhancers and silencers through a “positional-blocking mechanism”. The role of these sequences, both in modulation of the enhancers range of action (enhancer–promoter selectivity) and in the organization of the chromatin in functional domains, is emerging strongly in these last years. There is a great interest in identifying new insulators because deeper knowledge of these elements can help understand how cis-regulatory elements coordinate the expression of the target genes. However, while insulators are critical in gene regulation and genome functioning, only a few have been reported so far. Here, we describe a new insulator sequence that is located in the 5′UTR of the Drosophila retrotransposon ZAM. We have used an “enhancer–blocking assay” to test its effects on the activity of the enhancer in transiently transfected Drosophila S2R+ cell line. Moreover, we show that the new insulator is able to affect significantly the enhancer–promoter interaction in the human cell line HEK293. These results suggest the possibility of employing the ZAM insulator in gene transfer protocols from insects to mammals in order to counteract the transgene positional and genotoxic effects.

Early phase human clinical trials using several versions of oncolytic herpes simplex virus type 1 (oHSV) have shown promise in the treatment of GBM, but efficacy has been limited. Impediments to oHSV therapy include poor virus spread due in part to the tumor extracellular matrix, and insufficient replication in tumor cells as a result of attenuating mutations. Thus, the central goal of this project is to improve oncolytic vector delivery, replication and spread while maintaining safety and tumor specificity. We have already developed a new class of two stage tumor targeted oHSV combining (i) selective infection through tumor-specific receptors and (ii) selective replication based on differential expression of microRNAs (miRs) in tumor and normal cells. We further modify our vector by arming with the matrix metalloproteinase 9 (MMP9) as a means to reduce vector trapping in the tumor extracellular matrix. Here we show that MMP9 expression (i) enhances Oncolytic vector spreading in GBM neruospheres in vitro and (ii) improves tumor killing in a xenogeneic model of primary human GBM with significant long-term survival (≥50%) comparable to the control. Citation Format: Aofei Li, Marco Marzulli, Mingdi Zhang, William Goins, Balveen Kaur, Chelsea Bolyard, Nduka Amankulor, Daniela Leronni, Paola Sette, Justus Cohen, Joseph Glorioso, Paola Grandi. Arming a tumor targeted oncolytic herpes simplex sirus type 1 with matrix metalloproteinase 9 for enhanced vector distribution and killing activity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3546. doi:10.1158/1538-7445.AM2015-3546

Gene therapy often requires selective gene delivery to specific cellular subpopulations to provide the most effective therapeutic outcome and avoid off-target effects. This can be accomplished by viral vector targeting involving replacement of the natural virus-cell interactions that trigger virus entry, with novel cell-specific interactions, thereby permitting delivery of therapeutic transgenes to specific cell types. Strategies for full retargeting of HSV require (i) mutagenesis-mediated virus detargeting from its cognate receptors (HVEM and nectin1) recognized by the virus attachment/entry component glycoprotein D (gD) and (ii) the introduction of new ligands into gD that allow entry through recognition of cognate cellular receptors. To target an HSV vector for entry exclusively into cells expressing the receptors GFRa1 or TrkA, we employed the ligands GDNF and NGF, respectively. We replaced the signal peptide and HVEM binding domain of gD with pre-pro-(pp)GDNF to create a GFRa1 targeting protein, gD(Y38)_GDNF, that can still bind nectin1. Virus expressing gD(Y38)_GDNF was propagated on cells expressing nectin1 and purified virus was shown to enter nectin1-deficient cells in a GFRa1-dependent manner. Likewise, TrkA-dependent virus entry was observed using ppNGF as a targeting ligand. Although propagation of completely retargeted viruses (mutationally inactivated for both HVEM and nectin1 binding) can be achieved in complementing cell lines that express the target receptor, we observed differences among cell lines in retargeted virus spread. HSV receptor-deficient J1.1-2 and B78H1 cells engineered to express the target receptors allowed virus entry, but only B78H1 based cells displayed observable spread. U2OS cells engineered to express GFRa1 demonstrated the most robust virus entry and spread, but these cells express abundant nectin1, providing strong selective pressure for reversion of the nectin1 binding defect of fully retargeted viruses during propagation. In one approach to overcome this problem, we developed genetic selection methods to isolate retargeted virus variants that both entered and spread on J1.1-2 cells transduced with the target receptor. We found that selected variants had acquired mutations in other envelope glycoproteins, including one glycoprotein involved in envelope-cell fusion events (gH) and two that were previously shown to contribute to the spreading process (gE and gI). We expect that the combination of virus mutants and engineered cell lines for efficient production of stably retargeted HSV gene therapy vectors will allow us to achieve HSV retargeting to a broader range of receptors.

Early phase human clinical trials using several versions of oncolytic herpes simplex virus type 1 (oHSV) have shown promise in the treatment of GBM, but efficacy has been limited. Impediments to oHSV therapy include poor virus spread due in part to the tumor extracellular matrix, and insufficient replication in tumor cells as a result of attenuating mutations. Thus, the central goal of this project is to improve oncolytic vector delivery, replication and spread while maintaining safety and tumor specificity. We have already developed a new class of two stage tumor targeted oHSV combining (i) selective infection through tumor-specific receptors and (ii) selective replication based on differential expression of microRNAs (miRs) in tumor and normal cells. We further modify our vector by arming with the matrix metalloproteinase 9 (MMP9) as a means to reduce vector trapping in the tumor extracellular matrix. Here we show that MMP9 expression (i) enhances Oncolytic vector spreading in GBM neruospheres in vitro and (ii) improves tumor killing in a xenogeneic model of primary human GBM with significant long-term survival (≥50%) comparable to the control.