Dedifferentiation rescues senescence of progeria cells but only while pluripotent

Hutchinson-Gilford progeria syndrome (HGPS) is a genetic disease in which children develop pathologies associated with old age. HGPS is caused by a mutation in the LMNA gene, resulting in the formation of a dominant negative form of the intermediate filament, nuclear structural protein lamin A, termed progerin. Expression of progerin alters the nuclear architecture and heterochromatin, affecting cell cycle progression and genomic stability. Two groups recently reported the successful generation and characterization of induced pluripotent stem cells (iPSCs) from HGPS fibroblasts. Remarkably, progerin expression and senescence phenotypes are lost in iPSCs but not in differentiated progeny. These new HGPS iPSCs are valuable for characterizing the role of progerin in driving HGPS and aging and for screening therapeutic strategies to prevent or delay cell senescence

Tumor necrosis factor–Α contributes to below-level neuropathic pain after spinal cord injury

Objective Our objective was to elucidate the mechanisms responsible for below-level pain after partial spinal cord injury (SCI). Methods We used lateral hemisection to model central neuropathic pain and herpes simplex viral (HSV) vector–mediated transfer of the cleaved soluble receptor for tumor necrosis factor–Α (TNF-Α) to evaluate the role of TNF-Α in the pathogenesis of below-level pain. Results We found activation of microglia and increased expression of TNF-Α below the level of the lesion in the lumbar spinal cord after T13 lateral hemisection that correlated with emergence of mechanical allodynia in the hind limbs of rats. Lumbar TNF-Α had an apparent molecular weight of 27kDa, consistent with the full-length transmembrane form of the protein (mTNF-Α). Expression of the p55 TNF soluble receptor (sTNFRs) by HSV-mediated gene transfer resulted in reduced pain behavior and a decreased number of ED1-positive cells, as well as decreased phosphorylation of the p38 MAP kinase (p-p38) and diminished expression of mTNF-Α in the dorsal horn. Interpretation These results suggest that expression of mTNF-Α after injury is related to development of pain, and that reverse signaling through mTNF-Α by sTNFR at that level reduces cellular markers of inflammatory response and pain-related behavior. Ann Neurol 2006;59:843–851Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/50655/1/20855_ftp.pd

Gene therapy for pain: Results of a phase I clinical trial

Objective: Preclinical evidence indicates that gene transfer to the dorsal root ganglion using replication‐defective herpes simplex virus (HSV)‐based vectors can reduce pain‐related behavior in animal models of pain. This clinical trial was carried out to assess the safety and explore the potential efficacy of this approach in humans. Methods: We conducted a multicenter, dose‐escalation, phase I clinical trial of NP2, a replication‐defective HSV‐based vector expressing human preproenkephalin ( PENK ) in subjects with intractable focal pain caused by cancer. NP2 was injected intradermally into the dermatome(s) corresponding to the radicular distribution of pain. The primary outcome was safety. As secondary measures, efficacy of pain relief was assessed using a numeric rating scale (NRS), the Short Form McGill Pain Questionnaire (SF‐MPQ), and concurrent opiate usage. Results: Ten subjects with moderate to severe intractable pain despite treatment with >200mg/day of morphine (or equivalent) were enrolled into the study. Treatment was well tolerated with no study agent‐related serious adverse events observed at any point in the study. Subjects receiving the low dose of NP2 reported no substantive change in pain. Subjects in the middle‐ and high‐dose cohorts reported pain relief as assessed by NRS and SF‐MPQ. Interpretation: Treatment of intractable pain with NP2 was well tolerated. There were no placebo controls in this relatively small study, but the dose‐responsive analgesic effects suggest that NP2 may be effective in reducing pain and warrants further clinical investigation. ANN NEUROL 2011Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/86983/1/22446_ftp.pd

Adverse effects of adenovirus-mediated gene transfer of human transforming growth factor beta 1 into rabbit knees

To examine the effect of transforming growth factor (TGF)-β1 on the regulation of cartilage synthesis and other articular pathologies, we used adenovirus-mediated intra-articular gene transfer of TGF-β1 to both naïve and arthritic rabbit knee joints. Increasing doses of adenoviral vector expressing TGF-β1 were injected into normal and antigen-induced arthritis rabbit knee joints through the patellar tendon, with the same doses of an adenoviral vector expressing luciferase injected into the contralateral knees as the control. Intra-articular injection of adenoviral vector expressing TGF-β1 into the rabbit knee resulted in dose-dependent TGF-β1 expression in the synovial fluid. Intra-articular TGF-β1 expression in both naïve and arthritic rabbit knee joints resulted in significant pathological changes in the rabbit knee as well as in adjacent muscle tissue. The observed changes induced by elevated TGF-β1 included inhibition of white blood cell infiltration, stimulation of glycosaminoglycan release and nitric oxide production, and induction of fibrogenesis and muscle edema. In addition, induction of chondrogenesis within the synovial lining was observed. These results suggest that even though TGF-β1 may have anti-inflammatory properties, it is unable to stimulate repair of damaged cartilage, even stimulating cartilage degradation. Gene transfer of TGF-β1 to the synovium is thus not suitable for treating intra-articular pathologies

322. Benign Herpes Simplex Virus Vector Design for Efficient Delivery of Large or Multiple Transgenes To a Diversity of Cells

Viral vectors derived from herpes simplex virus (HSV) have the potential to revolutionize gene therapy due to their ability to accommodate large and multiple therapeutic transgenes. However, current HSV gene therapy vectors express toxic levels of an immediate-early (IE) protein, ICP0, whose function is required for robust and sustained transgene expression. Here we report the development of a new generation of HSV vectors that are IE-gene independent and non-toxic, yet capable of persistent transgene expression in a variety of human primary non-neuronal cell types. We identified a CTCF motif cluster upstream of the latency promoter and a known long-term regulatory region as key elements for the protection of transgene expression cassettes from global silencing of the viral genome in the absence of all viral IE gene products. Using this new HSV vector system, we have observed vigorous expression of full-length dystrophin cDNA (14 kb) for several weeks in a dystrophin-deficient muscle cell line. We further tested our vectors for transgene expression in rodent brain. While we detected variable persistence of gene expression from the latency locus, we were surprised to observe vigorous long-term reporter gene expression from one other locus despite the absence of gene expression from this locus in non-neuronal cells. These findings demonstrate that transgene expression in neurons is operatively different from that in non-neuronal cells and suggest that multiple loci can be used for expression of foreign genes in the nervous system. In addition, our data raise the prospect that our highly defective HSV vector system will be applicable as a safe delivery tool for large and multiple therapeutic genes to a wide range of non-neuronal tissues

Herpes simplex virus thymidine kinase gene is stably maintained and expressed in cells transformed by protoplast fusion

We examined a series of transformed cell lines resulting from transfer of the herpes simplex virus type 1 thymidine kinase gene to Ltk − cells by protoplast fusion gene transfer. We show that multiple copies of the transforming plasmid DNA, ranging from a minimum of two to greater than 20, were present in one or at most a few integration sites in each cell line. The TK + phenotype was stable in five independent transformed cell lines after growth in nonselective medium for over a year. Transforming plasmid DNA was stable in one cell line containing from two to five copies after a year of growth in nonselective medium. In another cell line initially containing about 20 copies, the transforming DNA became rearranged soon after growth to mass culture, resulting in a decrease to two to five copies which then remained stably maintained. This suggests that TK + transformants resulting from protoplast fusion are stable when the input DNA has integrated in a relatively low copy number.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/45531/1/11188_2005_Article_BF01534902.pd

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

Varicella-Zoster viruses associated with post-herpetic neuralgia induce sodium current density increases in the ND7-23 Nav-1.8 neuroblastoma cell line

Post-herpetic neuralgia (PHN) is the most significant complication of herpes zoster caused by reactivation of latent Varicella-Zoster virus (VZV). We undertook a heterologous infection in vitro study to determine whether PHN-associated VZV isolates induce changes in sodium ion channel currents known to be associated with neuropathic pain. Twenty VZV isolates were studied blind from 11 PHN and 9 non-PHN subjects. Viruses were propagated in the MeWo cell line from which cell-free virus was harvested and applied to the ND7/23-Nav1.8 rat DRG x mouse neuroblastoma hybrid cell line which showed constitutive expression of the exogenous Nav 1.8, and endogenous expression of Nav 1.6 and Nav 1.7 genes all encoding sodium ion channels the dysregulation of which is associated with a range of neuropathic pain syndromes. After 72 hrs all three classes of VZV gene transcripts were detected in the absence of infectious virus. Single cell sodium ion channel recording was performed after 72 hr by voltage-clamping. PHN-associated VZV significantly increased sodium current amplitude in the cell line when compared with non-PHN VZV, wild-type (Dumas) or vaccine VZV strains ((POka, Merck and GSK). These sodium current increases were unaffected by acyclovir pre-treatment but were abolished by exposure to Tetrodotoxin (TTX) which blocks the TTX-sensitive fast Nav 1.6 and Nav 1.7 channels but not the TTX-resistant slow Nav 1.8 channel. PHN-associated VZV sodium current increases were therefore mediated in part by the Nav 1.6 and Nav 1.7 sodium ion channels. An additional observation was a modest increase in message levels of both Nav1.6 and Nav1.7 mRNA but not Nav 1.8 in PHN virally infected cells