Neat1 lncRNA organizes the inflammatory gene expressions in the dorsal root ganglion in neuropathic pain caused by nerve injury.

Primary sensory neurons regulate inflammatory processes in innervated regions through neuro-immune communication. However, how their immune-modulating functions are regulated in concert remains largely unknown. Here, we show that Neat1 long non-coding RNA (lncRNA) organizes the proinflammatory gene expressions in the dorsal root ganglion (DRG) in chronic intractable neuropathic pain in rats. Neat1 was abundantly expressed in the DRG and was upregulated after peripheral nerve injury. Neat1 overexpression in primary sensory neurons caused mechanical and thermal hypersensitivity, whereas its knockdown alleviated neuropathic pain. Bioinformatics analysis of comprehensive transcriptome changes indicated the inflammatory response was the most relevant function of genes upregulated through Neat1. Consistent with this, upregulation of proinflammatory genes in the DRG following nerve injury was suppressed by Neat1 knockdown. Expression changes of these proinflammatory genes were regulated through Neat1-mRNA interaction-dependent and -independent mechanisms. Notably, Neat1 increased proinflammatory genes by stabilizing its interacting mRNAs in neuropathic pain. Finally, Neat1 in primary sensory neurons contributed to spinal inflammatory processes that mediated peripheral neuropathic pain. These findings demonstrate that Neat1 lncRNA is a key regulator of neuro-immune communication in neuropathic pain.

iPSC-derived mesenchymal stem cells attenuate cerebral ischemia-reperfusion injury by inhibiting inflammatory signaling and oxidative stress.

Induced pluripotent stem cell-derived mesenchymal stem cells (iMSCs) hold great promise as a cell source for transplantation into injured tissues to alleviate inflammation. However, the therapeutic efficacy of iMSC transplantation for ischemic stroke remains unknown. In this study, we evaluated the therapeutic effects of iMSC transplantation on brain injury after ischemia-reperfusion using a rat transient middle cerebral artery occlusion model and compared its therapeutic efficacy with that of bone marrow mesenchymal stem cells (BMMSCs). We showed that iMSCs and BMMSCs reduced infarct volumes after reperfusion and significantly improved motor function on days 3, 7, 14, 28, and 56 and cognitive function on days 28 and 56 after reperfusion compared with the vehicle group. Furthermore, immunological analyses revealed that transplantation of iMSCs and BMMSCs inhibited microglial activation and expression of proinflammatory cytokines and suppressed oxidative stress and neuronal cell death in the cerebral cortex at the ischemic border zone. No difference in therapeutic effect was observed between the iMSC and BMMSC groups. Taken together, our results demonstrate that iMSC therapy can be a practical alternative as a cell source for attenuation of brain injury and improvement of neurological function because of the unlimited supply of uniform therapeutic cells.

Characterization of Megabat-Favored, CA-Dependent Susceptibility to Retrovirus Infection.

The isolation of the Koala retrovirus-like virus from Australian megabats and the identification of endogenous retroviruses in the bat genome have raised questions on bat susceptibility to retroviruses in general. To answer this, we studied the susceptibility of 12 cell lines from 11 bat species to four well-studied retroviruses (human and simian immunodeficiency viruses [HIV and SIV] and murine leukemia viruses [B- and N-MLV]). Systematic comparison of retroviral susceptibility among bats revealed that megabat cell lines were overall less susceptible to the four retroviruses than microbat cell lines, particularly to HIV-1 infection, whereas lineage-specific differences were observed for MLV susceptibility. Quantitative PCR of reverse transcription (RT) products, infection in heterokaryon cells, and point mutation analysis of the capsid (CA) revealed that (i) HIV-1 and MLV replication were blocked at the nuclear transport of the pre-integration complexes and before and/or during RT, respectively, and (ii) the observed lineage-specific restriction can be attributed to a dominant cellular factor constrained by specific positions in CA. Investigation of bat homologs of the three previously reported post-entry restriction factors constrained by the same residues in CA, tripartite motif-protein 5α (TRIM5α), myxovirus resistance 2/B (Mx2/MxB), and carboxy terminus-truncated cleavage and polyadenylation factor 6 (CPSF6-358), demonstrated poor anti-HIV-1 activity in megabat cells, whereas megabat TRIM5α restricted MLV infection, suggesting that the major known CA-dependent restriction factors were not dominant in the observed lineage-specific susceptibility to HIV-1 in bat cells. Therefore, HIV-1 susceptibility of megabat cells may be determined in a manner distinct from that of primate cells. IMPORTANCE Recent studies have demonstrated the circulation of gammaretroviruses among megabats in Australia and the bats' resistance to HIV-1 infection; however, the origins of these viruses in megabats and the contribution of bats to retrovirus spread to other mammalian species remains unclear. To determine the intrinsic susceptibility of bat cells to HIV-1 infection, we investigated 12 cell lines isolated from 11 bat species. We report that lineage-specific retrovirus restriction in the bat cell lines can be attributed to CA-dependent factors. However, in the megabat cell lines examined, factors known to bind capsid and block infection in primate cell culture, including homologs of TRIM5α, Mx2/MxB, and CPSF6, failed to exhibit significant anti-HIV-1 activities. These results suggested that the HIV-1 susceptibility of megabat cells occurs in a manner distinct from that of primate cells, where cellular factors, other than major known CA-dependent restriction factors, with lineage-specific functions could recognize retroviral proteins in megabats.

Drinking hydrogen water improves photoreceptor structure and function in retinal degeneration 6 mice.

Retinitis pigmentosa (RP) is a genetically heterogeneous group of inherited retinal disorders involving the progressive dysfunction of photoreceptors and the retinal pigment epithelium, for which there is currently no treatment. The rd6 mouse is a natural model of autosomal recessive retinal degeneration. Given the known contributions of oxidative stress caused by reactive oxygen species (ROS) and selective inhibition of potent ROS peroxynitrite and OH·by H2 gas we have previously demonstrated, we hypothesized that ingestion of H2 water may delay the progression of photoreceptor death in rd6 mice. H2 mice showed significantly higher retinal thickness as compared to controls on optical coherence tomography. Histopathological and morphometric analyses revealed higher thickness of the outer nuclear layer for H2 mice than controls, as well as higher counts of opsin red/green-positive cells. RNA sequencing (RNA-seq) analysis of differentially expressed genes in the H2 group versus control group revealed 1996 genes with significantly different expressions. Gene and pathway ontology analysis showed substantial upregulation of genes responsible for phototransduction in H2 mice. Our results show that drinking water high in H2 (1.2-1.6 ppm) had neuroprotective effects and inhibited photoreceptor death in mice, and suggest the potential of H2 for the treatment of RP.

Evaluation of parameters for efficient purification and long-term storage of herpes simplex virus-based vectors.

Replication competent oncolytic herpes simplex virus (HSV) vectors have been used extensively to treat solid tumors with promising results. However, highly defective HSV vectors will be needed for applications that require sustained therapeutic gene expression in the absence of vector-related toxicity or inflammation. These vectors require complementing cell lines for their manufacture, creating significant challenges to achieve high yields of infectious virus particles. We recently described an improved upstream process for the production of a non-cytotoxic HSV vector for gene therapy applications. Here, we sought to optimize the downstream conditions for purification and long-term storage of the same vector, JΔNI5. We compared different methods to remove cellular impurities and concentrate the vector by monitoring both physical and biological titers, resulting in the establishment of optimal conditions for vector production. To optimize the long-term storage parameters for non-cytotoxic HSV vectors, we evaluated vector stability at low temperature and sensitivity to freeze-thaw cycles. We report that suboptimal purification and storage methods resulted in loss of vector viability. Our results describe effective and reproducible protocols for purification and storage of HSV vectors for pre-clinical studies.

A PCR-amplified transgene fragment flanked by a single copy of a truncated inverted terminal repeat for recombinant adeno-associated virus production prevents unnecessary plasmid DNA packaging.

The application of recombinant adeno-associated viruses (rAAVs) for gene therapy faces certain challenges, including genome packaging of non-vector sequences. Inverted terminal repeats (ITRs) flanking the rAAV genome, comprising three inverted repeat regions (A, B, and C) and a non-inverted repeat region (D), contribute to non-vector genome packaging. We aimed to circumvent this issue by comparing the properties of rAAV containing DNA plasmids and PCR-amplified transgenes, including a single copy of the AD sequence (rAAV-pAD/L-AD, respectively), which is a truncated form of ITR, with those of wild-type ITR genome (single-stranded and self-complementary AAV; ssAAV and scAAV). The packaging efficiency of rAAV-pAD/L-AD was found to be comparable to that of scAAV, whereas the transduction efficiency of rAAV-pAD/L-AD was lower than that of ss/scAAV. Remarkably, rAAV-L-AD reduced the plasmid backbone packaging contamination compared to ss/scAAV. Furthermore, to confirm the functionality of this system, we generated a rAAV-L-AD harboring a short hairpin RNA targeting ATP5B (rAAV-L-AD-shATP5B) and found that it caused a significant decrease in ATP5B mRNA levels when transduced into HEK293EB cells, suggesting that it was functional. Thus, our system successfully packaged L-AD into capsids with minimal contamination of plasmid DNA, offering a novel functional packaging platform without causing plasmid backbone encapsidation.

Generation of an Oncolytic Herpes Simplex Viral Vector Completely Retargeted to the GDNF Receptor GFRα1 for Specific Infection of Breast Cancer Cells.

Oncolytic herpes simplex viruses (oHSV) are under development for the treatment of a variety of human cancers, including breast cancer, a leading cause of cancer mortality among women worldwide. Here we report the design of a fully retargeted oHSV for preferential infection of breast cancer cells through virus recognition of GFRα1, the cellular receptor for glial cell-derived neurotrophic factor (GDNF). GFRα1 displays a limited expression profile in normal adult tissue, but is upregulated in a subset of breast cancers. We generated a recombinant HSV expressing a completely retargeted glycoprotein D (gD), the viral attachment/entry protein, that incorporates pre-pro-GDNF in place of the signal peptide and HVEM binding domain of gD and contains a deletion of amino acid 38 to eliminate nectin-1 binding. We show that GFRα1 is necessary and sufficient for infection by the purified recombinant virus. Moreover, this virus enters and spreads in GFRα1-positive breast cancer cells in vitro and caused tumor regression upon intratumoral injection in vivo. Given the heterogeneity observed between and within individual breast cancers at the molecular level, these results expand our ability to deliver oHSV to specific tumors and suggest opportunities to enhance drug or viral treatments aimed at other receptors.

Protocol Optimization for the Production of the Non-Cytotoxic JΔNI5 HSV Vector Deficient in Expression of Immediately Early Genes.

Non-toxic herpes simplex virus (HSV) vectors can be generated by functional deletion of all immediate-early (IE) genes, providing a benign vehicle with potential for gene therapy. However, deletion of multiple IE genes raises manufacturing concerns and thus limits clinical application of these vectors. To address this issue, we previously developed a novel production cell line, called U2OS-ICP4/27, by lentiviral transduction of human osteosarcoma U2OS cells with two essential HSV IE genes, ICP4 and ICP27. To optimize the process of vector manufacturing on this platform, we evaluated several cell culture parameters of U2OS-ICP4/27 for high-titer and -quality production of non-toxic HSV vectors, revealing that the yields and functionality of these vectors can be significantly influenced by culturing conditions. We also found that several chemical compounds can enhance the replication of non-toxic HSV vectors and their release from producer cells into the supernatants. Notably, the vector produced by our optimized protocol displayed a greatly improved vector yield and quality and showed elevated transgene expression in cultures of primary dorsal root ganglion neurons. Taken together, our optimized production approach emerges as a relevant protocol for high-yield and high-quality preparation of non-toxic HSV-based gene therapy vectors.

Epstein-Barr virus-associated hemophagocytic syndrome in a patient with ulcerative colitis during treatment with azathioprine: A case report and review of literature.

A 19-year-old female was diagnosed with ulcerative colitis when she presented with persistent melena, and has been treated with 5-aminosalicylic acid for 4 years, with additional azathioprine for 2 years at our hospital. The patient experienced high-grade fevers, chills, and cough five d prior to presenting to the outpatient unit. At first, the patient was suspected to have developed neutropenic fever; however, she was diagnosed with Epstein-Barr virus-associated hemophagocytic syndrome (EB-VAHS) upon fulfilling the diagnostic criteria after bone marrow aspiration. When patients with inflammatory bowel disease treated with immunomodulators, such as thiopurine preparations, develop fever, EB-VAHS should be considered in the differential diagnosis.

Highly Efficient Ultracentrifugation-free Chromatographic Purification of Recombinant AAV Serotype 9.

Recombinant adeno-associated virus serotype 9 (rAAV9) can specifically transduce muscle and neuronal tissues; thus, rAAV9 can potentially be used in gene therapy. However, rAAV9 is the most challenging rAAV serotype to purify. Traditionally, rAAV9 has been purified by ultracentrifugation, which is not scalable. We recently described a chromatographic purification protocol for rAAV1; this protocol can achieve scalable purifications. In this study, we attempted to optimize this protocol for purifying rAAV9 preparations, and we developed a novel, effective method for high-yield purification of rAAV9 using quaternary ammonium anion exchangers and size-exclusion chromatography. The final purified rAAV9 contained mainly three capsid proteins, as observed by SDS-PAGE. Furthermore, negative-stain electron microscopy demonstrated that 96.1% ± 1.1% of rAAV9 particles carried the viral genome containing the EGFP transgene, indicating that impurities and empty capsids can be eliminated with our purification protocol. The final rAAV9 titer obtained by our protocol totaled 2.5 ± 0.4 × 1015 viral genomes produced from ∼3.2 × 109 HEK293EB cells. We confirmed that our protocol can also be applied to purify other varied AAV genome constructs. Our protocol can scale up production of pure rAAV9, in compliance with current good manufacturing practice, for clinical applications in human gene therapy.

Herpes Simplex Virus Vectors for Gene Transfer to the Central Nervous System.

Neurodegenerative diseases (NDs) have a profound impact on human health worldwide and their incidence is predicted to increase as the population ages. ND severely limits the quality of life and leads to early death. Aside from treatments that may reduce symptoms, NDs are almost completely without means of therapeutic intervention. The genetic and biochemical basis of many NDs is beginning to emerge although most have complex etiologies for which common themes remain poorly resolved. Largely relying on progress in vector design, gene therapy is gaining increasing support as a strategy for genetic treatment of diseases. Here we describe recent developments in the engineering of highly defective herpes simplex virus (HSV) vectors suitable for transfer and long-term expression of large and/or multiple therapeutic genes in brain neurons in the complete absence of viral gene expression. These advanced vector platforms are safe, non-inflammatory, and persist in the nerve cell nucleus for life. In the near term, it is likely that HSV can be used to treat certain NDs that have a well-defined genetic cause. As further information on disease etiology becomes available, these vectors may take on an expanded role in ND therapies, including gene editing and repair.

Cellular Antisilencing Elements Support Transgene Expression from Herpes Simplex Virus Vectors in the Absence of Immediate Early Gene Expression.

Inactivation of all herpes simplex virus (HSV) immediate early (IE) genes to eliminate vector cytotoxicity results in rapid silencing of the viral genome, similar to the establishment of HSV latency. We recently reported that silencing of a nonviral reporter cassette could be overcome in nonneuronal cells by positioning the cassette in the viral latency (LAT) locus between resident chromatin boundary elements. Here, we tested the abilities of the chicken hypersensitive site 4 insulator and the human ubiquitous chromatin opening element A2UCOE to promote transgene expression from an IE-gene-inactivated HSV vector. We found that A2UCOE was particularly active in nonneuronal cells and reduced reporter promoter occupancy by a repressive histone mark. We determined whether multiple transgenes could be expressed under the control of different promoters from different loci of the same virus. The results showed abundant coexpression of LAT-embedded and A2UCOE-flanked genes in nonneuronal cells. In addition, a third reporter gene without known protective elements was active in cultured rat sensory neurons. These findings indicate that cellular antisilencing sequences can contribute to the expression of multiple genes from separate promoters in fully IE gene-disabled HSV vectors, providing an opportunity for therapeutic applications requiring mutually independent expression of different gene products from a single vector.IMPORTANCE Gene therapy has now entered a phase of development in which a growing number of recessive single gene defects can be successfully treated by vector-mediated introduction of a wild-type copy of the gene into the appropriate tissue. However, many disease conditions, such as neurodegeneration, cancer, and inflammatory processes, are more complex, requiring either multiple gene corrections or provision of coordinated gene activities to achieve a therapeutic outcome. Although herpes simplex virus (HSV) vectors have the capacity to meet this need, the challenge has been to genetically engineer the HSV genome in a manner to prevent expression of any viral genes while retaining the ability to express multiple therapeutic transgenes under independent transcriptional control. Here, we show that non-HSV insulator elements can be applied to retain at least transient transgene activity from multiple viral loci, thereby opening the door for more complex gene therapy applications in the future.

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.

Engineered HSV vector achieves safe long-term transgene expression in the central nervous system.

Previously we reported a new series of highly defective herpes simplex virus type 1 (HSV-1) vectors that were functionally devoid of all viral immediately early (IE) genes, resulting in virtual absence of viral gene expression. Nevertheless, a reporter gene cassette inserted into the vector flanked by boundary elements from the viral latency locus showed high, persistent reporter gene activity in non-neuronal cells while an independent expression cassette inserted into a deleted ICP4 locus remained almost silent. In contrast to non-neuronal cells, we show here that the ICP4 locus cassette permitted robust reporter gene expression in a diversity of neurons following stereotactic injection of different rat brain regions; transgene expression in the hippocampus lasted up to 6 months and was essentially restricted to neurons. No evidence of neuronal cell toxicity or induction of inflammatory cell infiltrates was observed. An independent reporter gene cassette located in an intergenic region remained silent, indicating that the transgene promoter and/or insertion site are critical for sustained expression. These findings suggest the suitability of this vector for therapeutic intervention into diseases of the central nervous system that require the expression of large and/or multiple therapeutic transgenes.

Wnt signaling regulates hepatobiliary repair following cholestatic liver injury in mice.

Hepatic repair is directed chiefly by the proliferation of resident mature epithelial cells. Furthermore, if predominant injury is to cholangiocytes, the hepatocytes can transdifferentiate to cholangiocytes to assist in the repair and vice versa, as shown by various fate-tracing studies. However, the molecular bases of reprogramming remain elusive. Using two models of biliary injury where repair occurs through cholangiocyte proliferation and hepatocyte transdifferentiation to cholangiocytes, we identify an important role of Wnt signaling. First we identify up-regulation of specific Wnt proteins in the cholangiocytes. Next, using conditional knockouts of Wntless and Wnt coreceptors low-density lipoprotein-related protein 5/6, transgenic mice expressing stable ß-catenin, and in vitro studies, we show a role of Wnt signaling through ß-catenin in hepatocyte to biliary transdifferentiation. Last, we show that specific Wnts regulate cholangiocyte proliferation, but in a ß-catenin-independent manner. CONCLUSION: Wnt signaling regulates hepatobiliary repair after cholestatic injury in both ß-catenin-dependent and -independent manners. (Hepatology 2016;64:1652-1666).

Glycolipid dynamics in generation and differentiation of induced pluripotent stem cells.

Glycosphingolipids (GSLs) are glycoconjugates that function as mediators of cell adhesion and modulators of signal transduction. Some well-defined markers of undifferentiated human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) are glycoconjugates, such as SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81. However, Comprehensive GSL profiles of hiPSCs have not yet been elucidated. The global images of GSLs from the parental cells, hiPSCs, and differentiated cells revealed that there are parental cell-independent specific glycolipids, including Globo H (fucosyl-Gb5Cer) and H type1 antigen (fucosyl-Lc4Cer) that are novel markers for undifferentiated hiPSCs. Interestingly, undifferentiated hiPSCs expressed H type 1 antigen, specific for blood type O, regardless of the cells' genotypes. Thus, in this study, we defined the dynamics of GSL remodeling during reprogramming from parental cell sets to iPSC sets and thence to iPSC-neural cells.

Herpes simplex viral-vector design for efficient transduction of nonneuronal cells without cytotoxicity.

The design of highly defective herpes simplex virus (HSV) vectors for transgene expression in nonneuronal cells in the absence of toxic viral-gene activity has been elusive. Here, we report that elements of the latency locus protect a nonviral promoter against silencing in primary human cells in the absence of any viral-gene expression. We identified a CTCF motif cluster 5' to the latency promoter and a known long-term regulatory region as important elements for vigorous transgene expression from a vector that is functionally deleted for all five immediate-early genes and the 15-kb internal repeat region. We inserted a 16.5-kb expression cassette for full-length mouse dystrophin and report robust and durable expression in dystrophin-deficient muscle cells in vitro. Given the broad cell tropism of HSV, our design provides a nontoxic vector that can accommodate large transgene constructs for transduction of a wide variety of cells without vector integration, thereby filling an important void in the current arsenal of gene-therapy vectors.

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.

Removal of reprogramming transgenes improves the tissue reconstitution potential of keratinocytes generated from human induced pluripotent stem cells.

Human induced pluripotent stem cell (hiPSC) lines have a great potential for therapeutics because customized cells and organs can be induced from such cells. Assessment of the residual reprogramming factors after the generation of hiPSC lines is required, but an ideal system has been lacking. Here, we generated hiPSC lines from normal human dermal fibroblasts with piggyBac transposon bearing reprogramming transgenes followed by removal of the transposon by the transposase. Under this condition, we compared the phenotypes of transgene-residual and -free hiPSCs of the same genetic background. The transgene-residual hiPSCs, in which the transcription levels of the reprogramming transgenes were eventually suppressed, were quite similar to the transgene-free hiPSCs in a pluripotent state. However, after differentiation into keratinocytes, clear differences were observed. Morphological, functional, and molecular analyses including single-cell gene expression profiling revealed that keratinocytes from transgene-free hiPSC lines were more similar to normal human keratinocytes than those from transgene-residual hiPSC lines, which may be partly explained by reactivation of residual transgenes upon induction of keratinocyte differentiation. These results suggest that transgene-free hiPSC lines should be chosen for therapeutic purposes.