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Dopaminergic neurons are the predominant brain cells affected in Parkinson's disease. With the limited availability of live human brain dopaminergic neurons to study pathological mechanisms of Parkinson's disease, dopaminergic neurons have been generated from human-skin-cell-derived induced pluripotent stem cells. Originally, induced pluripotent stem-cell-derived dopaminergic neurons were generated using small molecules. These neurons took more than two months to mature. However, the transcription-factor-mediated differentiation of induced pluripotent stem cells has revealed quicker and cheaper methods to generate dopaminergic neurons. In this study, we compared and contrasted three protocols to generate induced pluripotent stem-cell-derived dopaminergic neurons using transcription-factor-mediated directed differentiation. We deviated from the established protocols using lentivirus transduction to stably integrate different transcription factors into the AAVS1 safe harbour locus of induced pluripotent stem cells. We used different media compositions to generate more than 90% of neurons in the culture, out of which more than 85% of the neurons were dopaminergic neurons within three weeks. Therefore, from our comparative study, we reveal that a combination of transcription factors along with small molecule treatment may be required to generate a pure population of human dopaminergic neurons.

Host antiviral proteins inhibit primate lentiviruses and other retroviruses by targeting many features of the viral life cycle. The lentiviral capsid protein and the assembled viral core are known to be inhibited through multiple, directly acting antiviral proteins. Several phenotypes, including those known as Lv1 through Lv5 , have been described as cell type-specific blocks to infection against some but not all primate lentiviruses. Here we review important features of known capsid-targeting blocks to infection together with several blocks to infection for which the genes responsible for the inhibition still remain to be identified. We outline the features of these blocks as well as how current methodologies are now well suited to find these antiviral genes and solve these long-standing mysteries in the HIV and retrovirology fields., Competing Interests: The authors declare no conflict of interest.

Deficiency of iduronate 2-sulfatase (IDS) causes Mucopolysaccharidosis type II (MPS II), a lysosomal storage disorder characterized by systemic accumulation of glycosaminoglycans (GAGs), leading to a devastating cognitive decline and life-threatening respiratory and cardiac complications. We previously found that hematopoietic stem and progenitor cell-mediated lentiviral gene therapy (HSPC-LVGT) employing tagged IDS with insulin-like growth factor 2 (IGF2) or ApoE2, but not receptor-associated protein minimal peptide (RAP12x2), efficiently prevented brain pathology in a murine model of MPS II. In this study, we report on the effects of HSPC-LVGT on peripheral pathology and we analyzed IDS biodistribution. We found that HSPC-LVGT with all vectors completely corrected GAG accumulation and lysosomal pathology in liver, spleen, kidney, tracheal mucosa, and heart valves. Full correction of tunica media of the great heart vessels was achieved only with IDS.IGF2co gene therapy, while the other vectors provided near complete ( IDS.ApoE2co ) or no ( IDSco and IDS.RAP12x2co ) correction. In contrast, tracheal, epiphyseal, and articular cartilage remained largely uncorrected by all vectors tested. These efficacies were closely matched by IDS protein levels following HSPC-LVGT. Our results demonstrate the capability of HSPC-LVGT to correct pathology in tissues of high clinical relevance, including those of the heart and respiratory system, while challenges remain for the correction of cartilage pathology.

Drug-based antiretroviral therapies (ART) efficiently suppress HIV replication in humans, but the virus persists as integrated proviral reservoirs in small numbers of cells. Importantly, ART cannot eliminate HIV from an infected individual, since it does not target the integrated provirus. Therefore, genome editing-based strategies that can inactivate or excise HIV genomes would provide the technology for novel curative therapies. In fact, the HIV-1 LTR-specific designer-recombinase Brec1 has been shown to remove integrated proviruses from infected cells and is highly efficacious on clinical HIV-1 isolates in vitro and in vivo, suggesting that Brec1 has the potential for clinical development of advanced HIV-1 eradication strategies in people living with HIV. In line with the preparation of a first-in-human advanced therapy medicinal product gene therapy trial, we here present an extensive preclinical evaluation of Brec1 and lentiviral vectors expressing the Brec1 transgene. This included detailed functional analysis of potential genomic off-target sites, assessing vector safety by investigating vector copy number (VCN) and the risk for potential vector-related insertional mutagenesis, as well as analyzing the potential of Brec1 to trigger an undesired strong T cell immune response. In conclusion, the antiviral designer-recombinase Brec1 is shown to lack any detectable cytopathic, genotoxic or T cell-related immunogenic effects, thereby meeting an important precondition for clinical application of the therapeutic lentiviral vector LV-Brec1 in novel HIV-1 curative strategies., Competing Interests: The author’s have read the journal’s policy and have the following competing interests: Jan Chemnitz, Joachim Hauber, Frank Buchholz, Ilona Hauber, Niklas Beschorner and Maike Voges are founding partners of PROVIREX Genome Editing Therapies GmbH, which is commercially developing Brec1 technology. Niklas Beschorner and Maike Voges are full-time employees of PROVIREX Genome Editing Therapies GmbH since 2022, and Jan Chemnitz, Frank Buchholz, Joachim Hauber and Ilona Hauber are part-time employees. This does not alter our adherence to PLOS ONE policies on sharing data and materials. There are no patents, products in development or marketed products associated with this research to declare., (Copyright: © 2024 Beschorner et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Mucopolysaccharidosis type II (MPS II), or Hunter syndrome, is a rare X-linked recessive lysosomal storage disorder due to a mutation in the lysosomal enzyme iduronate-2-sulfatase (IDS) gene. IDS deficiency leads to a progressive, multisystem accumulation of glycosaminoglycans (GAGs) and results in central nervous system (CNS) manifestations in the severe form. We developed up to clinical readiness a new hematopoietic stem cell (HSC) gene therapy approach for MPS II that benefits from a novel highly effective transduction protocol. We first provided proof of concept of efficacy of our approach aimed at enhanced IDS enzyme delivery to the CNS in a murine study of immediate translational value, employing a lentiviral vector (LV) encoding a codon-optimized human IDS cDNA. Then the therapeutic LV was tested for its ability to efficiently and safely transduce bona fide human HSCs in clinically relevant conditions according to a standard vs. a novel protocol that demonstrated superior ability to transduce bona fide long-term repopulating HSCs. Overall, these results provide strong proof of concept for the clinical translation of this approach for the treatment of Hunter syndrome., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

The human lymphoblastoid cell line TK6 stands out as the most widely employed human cell line in genotoxicity testing, as recommended by various testing guidelines for in vitro assessments. Nevertheless, like many testing cell lines, TK6 lacks functional phase I drug-metabolizing enzymes crucial for chemical genotoxicity evaluations. This protocol introduces a lentivirus-based methodology for establishing a panel of TK6-derived cell lines, each expressing one of 14 cytochrome P450s (CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A5, and CYP3A7). The utilization of a lentiviral expression system ensures stable transduction, offering notable advantages such as sustained transgene expression, high transduction efficiency, positive selection feasibility, and user-friendly application. Additionally, we present a detailed procedure for validating the enhanced expression of each CYP in the established cell lines through real-time PCR, western blotting, and mass spectrometry analysis. Lastly, we exemplify the application of these CYP-expressing TK6 cell lines in genotoxicity testing, employing a flow-cytometry-based in vitro micronucleus test. Published 2024. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol 1: Lentivirus production and transduction for TK6 cells Support Protocol: Selecting a single clone of CYP-expressing TK6 cells Basic Protocol 2: Validation of CYP expression in TK6 cell lines Basic Protocol 3: Application of transduced cell lines in flow-cytometry-based micronucleus assay., (Published 2024. This article is a U.S. Government work and is in the public domain in the USA.)

The recent uptick in the approval of ex vivo cell therapies highlights the relevance of lentivirus (LV) as an enabling viral vector of modern medicine. As labile biologics, however, LVs pose critical challenges to industrial biomanufacturing. In particular, LV purification-currently reliant on filtration and anion-exchange or size-exclusion chromatography-suffers from long process times and low yield of transducing particles, which translate into high waiting time and cost to patients. Seeking to improve LV downstream processing, this study introduces peptides targeting the enveloped protein Vesicular stomatitis virus G (VSV-G) to serve as affinity ligands for the chromatographic purification of LV particles. An ensemble of candidate ligands was initially discovered by implementing a dual-fluorescence screening technology and a targeted in silico approach designed to identify sequences with high selectivity and tunable affinity. The selected peptides were conjugated on Poros resin and their LV binding-and-release performance was optimized by adjusting the flow rate, composition, and pH of the chromatographic buffers. Ligands GKEAAFAA and SRAFVGDADRD were selected for their high product yield (50%-60% of viral genomes; 40%-50% of HT1080 cell-transducing particles) upon elution in PIPES buffer with 0.65 M NaCl at pH 7.4. The peptide-based adsorbents also presented remarkable values of binding capacity (up to 3·10 9 TU per mL of resin, or 5·10 11 vp per mL of resin, at the residence time of 1 min) and clearance of host cell proteins (up to a 220-fold reduction of HEK293 HCPs). Additionally, GKEAAFAA demonstrated high resistance to caustic cleaning-in-place (0.5 M NaOH, 30 min) with no observable loss in product yield and quality., (© 2023 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LLC.)

Pompe disease is a lysosomal storage disorder caused by deficiency of acid alpha-glucosidase (GAA), resulting in glycogen accumulation with profound pathology in skeletal muscle. We recently developed an optimized form of lentiviral gene therapy for Pompe disease in which a codon-optimized version of the GAA transgene (LV-GAAco) was fused to an insulin-like growth factor 2 (IGF2) peptide (LV-IGF2.GAAco), to promote cellular uptake via the cation-independent mannose-6-phosphate/IGF2 receptor. Lentiviral gene therapy with LV-IGF2.GAAco showed superior efficacy in heart, skeletal muscle, and brain of Gaa -/- mice compared to gene therapy with untagged LV-GAAco. Here, we used quantitative mass spectrometry using TMT labeling to analyze the muscle proteome and the response to gene therapy in Gaa -/- mice. We found that muscle of Gaa -/- mice displayed altered levels of proteins including those with functions in the CLEAR signaling pathway, autophagy, cytoplasmic glycogen metabolism, calcium homeostasis, redox signaling, mitochondrial function, fatty acid transport, muscle contraction, cytoskeletal organization, phagosome maturation, and inflammation. Gene therapy with LV-GAAco resulted in partial correction of the muscle proteome, while gene therapy with LV-IGF2.GAAco resulted in a near-complete restoration to wild type levels without inducing extra proteomic changes, supporting clinical development of lentiviral gene therapy for Pompe disease. SIGNIFICANCE: Lysosomal glycogen accumulation is the primary cause of Pompe disease, and leads to a cascade of pathological events in cardiac and skeletal muscle and in the central nervous system. In this study, we identified the proteomic changes that are caused by Pompe disease in skeletal muscle of a mouse model. We showed that lentiviral gene therapy with LV-IGF2.GAAco nearly completely corrects disease-associated proteomic changes. This study supports the future clinical development of lentiviral gene therapy with LV-IGF2.GAAco as a new treatment option for Pompe disease., Competing Interests: Declaration of Competing Interest A.T. van der Ploeg received funding for research, clinical trials and as advisor from Sanofi-Genzyme, Amicus Therapeutics, Biomarin, Ultragenix, Sarepta, Audentes and Spark Therapeutics on enzyme replacement therapy or next-generation therapies in the field of Pompe disease, and other lysosomal storage diseases or neuromuscular disorders, under agreements with Erasmus MC University Medical Center. N. van Til has been employee of and advisor to Avrobio. W.W.M. Pim Pijnappel and A.T. van der Ploeg are advisors of LentiCure B.V. W.W.M.Pijnappel, E. Vlaar, and A.T. van der Ploeg are inventors on patents in the field of gene therapy., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Tau pathology is a major hallmark of many neurodegenerative diseases summarized under the term tauopathies. In most of these disorders,  such as Alzheimer's disease, the neuronal axonal microtubule-binding Tau protein becomes mislocalized to the somatodendritic compartment. In human disease, this missorting of Tau is accompanied by an abnormally high phosphorylation state of the Tau protein, and several downstream pathological consequences (e.g., loss of microtubules, degradation of postsynaptic spines, impaired synaptic transmission, neuronal death). While some mechanisms of Tau sorting, missorting, and associated pathologies have been addressed in rodent models, few studies have addressed human Tau in physiological disease-relevant human neurons. Thus, suitable human-derived in vitro models are necessary. This protocol provides a simple step-by-step protocol for generating homogeneous cultures of cortical glutamatergic neurons using an engineered Ngn2 transgene-carrying WTC11 iPSC line. We further demonstrate strategies to improve neuronal maturity, that is, synapse formation, Tau isoform expression, and neuronal activity by co-culturing hiPSC-derived glutamatergic neurons with mouse-derived astrocytes. Finally, we describe a simple protocol for high-efficiency lentiviral transduction of hiPSC-derived neurons at almost all stages of differentiation., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The TGF signaling pathway is a key regulator of cancer progression. In this work, we report for the first time the antitumor activity of TβRII-SE/Fc, a novel peptibody whose targeting domain is comprised of the soluble endogenous isoform of the human TGF-β type II receptor (TβRII-SE). Overexpression of TβRIISE/Fc reduces in vitro cell proliferation and migration while inducing cell cycle arrest and apoptosis in human colorectal cancer-derived cell lines. Moreover, TβRII-SE/Fc overexpression reduces tumorigenicity in BALB/c nude athymic mice. Our results revealed that TRII-SE/Fc-expressing tumors were significantly reduced in size or were even incapable of developing. We also demonstrated that the novel peptibody has the ability to inhibit the canonical TGF-β and BMP signaling pathways while identifying SMAD-dependent and independent proteins involved in tumor progression that are modulated by TβRII-SE/Fc. These findings provide insights into the underlying mechanism responsible for the antitumor activity of TβRII-SE/Fc. Although more studies are required to demonstrate the effectiveness and safety of the novel peptibody as a new therapeutic for the treatment of cancer, our initial in vitro and in vivo results in human colorectal tumor-derived cell lines are highly encouraging. Our results may serve as the foundation for further research and development of a novel biopharmaceutical for oncology., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Usher syndrome 1B (USH1B) is a devastating genetic disorder with congenital deafness, loss of balance, and blindness caused by mutations in the myosin-VIIa (MYO7A) gene, for which there is currently no cure. We developed a gene therapy approach addressing the vestibulo-cochlear deficits of USH1B using a third-generation, high-capacity lentiviral vector system capable of delivering the large 6,645-bp MYO7A cDNA. Lentivirally delivered MYO7A and co-encoded dTomato were successfully expressed in the cochlear cell line HEI-OC1. In normal-hearing mice, both cochlea and the vestibular organ were efficiently transduced, and ectopic MYO7A overexpression did not show any adverse effects. In Shaker-1 mice, an USH1B disease model based on Myo7a mutation, cochlear and vestibular hair cells, the main inner ear cell types affected in USH1B, were successfully transduced. In homozygous mutant mice, delivery of MYO7A at postnatal day 16 resulted in a trend for partial recovery of auditory function and in strongly reduced balance deficits. Heterozygous mutant mice were found to develop severe hearing loss at 6 months of age without balance deficits, and lentiviral MYO7A gene therapy completely rescued hearing to wild-type hearing thresholds. In summary, this study demonstrates improved hearing and balance function through lentiviral gene therapy in the inner ear., Competing Interests: Declaration of interests H.S. is a shareholder in Rescue Hearing Inc. and on the advisory board of MedEl GmbH. J.W.S., M.M., H.B., A.W., A.S., and H.S. have submitted patent applications for lentiviral gene therapy for the inner ear., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Ex-vivo gene therapy has been shown to be an effective method for treating bone defects in pre-clinical models. As gene therapy is explored as a potential treatment option in humans, an assessment of the safety profile becomes an important next step. The purpose of this study was to evaluate the biodistribution of viral particles at the defect site and various internal organs in a rat femoral defect model after implantation of human ASCs transduced with lentivirus (LV) with two-step transcriptional activation (TSTA) of bone morphogenetic protein-2 (LV-TSTA-BMP-2). Animals were sacrificed at 4-, 14-, 56-, and 84-days post implantation. The defects were treated with either a standard dose (SD) of 5 million cells or a high dose (HD) of 15 million cells to simulate a supratherapeutic dose. Treatment groups included (1) SD LV-TSTA-BMP-2 (2) HD LV-TSTA-BMP-2, (3) SD LV-TSTA-GFP (4) HD LV-TSTA-GFP and (5) SD nontransduced cells. The viral load at the defect site and ten organs was assessed at each timepoint. Histology of all organs, ipsilateral tibia, and femur were evaluated at each timepoint. There were nearly undetectable levels of LV-TSTA-BMP-2 transduced cells at the defect site at 84-days and no pathologic changes in any organ at all timepoints. In conclusion, human ASCs transduced with a lentiviral vector were both safe and effective in treating critical size bone defects in a pre-clinical model. These results suggest that regional gene therapy using lentiviral vector to treat bone defects has the potential to be a safe and effective treatment in humans., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

We attempted to construct a myeloid leukemia cell strain for stable overexpression and knock-down of miR-217 and explored the possible mechanism underlying miR-217 in chronic myeloid leukemia (CML). MiR-217 overexpression and the knock-down lentiviral vector with puromycin resistance were constructed and packaged within recombinant lentivirus. Stably transfected K562 cells were obtained through puromycin screening, and the qPCR assay detected the relative expression of the target gene. The proliferation, apoptosis, and methylation level of PER2 within cultured cells were detected using the CCK-8 assay, flow cytometry, and TaqMan real‑time fluorescence quantitative methylation-specific PCR. qPCR and Western blot detected the expression of miR-217-related genes within the constructed K562 cell model. Colony PCR and sequencing proved that recombinant lentivirus expression vectors pSE16 and pSE17 were correctly constructed. The lentivirus titer was 2.95 × 10 9 and 2.61 × 10 9  IU/mL. The miR-217 expression level was high in pSE5316-K562 cells, and that of the miR-217 sponge was high in pSE5317-K562 cells. Overexpressed miR-217 could inhibit the K562 cell proliferation and induce apoptosis. Inhibition of miR-217 enhanced the expression of DNMT3A, decreased the PER2 expression, and elevated the degree of PER2 methylation. The miR-217 overexpression and knock-down of the K562 cell line were successfully constructed, providing a tool for further exploring the miR-217 mechanism in CML. DNMT3A could be the molecular target of miR-217 by regulating PER2 gene methylation and getting involved with the occurrence and development of CML., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

HIV-1 replication in primary monocyte-derived macrophages (MDMs) is kinetically restricted at the reverse transcription step due to the low deoxynucleoside triphosphates (dNTP) pools established by host dNTPase, SAM and HD domain containing protein 1 (SAMHD1). Lentiviruses such as HIV-2 and some Simian immunodeficiency virus counteract this restriction using viral protein X (Vpx), which proteosomally degrades SAMHD1 and elevates intracellular dNTP pools. However, how dNTP pools increase after Vpx degrades SAMHD1 in nondividing MDMs where no active dNTP biosynthesis is expected to exists remains unclear. In this study, we monitored known dNTP biosynthesis machinery during primary human monocyte differentiation to MDMs and unexpectedly found MDMs actively express dNTP biosynthesis enzymes such as ribonucleotide reductase, thymidine kinase 1, and nucleoside-diphosphate kinase. During differentiation from monocytes the expression levels of several biosynthesis enzymes are upregulated, while there is an increase in inactivating SAMHD1 phosphorylation. Correspondingly, we observed significantly lower levels of dNTPs in monocytes compared to MDMs. Without dNTP biosynthesis availability, Vpx failed to elevate dNTPs in monocytes, despite SAMHD1 degradation. These extremely low monocyte dNTP concentrations, which cannot be elevated by Vpx, impaired HIV-1 reverse transcription in a biochemical simulation. Furthermore, Vpx failed to rescue the transduction efficiency of a HIV-1 GFP vector in monocytes. Collectively, these data suggest that MDMs harbor active dNTP biosynthesis and Vpx requires this dNTP biosynthesis to elevate dNTP levels to effectively counteract SAMHD1 and relieve the kinetic block to HIV-1 reverse transcription in MDMs., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Lentivirus is an efficient gene transfer system that is widely used in basic science. We aimed to improve viral titer by applying an ultra-expression vectors to lentiviral packaging. Application of the ultra-expression vectors increased biological titer 4 times for standard preparation. We also evaluated the efficacy of the ultra-expression vectors to relatively longer insert fragments, such as CSII-CMV-CNROE containing 5 genes in multiple cloning sites. Packaging of the ultra-expression vectors showed 3.5 times higher biological titer compared with the original method. Our improved packaging system could be applied to lentivirus to produce higher titers., Competing Interests: Declaration of competing interest All authors do not have any conflict of interest and competing interest to disclose., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The area postrema (AP) of the brain is exposed to circulating metabolites and hormones. However, whether AP detects glucose changes to exert biological responses remains unknown. Its neighboring nuclei, the nucleus tractus solitarius (NTS), responds to acute glucose infusion by inhibiting hepatic glucose production, but the mechanism also remains elusive. Herein, we characterized AP and NTS glucose-sensing mechanisms. Infusion of glucose into the AP, like the NTS, of chow rats suppressed glucose production during the pancreatic (basal insulin)-euglycemic clamps. Glucose transporter 1 or pyruvate kinase lentiviral-mediated knockdown in the AP negated AP glucose infusion to lower glucose production, while the glucoregulatory effect of NTS glucose infusion was also negated by knocking down glucose transporter 1 or pyruvate kinase in the NTS. Furthermore, we determined that high-fat (HF) feeding disrupts glucose infusion to lower glucose production in association with a modest reduction in the expression of glucose transporter 1, but not pyruvate kinase, in the AP and NTS. However, pyruvate dehydrogenase activator dichloroacetate infusion into the AP or NTS that enhanced downstream pyruvate metabolism and recapitulated the glucoregulatory effect of glucose in chow rats still failed to lower glucose production in HF rats. We discovered that a glucose transporter 1- and pyruvate kinase-dependent glucose-sensing mechanism in the AP (as well as the NTS) lowers glucose production in chow rats and that HF disrupts the glucose-sensing mechanism that is downstream of pyruvate metabolism in the AP and NTS. These findings highlight the role of AP and NTS in mediating glucose to regulate hepatic glucose production., Competing Interests: Conflict of interest The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Primary human mammary epithelial cells (pHMECs) are known to be remarkably difficult to engineer genetically. Here, we present a protocol for efficient transduction of pHMECs using a baboon retroviral envelope glycoprotein for pseudotyping of lentiviral vectors (BaEV-LVs). We describe the preparation of the BaEV-LVs, the isolation of pHMECs from breast samples, and the subsequent transduction of pHMECs. We also detail the use of CRISPRi technology to efficiently silence gene expression in pHMECs, which can then be used for functional assays. For complete details on the use and execution of this protocol, please refer to Richart et al. (2022). 1 ., Competing Interests: Declaration of interests E.V. is the inventor of the patent on pseudotyping of retroviral particles with BaEV envelope glycoproteins (patent WO 07290918.7)., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Members of deltacoronavirus (DCoV) have mostly been identified in diverse avian species as natural reservoirs, though the porcine DCoV (PDCoV) is a major swine enteropathogenic virus with global spread. The important role of aminopeptidase N (APN) orthologues from various mammalian and avian species in PDCoV cellular entry and interspecies transmission has been revealed recently. In this study, comparative analysis indicated that three avian DCoVs, bulbul DCoV HKU11, munia DCoV HKU13, and sparrow DCoV HKU17 (Chinese strain), and PDCoV in the subgenera Buldecovirus are grouped together at whole-genome levels; however, the spike (S) glycoprotein and its S1 subunit of HKU17 are more closely related to night heron DCoV HKU19 in Herdecovirus . Nevertheless, the S1 protein of HKU11, HKU13, or HKU17 bound to or interacted with chicken APN (chAPN) or porcine APN (pAPN) by flow cytometry analysis of cell surface expression of APN and by coimmunoprecipitation in APN-overexpressing cells. Expression of chAPN or pAPN allowed entry of pseudotyped lentiviruses with the S proteins from HKU11, HKU13 and HKU17 into nonsusceptible cells and natural avian and porcine cells, which could be inhibited by the antibody against APN or anti-PDCoV-S1. APN knockdown by siRNA or knockout by CRISPR/Cas9 in chicken or swine cell lines significantly or almost completely blocked infection of these pseudoviruses. Hence, we demonstrate that HKU11, HKU13, and HKU17 with divergent S genes likely engage chAPN or pAPN to enter the cells, suggesting a potential interspecies transmission from wild birds to poultry and from birds to mammals by certain avian DCoVs. IMPORTANCE The receptor usage of avian deltacoronaviruses (DCoVs) has not been investigated thus far, though porcine deltacoronavirus (PDCoV) has been shown to utilize aminopeptidase N (APN) as a cell receptor. We report here that chicken or porcine APN also mediates cellular entry by three avian DCoV (HKU11, HKU13, and HKU17) spike pseudoviruses, and the S1 subunit of three avian DCoVs binds to APN in vitro and in the surface of avian and porcine cells. The results fill the gaps in knowledge about the avian DCoV receptor and elucidate important insights for the monitoring and prevention of potential interspecies transmission of certain avian DCoVs. In view of the diversity of DCoVs, whether this coronavirus genus will cause novel virus to emerge in other mammals from birds, are worthy of further surveillance and investigation.

Background: Amyotrophic lateral sclerosis (ALS) is irreversible and fatal within 3-5 years, with limited options for treatment. It is imperative to develop a symptom-based treatment that may increase the survival of ALS patients and improve their quality of life. Inflammation status, especially elevated interleukin 1β (IL1β), has been reported to play a critical role in ALS progression. Our study determined that neutralizing circulating IL1β slows down the progression of ALS in an ALS mouse model., Methods: The ALS mouse model was developed by microinjection of lentivirus-carrying OPTN E478G (optineurin, a mutation from ALS patients) into the intra-motor cortex of mice. Peripheral circulating IL1β was neutralized by injecting anti-IL1β antibody into the tail vein. Enzyme-linked immunosorbent assay (ELISA) and real-time polymerase chain reaction (RT-PCR) were carried out to determine the protein and gene expression levels of IL1β. TUNEL assay was used to assess the neural cell death. Immunofluorescent staining of MAP2 and CASP3 was accomplished to evaluate neuronal cell apoptosis. Glial fibrillary acidic protein staining was performed to analyze the number of astrocytes. Rotarod test, grip strength test, balance beam test, and footprint test were conducted to assess the locomotive function after anti-IL1β treatment., Results: The model revealed that neuroinflammation contributes to ALS progression. ALS mice exhibited elevated neuroinflammation and IL1β secretion. After anti-IL1β treatment, ALS mice revealed decreased neural cell death and astrogliosis and gained improved muscle strength and motor ability., Conclusions: Blocking IL1β is a promising strategy to slow down the progression of ALS., (© 2022 The Authors. Animal Models and Experimental Medicine published by John Wiley & Sons Australia, Ltd on behalf of The Chinese Association for Laboratory Animal Sciences.)

Hypoxia-inducible factor (HIF) plays a crucial role in regulating the hypoxia-inducible state of nucleus pulposus cells in the intervertebral disc. In addition, the oxygen-dependent conversion of HIF-1α in nucleus pulposus cells is controlled by the protein proline 4-hydroxylase domain (PHD) family. To explore whether HIF-1α can be regulated by modulating PHD homologs to inhibit nucleus pulposus degeneration, PHD2-shRNAs were designed and a PHD2 interference vector was constructed. The expression of HIF-1α and PHD2 genes in the nucleus pulposus cells in the experimental group was detected by RT-PCR, and the expression of HIF-1α, MMP-2, Aggrecan, and Col II proteins in the P0-P3 cells in the experimental group and the control group was detected by Western blotting. The apoptosis of P0-P3 nucleus pulposus cells was detected by flow cytometry. After lentivirus infection, the interference efficiency of the PHD2 gene decreased with cell passage. The apoptosis of P1-P3 cells in the experimental group was significantly lower than that in the control group or degeneration group. Compared to the control group, the expression of HIF-1α, Aggrecan, and Col II proteins increased significantly, and the expression of MMP-2 protein decreased significantly. In conclusion, interference with PHD2 can upregulate the expression of HIF-1α, accelerate anabolism, reduce catabolism, inhibit apoptosis of nucleus pulposus cells, and then these can inhibit degeneration of nucleus pulposus cells. Our results can provide an effective therapeutic target in intervertebral discs during intervertebral disc degeneration, and this may have important clinical significance., (© 2021 S. Karger AG, Basel.)

Lentiviral transduction of human mesenchymal stem cells (MSCs) induces long-term transgene expression and holds great promise for multiple gene therapy applications. Polybrene is the most commonly used reagent to improve viral gene transfer efficiency in laboratory research; however, it is not approved for human use and has also been shown to impair MSC proliferation and differentiation. Therefore, there is a need for optimized transduction protocols that can also be adapted to clinical settings. LentiBOOST (LB) and protamine sulfate are alternative transduction enhancers (TEs) that can be manufactured to current Good Manufacturing Practice standards, are easily applied to existing protocols, and have been previously studied for the transduction of human CD34 + hematopoietic stem cells. In this study, we investigated these reagents for the enhancement of lentiviral transduction of adipose-derived MSCs. We found that the combination of LB and protamine sulfate could yield comparable or even superior transduction efficiency to polybrene, with no dose-dependent adverse effects on cell viability or stem cell characteristics. This combination of TEs represents a valuable clinically compatible alternative to polybrene with the potential to significantly improve the efficiency of lentiviral transduction of MSCs for gene therapy applications.

Background: Apoptosis plays an essential role in the development and treatment of tumors, and caspase-8 ( CASP8 ) plays an important role in the enzyme cascade reaction that leads to apoptosis. Human epidermal growth factor receptor 2 (HER-2) overexpressing breast cancer is highly aggressive and has a high recurrence rate and poor prognosis. This study investigated whether lentivirus-mediated Gag-CASP8 can effectively deliver activated CASP8 into primary human breast cancer cells overexpressing HER-2 to induce apoptosis and explore the underlying mechanism. Materials and Methods: HER-2 overexpressing primary human breast cancer cells were infected with lentivirus-like particles carrying Gag-CASP8. Results: After a 48h infection of primary human breast cancer cells with HER-2 by lentivirus-mediated Gag-CASP8, significant differences were observed in the survival rate, migration ability, S-phase number of cells, apoptosis rate, and intracellular activated CASP8 and caspase-3 levels in tumor cells compared with those in the control group ( p  < 0.05). Conclusions: Lentivirus-mediated Gag-CASP8 can deliver activated CASP8 into HER-2 overexpressing primary human breast cancer cells and induce apoptosis by activating caspase-3, a downstream apoptotic executive molecule. By blocking the S-phase to inhibit cell proliferation and migration, lentivirus-mediated Gag-CASP8 provides a reference for tumor gene therapy.

Purpose: The side effects of immune suppressants on immune rejection have become increasingly apparent after keratoplasty. To find out new alternative immunotherapy strategies, we studied the role of programmed death-1 (PD-1) and its ligand (PD-L1) co-stimulatory pathway in inducing immune tolerance of rat keratoplasty., Methods: The PD-L1 protein was constitutively overexpressed via lentiviral transduction in bone marrow-derived dendritic cells (BMDCs) from rats, then infused via the tail vein into rats before undergoing keratoplasty. Western blot analysis of PD-L1 protein confirmed the effectiveness of lentivirus-mediated. The phenotype of immature BMDC was confirmed by flow cytometry analysis with CD80, CD86, CD11c and MHC-II antibodies. To investigate the mechanism of the immune tolerance induced by BMDCs transfusion, PD-L1, IFN-γ and IL-17 in serum and cell culture supernatant were assessed by ELISA and qPCR., Results: After LPS stimulation, immature dendritic cells with over-expression of PD-L1 still showed high expression of PD-L1(p < 0.001), and low expression of IL-17 and IFN-γ (p < 0.001), which reduced neovascularization (p < 0.05), and prolonged the survival after corneal implants., Conclusion: Immature DC cells with overexpression of PD-L1 have low ability to activate T cells，which is a potential treatment for avoiding graft rejection by promoting natural immunosuppression. This cellular treatment is expected to reduce the use of immune suppressants and the occurrence of side effects., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Numerous genes exert multifaceted roles in hematopoiesis. Therefore, we generated novel lineage-specific RNA interference (RNAi) lentiviral vectors, H23B-Ery-Lin-shRNA and H234B-Ery-Lin-shRNA, to probe the functions of these genes in erythroid cells without affecting other hematopoietic lineages. The lineage specificity of these vectors was confirmed by transducing multiple hematopoietic cells to express a fluorescent protein. Unlike the previously reported erythroid lineage RNAi vector, our vectors were designed for cloning the short hairpin RNAs (shRNAs) for any gene, and they also provide superior knockdown of the target gene expression with a single shRNA integration per cell. High-level lineage-specific downregulation of BCL11A and ZBTB7A, two well-characterized transcriptional repressors of HBG in adult erythroid cells, was achieved with substantial induction of fetal hemoglobin with a single-copy lentiviral vector integration. Transduction of primary healthy donor CD34 +  cells with these vectors resulted in >80% reduction in the target protein levels and up to 40% elevation in the γ-chain levels in the differentiated erythroid cells. Xenotransplantation of the human CD34 +  cells transduced with H23B-Ery-Lin-shBCL11A LV in immunocompromised mice showed ~ 60% reduction in BCL11A protein expression with ~ 40% elevation of γ-chain levels in the erythroid cells derived from the transduced CD34 +  cells. Overall, the novel erythroid lineage-specific lentiviral RNAi vectors described in this study provide a high-level knockdown of target gene expression in the erythroid cells, making them suitable for their use in gene therapy for hemoglobinopathies. Additionally, the design of these vectors also makes them ideal for high-throughput RNAi screening for studying normal and pathological erythropoiesis., (© 2022. The Author(s).)

A promising treatment for β-hemoglobinopathies is the de-repression of γ-globin expression leading to increased fetal hemoglobin (HbF) by targeting BCL11A. Here, we aim to improve a lentivirus vector (LV) containing a single BCL11A shmiR (SS) to further increase γ-globin induction. We engineered a novel LV to express two shmiRs simultaneously targeting BCL11A and the γ-globin repressor ZNF410. Erythroid cells derived from human HSCs transduced with the double shmiR (DS) showed up to a 70% reduction of both BCL11A and ZNF410 proteins. There was a consistent and significant additional 10% increase in HbF compared to targeting BCL11A alone in erythroid cells. Erythrocytes differentiated from SCD HSCs transduced with the DS demonstrated significantly reduced in vitro sickling phenotype compared to the SS. Erythrocytes differentiated from transduced HSCs from β-thalassemia major patients demonstrated improved globin chain balance by increased γ-globin with reduced microcytosis. Reconstitution of DS-transduced cells from Berkeley SCD mice was associated with a statistically larger reduction in peripheral blood hemolysis markers compared with the SS vector. Overall, these results indicate that the DS LV targeting BCL11A and ZNF410 can enhance HbF induction for treating β-hemoglobinopathies and could be used as a model to simultaneously and efficiently target multiple gene products., Competing Interests: Declaration of interests D.A.W. has received research funding from bluebird bio for research in hemoglobinopathies. Boston Children’s Hospital has licensed certain intellectual property (IP) relevant to hemoglobinopathies to bluebird bio., (Copyright © 2022. Published by Elsevier Inc.)

Background: Traumatic brain injury (TBI) is a common clinical condition caused by external force. Aquaporin-4 (AQP4) in astrocytes participates in the generation of cell swelling in TBI., Methods: This research explored the effect of AQP4 gene silencing in a TBI rat model. A hydraulic craniocerebral trauma instrument was employed for establishing the TBI rat model. AQP4 expression in the brain was inhibited by the injection of AQP4 shRNA-lentiviral vector. The expression of relative genes was evaluated by Western blot and qRT-PCR. Neuronal apoptosis was analyzed by TUNEL assay., Results: AQP4 shRNA treatment inhibited AQP4 expression in the brain of rats with TBI. AQP4 shRNA alleviated TBI-induced brain edema and neurological deficit in rats. Neuronal apoptosis and astrocyte activation in TBI rats were reduced by AQP4 silencing., Conclusion: This research demonstrated that AQP4 shRNA-induced silencing of AQP4 in the TBI rat model reduced the expression of AQP4 and GFAP, alleviated brain edema, neurological deficit, neuronal apoptosis and inhibited astrocyte activation.

Background and Objective: Periodontitis is a chronic progressive inflammation that invades periodontal supporting tissues, in which periodontal tissue regeneration engineering offers new hope for prevention and treatment, including seed cells, scaffolds, and growth factors. In recent years, scholars have shown that autologous teeth can be used as new bone tissue repair materials for periodontal regeneration and bone tissue repair. The aim of this study was to establish a human periodontal ligament cell line that expresses the human bone morphogenetic protein 2 gene (BMP2) in a stable manner using lentiviral mediation in order to explore the effect of BMP2 from autologous tooth on the proliferative and osteogenic capacity of human periodontal ligament cells (hPDLCs)., Materials and Methods: Human periodontal ligament cells were cultured, subcultured, and identified, and then homologous recombinant lentivirus plasmid plv-BMP2 was constructed and transfected into the third passage (P 3 ) hPDLCs. After that, the effect of BMP2 on its proliferation was detected by CCK-8, at the same time, the osteogenic induction of hPDLCs was carried out at 7, 14, and 21 days, and then the effect of BMP2 on its osteogenic ability was detected by alizarin red staining, alkaline phosphatase activity determination, and the mRNA expression levels of osteogenic-related genes using real-time fluorescence quantitative PCR, including alkaline phosphatase, runt-related transcription factor 2, bone sialoprotein, osteocalcin, osteopontin, and collagen I. Finally, spss26.0 software was used for statistical processing., Results: The results showed that cells transfected with the homologous recombinant lentiviral plasmid pLV-BMP2 had a similar morphology to normal hPDLCs, showing a typical radial arrangement; the cell proliferative capacity of the pLV-BMP2 group as measured by CCK-8 was enhanced compared with the control group and the pLV-puro group (p < .05); alizarin red staining and alkaline phosphatase activity assay showed that the osteogenic ability of pLV-BMP2 was significantly enhanced compared with the control and pLV-puro groups (p < .01), and the findings of real-time fluorescence-based quantitative PCR showed high expression of osteogenic-related genes in pLV-BMP2 group (p < .01)., Conclusion: In conclusion, a stable periodontal ligament cell line overexpressing BMP2 was successfully established by a lentivirus-mediated method, which proved that BMP2 has a strong ability to promote the proliferation and osteogenesis of hPDLCs, thereby providing an opportunity for the study of periodontal tissue regeneration as well as providing an experimental basis for the application of autologous teeth as a new type of bone repair material for periodontal therapy and even for maxillofacial bone tissue repair., (© 2022 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Objective To investigate the effect of the gene vaccine in anti-tuberculosis immunity by constructing immunity-related p47 GTPase/ezrin-radixin-moesin-binding phosphoprotein 50 (LRG47/EBP50) gene co-expression recombinant lentivirus targeting vector. Methods Recombinant lentiviral plasmid vector pLenti-EBP50-LRG47 was established by using molecular cloning and packaged as lentivirus LV-EBP50-LRG47 and H37Rv infect macrophages. Then their bactericidal ability was tested by colony-forming units while the cellular autophagy and apoptosis was detected by flow cytometry. iNOS protein was detected by Western blotting and the expression level of nitric oxide (NO) was detected by ultraviolet spectrophotometer. Results The recombinant lentivirus LV-EBP50-LRG47 successfully up-regulated the expression of EBP50 and LRG47 after infecting macrophages. Compared with the control group, LV-EBP50-LRG47 can significantly inhibit the growth of intracellular H37Rv. The autophagy and apoptosis levels of LV-EBP50-LRG47 infected macrophages increased significantly, and the expression levels of iNOS and NO were significantly up-regulated. Conclusion LRG47/EBP50 gene co-expression enhances macrophages autophagy and apoptosis, and increases generation of iNOS and NO, which significantly inhibites the growth of intracellular H37Rv.

Objective: To construct a recombinant lentiviral vector for mouse miR-204 overexpression, and to verify the targeted regulation of miR-204 and DVL3 in silica (SiO(2)) -induced mouse lung epithelial cells (MLE-12 cells) . Methods: In October 2019, the pre-miR-204 gene was amplified from the mouse genome by the polymerase chain reaction (PCR) method. After sequencing, the amplified product was cloned into the pLenti-CMV-EGFP lentiviral vector. The positive clones were identified by PCR screening and sequencing. The miR-204 overexpressed lentiviral vector was transfected into 293T cells, and lentiviral packaging and titer determination were performed. The experiment was divided into SiO(2) control group, virus control group, and miR-204 virus group, and the expressions of miR-204 and DVL3 gene were detected by real-time PCR. Results: The miR-204 lentiviral expression vector Lv-miR-204-5p was constructed and identified correctly by PCR and sequencing, and a virus dilution with a titer of 9.57×10(8) IU/ml was obtained. The results of real-time PCR showed that the expression of miR-204 in MLE-12 cells of the miR-204 virus group was higher than that of SiO(2) control group and virus control group, and the expression of DVL3 gene was lower than that of SiO(2) control group and virus control group, the differences were statistically significant ( P <0.05) . Conclusion: Overexpression of miR-204 by lentiviral vector may inhibit the expression of DVL3 gene in silica-induced mouse lung epithelial cells.

A multimer of retroviral integrase (IN) synapses viral DNA ends within a stable intasome nucleoprotein complex for integration into a host cell genome. Reconstitution of the intasome from the maedi-visna virus (MVV), an ovine lentivirus, revealed a large assembly containing sixteen IN subunits 1 . Herein, we report cryo-EM structures of the lentiviral intasome prior to engagement of target DNA and following strand transfer, refined at 3.4 and 3.5 Å resolution, respectively. The structures elucidate details of the protein-protein and protein-DNA interfaces involved in lentiviral intasome formation. We show that the homomeric interfaces involved in IN hexadecamer formation and the α-helical configuration of the linker connecting the C-terminal and catalytic core domains are critical for MVV IN strand transfer activity in vitro and for virus infectivity. Single-molecule microscopy in conjunction with photobleaching reveals that the MVV intasome can bind a variable number, up to sixteen molecules, of the lentivirus-specific host factor LEDGF/p75. Concordantly, ablation of endogenous LEDGF/p75 results in gross redistribution of MVV integration sites in human and ovine cells. Our data confirm the importance of the expanded architecture observed in cryo-EM studies of lentiviral intasomes and suggest that this organization underlies multivalent interactions with chromatin for integration targeting to active genes., (© 2022. The Author(s).)

Alzheimer type of dementia is accompanied with progressive loss of cognitive function that directly correlates with accumulation of amyloid beta plaques. It is known that Fibroblast growth factor 21 (FGF21), a metabolic hormone, with strong neuroprotective potential, is induced during oxidative stress in Alzheimer's disease. Interestingly, FGF21 cross-talks with autophagy, a mechanism involved in the clearance of abnormal protein aggregate. Moreover, autophagy activation by Rapamycin delivers neuroprotective role in Alzheimer's disease. However, the synergistic neuroprotective efficacy of overexpressed FGF21 along with Rapamycin is not yet investigated. Therefore, the present study examined whether overexpressed FGF21 along with autophagy activation ameliorated neurodegenerative pathology in Alzheimer's disease. We found that cognitive deficits in rats with intracerebroventricular injection of Amyloid beta 1-42 oligomers were restored when injected with FGF21-expressing lentiviral vector combined with Rapamycin. Furthermore, overexpression of FGF21 along with Rapamycin downregulated protein levels of Amyloid beta 1-42 and phosphorylated tau and expression of major autophagy proteins along with stabilization of oxidative stress. Moreover, FGF21 overexpressed rats treated with Rapamycin revamped the neuronal density as confirmed by histochemical, cresyl violet and immunofluorescence analysis. These results generate compelling evidence that Alzheimer's disease pathology exacerbated by oligomeric amyloid beta may be restored by FGF21 supplementation combined with Rapamycin and thus present an appropriate treatment paradigm for people affected with Alzheimer's disease., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Yak is a unique species of cattle that is adapted to the harsh natural environment of the Qinghai-Tibet Plateau. Research on the function of the yak rumen is limited to animal experiments, and the cell molecular mechanism is very limited. The high cost of isolation and culture of adult yak rumen epithelial cells (YRECs), low success rate, and limited cell life limit the scope of long-term physiological functions and nutrient absorption mechanisms of yak rumen epithelium in vitro studies. This study aimed to explore the isolation and immortal culture methods of primary YRECs and establish a new cell line model for studying cell molecular mechanisms. The human telomerase reverse transcriptase gene (hTERT) and simian virus 40 large T antigen (SV40T) were transferred into primary YDECs using mammalian gene expression lentiviral vectors. The immortalized cell line (SV40T-YREC-hTERT) retains the morphological and functional characteristics of primary cells. The epithelial cell marker protein cytokeratin 18 of the immortalized cell lines was positive, and the cell proliferation and karyotype were normal. The SV40T and hTERT genes were successfully transferred into immortalized cell lines and maintained high expression. Simultaneously, the immortalized cell lines had normal function of short-chain fatty acid (SCFA) transport and absorption, and the immortalized yak rumen epithelial cell lines were successfully established. In addition, the transepithelial electrical resistance value gradually increased with culture time, and the permeability of epithelial cells decreased by culturing epithelial cells in Transwell culture chambers. Transmission electron microscopy demonstrated the submicroscopic structure of cells in the integrity barrier model and established the YREC barrier model in vitro., Competing Interests: The authors declare that there are no conflicts of interest regarding the publication of this paper., (Copyright © 2022 JunMei Wang et al.)

Extracellular vesicles have become a research focus for their potential as therapeutic vehicles that carry cargo substances. Extracellular vesicles may origin from the endosomal compartment and share several characteristics with the envelope of lentiviruses. A previous study reported that constitutive expression of the tetraspanin CD9, an extracellular vesicle marker, not only increases vesicle secretion from cells, but has also a positive effect on lentiviral transduction efficiency. Moreover, it was shown that expression of CD9 on the viral envelope in absence of viral glycoproteins was sufficient for the transduction of mammalian cells. In this study, we investigate the effect of CD9 and folate receptor alpha, a GPI-anchored protein, on biosynthesis and transduction efficiency of vesicles carrying lentiviral vectors. We demonstrate that neither CD9 nor FRα nor the combination of both were able to mediate a significant transduction of therapeutic vesicles carrying lentiviral RNA. Further studies are required to identify endogenous mammalian proteins that can be used for pseudotyping of viral envelopes to improve viral targeting without inducing immune responses., Competing Interests: The authors have declared that no competing interests exist.

Nucleic acid tests to detect the SARS-CoV-2 virus have been performed worldwide since the beginning of the COVID-19 pandemic. For the quality assessment of testing laboratories and the performance evaluation of molecular diagnosis products, reference materials (RMs) are required. In this work, we report the production of a lentiviral SARS-CoV-2 RM containing approximately 12 kilobases of its genome including common diagnostics targets such as RdRp, N, E, and S genes. The RM was measured with multiple assays using two different digital PCR platforms. To measure the homogeneity and stability of the lentiviral SARS-CoV-2 RM, reverse transcription droplet digital PCR (RT-ddPCR) was used with in-house duplex assays. The copy number concentration of each target gene in the extracted RNA solution was then converted to that of the RM solution. Their copy number values are measured to be from 1.5 × 10 5 to 2.0 × 10 5 copies/mL. The RM has a between-bottle homogeneity of 4.80-8.23% and is stable at 4 °C for 1 week and at -70 °C for 6 months. The lentiviral SARS-CoV-2 RM closely mimics real samples that undergo identical pre-analytical processes for SARS-CoV-2 molecular testing. By offering accurate reference values for the absolute copy number of viral target genes, the developed RM can be used to improve the reliability of SARS-CoV-2 molecular testing., (© 2021. The Author(s).)

Purpose: Viral keratitis caused by herpes simplex virus 1 (HSV-1) is a lifelong recurring disease and an unignored cause of blindness worldwide. Current antiviral therapy cannot eliminate the transcriptionally silent HSV-1 in latently infected patients. With the explosive applications of the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease (Cas) 9 gene-editing system in recent years, we aim to develop a CRISPR/Cas9 system targeting down the major HSV receptor, NECTIN-1 on human corneal epithelial cells (HCECs), to provide a novel strategy for herpes simplex keratitis (HSK) treatment., Methods: The selected single guide RNAs (sgRNAs) targeting human nectin cell adhesion molecule 1 (NECTIN-1), together with Cas-9, were assembled into lentivirus. HCECs were infected with Lenti-Cas9-gRNAs to establish NECTIN-1 knockdown cells. Following HSV-green fluorescent protein (GFP) infection, cell survival and virus infection were determined by fluorescence microscopy and flow cytometry. Relative HSV DNA amount was also compared through quantitative reverse transcriptase-polymerase chain reaction., Results: Lentivirus packaged with the CRISPR/Cas9 system and the two selected sgRNAs both successfully edited down the protein levels of NECTIN-1 of HCECs. After HSV-GFP infection, the infection rate of HCECs in knockdown groups dramatically decreased, especially in the NECTIN-1 knockdown group 1. In addition, the relative HSV DNA amount of both knockdown groups was only 30% when compared with the control group., Conclusions: We successfully knocked down the NECTIN-1 expression in vitro by the CRISPR/Cas9 system, which alleviated the HSV infection in HCECs., Translational Relevance: This study offered a promising target for the cure of HSK.

Genetically encoded biosensors are powerful tools to monitor cellular behavior, but the difficulty in generating appropriate reporters for chromatin factors hampers our ability to dissect epigenetic pathways. Here, we present TRACE (transgene reporters across chromatin environments), a high-throughput, genome-wide technique to generate fluorescent human reporter cell lines responsive to manipulation of epigenetic factors. By profiling GFP expression from a large pool of individually barcoded lentiviral integrants in the presence and absence of a perturbation, we identify reporters responsive to pharmacological inhibition of the histone lysine demethylase LSD1 and genetic ablation of the PRC2 subunit SUZ12. Furthermore, by manipulating the HIV-1 host factor LEDGF through targeted deletion or fusion to chromatin reader domains, we alter lentiviral integration site preferences, thus broadening the types of chromatin examined by TRACE. The phenotypic reporters generated through TRACE will allow the genetic interrogation of a broad range of epigenetic pathways, furthering our mechanistic understanding of chromatin biology., Competing Interests: Declaration of interests R.E.K. is a member of Molecular Cell’s Advisory Board., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Dermal white adipose tissue (dWAT) is involved in the maintenance of skin homeostasis. However, the studies concerning its molecular regulation are limited. In the present paper, we ask whether the introduction of two transcription factors, Foxn1 and Hif-1α, into the post-wounded skin of Foxn1 -/- mice regulates dWAT during wound healing (days 3 and 6). We have chosen lentivirus vectors (LVs) as a tool to deliver Foxn1 and Hif-1α into the post-wounded skin. We documented that combinations of both transgenes reduces the number, size and diameter of dermal adipocytes at the wound bed area. The qRT-PCR analysis of pro-adipogenic genes, revealed that LV-Hif-1α alone, or combined with LV-Foxn1, increases the mRNA expression of Pparγ , Glut 4 and Fasn at post-wounding day 6. However, the most spectacular stimulatory effect of Foxn1 and/or Hif-1α was observed for Igf2, the growth factor participating in adipogenic signal transduction. Our data also shows that Foxn1/Hif-1α, at post-wounding day 3, reduces levels of CD68 and MIP-1γ mRNA expression and the percentage of CD68 positive cells in the wound site. In conclusion, the present data are the first to document that Foxn1 and Hif-1α cooperatively (1) regulate dWAT during the proliferative phase of skin wound healing through the Igf2 signaling pathway, and (2) reduce the macrophages content in the wound site.

Objective: To observe the effects of lentivirus-mediated RNA interference (RNAi) of hypoxia-inducible factor 1α (HIF-1α) and phosphatase and tensin homolog on chromosome ten (PTEN) on oxygen-glucose deprivation (OGD) injury in primary cultured rat neurons., Methods: Primary cultures of neonatal SD rat neurons were infected by lentiviral vectors carrying short hairpin RNA (shRNA) targeting HIF-1α or PTEN followed 4 days later by hypoxic exposure, and the control neurons were infected with the empty virus only with or without subsequent hypoxic exposure. Twenty-four hours after hypoxia, the interference efficiency was assessed with qRT-PCR, and lactate dehydrogenase (LDH) assay and AnnexinV-FITC/ PI assay were performed to detect neuronal damage and apoptosis. The expressions of the related proteins were determined with Western blotting., Results: Lentivirus-mediated RNAi effectively silenced the mRNA expression of the target genes. HIF-1α silencing obviously aggravated the hypoxia-induced damage and apoptosis of the neurons, enhanced the expression of PTEN protein and significantly lowered the expressions of p-PTEN, p-AKT, NR2A and VEGFa ( P < 0.05). PTEN silencing significantly alleviated hypoxia-induced damage and apoptosis of the neurons and increased the cellular expressions of p-PTEN and p-AKT ( P < 0.05) without obviously affecting the expressions of HIF-1α, NR2A or VEGFa ( P >0.05)., Conclusion: An up-regulated expression of HIF-1α causes down-regulation of PTEN expression to protect primary cultured rat neurons against OGD injury.

Activation of the type I angiotensin receptor (AT1-R) in vascular smooth muscle cells (VSMCs) plays a crucial role in the regulation of blood pressure; however, it is also responsible for the development of pathological conditions such as vascular remodeling, hypertension and atherosclerosis. Stimulation of the VSMC by angiotensin II (AngII) promotes a broad variety of biological effects, including gene expression changes. In this paper, we have taken an integrated approach in which an analysis of AngII-induced gene expression changes has been combined with the use of small-molecule inhibitors and lentiviral-based gene silencing, to characterize the mechanism of signal transduction in response to AngII stimulation in primary rat VSMCs. We carried out Affymetrix GeneChip experiments to analyze the effects of AngII stimulation on gene expression; several genes, including DUSP5 , DUSP6 , and DUSP10 , were identified as upregulated genes in response to stimulation. Since various dual-specificity MAPK phosphatase (DUSP) enzymes are important in the regulation of mitogen-activated protein kinase (MAPK) signaling pathways, these genes have been selected for further analysis. We investigated the kinetics of gene-expression changes and the possible signal transduction processes that lead to altered expression changes after AngII stimulation. Our data shows that the upregulated genes can be stimulated through multiple and synergistic signal transduction pathways. We have also found in our gene-silencing experiments that epidermal growth factor receptor (EGFR) transactivation is not critical in the AngII-induced expression changes of the investigated genes. Our data can help us understand the details of AngII-induced long-term effects and the pathophysiology of AT1-R. Moreover, it can help to develop potential interventions for those symptoms that are induced by the over-functioning of this receptor, such as vascular remodeling, cardiac hypertrophy or atherosclerosis.

As gene delivery tools, lentiviral vectors (LV) have broad applications in chimeric antigen receptor therapy (CAR-T). Large-scale production of functional LV is limited by the adherent, serum-dependent nature of HEK293T cells used in the manufacturing. HEK293T adherent cells were adapted to suspension cells in a serum-free medium to establish large-scale processes for functional LV production in a stirred bioreactor without micro-carriers. The results showed that 293 T suspension was successfully cultivated in F media (293 CD05 medium and SMM293-TII with 1:1 volume ratio), and the cells retained the capacity for LV production. After cultivation in a 5.5 L bioreactor for 4 days, the cells produced 1.5 ± 0.3 × 10 7 TU/mL raw LV, and the lentiviral transduction efficiency was 48.6 ± 2.8% in T Cells. The yield of LV equaled to the previous shake flask. The critical process steps were completed to enable a large-scale LV production process. Besides, a cryopreservation solution was developed to reduce protein involvement, avoid cell grafting and reduce process cost. The process is cost-effective and easy to scale up production, which is expected to be highly competitive.

New insights into the structure of the hybrid κ/β-carrageenan (κ/β-CRG) of the red alga Tichocarpus crinitus have been obtained. Carrageenan oligosaccharides were prepared through the chemical and enzymatic depolymerization of κ/β-CRG with κ-carrageenase and its the enzyme-resistant fraction. The composition and distribution of the repetition units of κ/β- CRG were investigated by using the negative ion tandem MALDI-TOFMS and ESIMS method, which made it possible to prove and characterize the hybrid structure of this polysaccharide. An analysis revealed the blockwise distribution of the long β-blocks along the polysaccharide chain, with the inclusion of κ/β, μ/ν-blocks and some ι-blocks. Furthermore, the desulfated κ/β-CRG was shown to contain of -G-D- repeating units up to 3.5 kDa. Previous studies have demonstrated that CRGs suppress the replication of several viruses. Here, we established that κ/β-CRG and its oligosaccharides significantly inhibit the transduction efficiency of replication-defective lentiviral particles pseudotyped with the envelope proteins of three different viruses. We found that the polysaccharide and its oligosaccharides strongly reduced the transduction efficiency of lentiviral particles pseudotyped with GP160-the envelope protein of the human immunodeficiency virus HIV-1-when added to T-lymphocyte Jurkat cells. The CRG oligosaccharides displayed significantly higher antiviral activity.

An alternative treatment to liver transplantation includes the use of differentiated stem cells. Hypoxia has been shown to endow human-induced pluripotent stem cells (hiPSCs) with enhanced hepatic differentiation. We have investigated a new strategy for hepatocyte differentiation from hiPSCs using a three-step differentiation protocol with lentiviral overexpression of hypoxia-microRNA-210 of cells grown on a hybrid scaffold. We analyzed the transduction of the miR-210 lentiviral and definitive endoderm and pluripotency gene markers, including SRY-box 17 (SOX17), forkhead box A2 (FOXA2), and octamer-binding transcription factor 4 (OCT-4) by Real-Time PCR and fluorescent microscope. The scanning electron microscopy (SEM) examined the 3D cell morphological changes. Immunocytochemistry staining was used together with assays for aspartate aminotransferase, alanine aminotransferase, and urea secretion to analyze hepatocyte biomarkers and functional markers consisting of α -fetoprotein (AFP), low-density lipoprotein (LDL) uptake, fat accumulation, and glycogen. The flow cytometry analyzed the generation of reactive oxygen species (ROS). Compared to cells transfected with the blank lentiviral vectors as a control, overexpressing miR-210 was at higher levels in hiPSCs. The expression of endodermal genes and glycogen synthesis significantly increased in the differentiated lentiviral miR-210 cells without any differences regarding lipid storage level. Additionally, cells containing miR-210 showed a greater expression of ALB, LDL, AST, ALT, urea, and insignificant lower AFP and ROS levels after 18 days. However, SEM showed no significant differences between cells under the differentiation process and controls. In conclusion, the differentiation of hiPSCs to hepatocyte-like cells under hypoxia miR-210 may be a suitable method for cell therapy and regenerative medicine., Competing Interests: The authors declare no conflict of interest., (Copyright © 2021 Naser Mobarra et al.)

Aims: Notch signaling is closely related to a variety of diseases, but the role of Notch2 in allergic rhinitis (AR) remain unclear. This study was performed to investigate the effects of Notch2 on the differentiation of Treg cells and on the inflammatory response of AR., Materials and Methods: Peripheral blood (including 101 AR patients and 66 Controls) and nasal mucosa (including 19 AR patients and 17 Controls) were collected to detect the expression levels of Notch2, NICD2 and FOXP3. CD4+ T cells of human origin were selected to detect the effects of Notch2 on the differentiation of Treg cells and FOXP3. An AR mouse model was established, and lentiviruses overexpressing Notch2 were administered. Then, allergic symptoms, OVA-sIgE titers, nasal mucosal inflammation, Th1/Th2/Th17 cytokines and splenic Treg cells were assessed., Key Findings: Compared with that in the Control group, the expression of Notch2 in the AR group was decreased, and Notch2 expression was negatively correlated with the degree of allergy (P < 0.01). The expression levels of Notch2, NICD2 and FOXP3 were decreased in the nasal mucosa of AR patients. Notch2 can promote the differentiation of human Treg cells in vitro (P < 0.05), and Notch2 can directly promote FOXP3 transcription. Animal experiments showed after the upregulation of Notch2 expression, the allergic inflammatory of mice with AR was reduced, the differentiation of Treg cells was increased, and the imbalance of T cells was reversed (P < 0.05)., Significance: Notch2 promotes the differentiation of Treg cells by upregulating FOXP3 expression, thus significantly inhibiting the inflammatory response of AR., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Novel therapies for the treatment of early steroid-induced osteonecrosis of the femoral head (SONFH) are urgently needed in orthopedics. Transplantation of bone marrow mesenchymal stem cells (BMSCs) provides new strategies for treating this condition at the early stage. However, stress-induced apoptosis of BMSCs transplanted into the femoral head necrotic area limits the efficacy of BMSC transplantation. Inhibiting BMSC apoptosis is key to improving the efficacy of this procedure. In our previous studies, we confirmed that Parkinson disease protein 7 (PARK7) is active in antioxidant defense and can clear reactive oxygen species (ROS), protect the mitochondria, and impart resistance to stress-induced apoptosis in BMSCs. In this study, we investigated the mechanism driving this PARK7-mediated resistance to apoptosis in BMSCs. Our results indicate that PARK7 promoted the disintegration of nuclear factor (erythroid-derived 2)-like 2 (Nrf2)/Kelch-like echinacoside-associated protein 1 (Keap1) complex. The free Nrf2 then entered the nucleus and activated the genetic expression of manganese superoxide dismutase (MnSOD), catalase (CAT), glutathione peroxidase (GPx), and other antioxidant enzymes that clear excessive ROS, thereby protecting BMSCs from stress-induced apoptosis. To further explore whether PARK7-mediated resistance to stress-induced apoptosis could improve the efficacy of BMSC transplantation in early-stage SONFH, we transplanted BMSCs-overexpressing PARK7 into rats with early-stage SONFH. We then evaluated the survival of transplanted BMSCs and bone regeneration in the femoral head necrotic area of these rats. The results indicated that PARK7 promoted the survival of BMSCs in the osteonecrotic area and improved the transplantation efficacy of BMSCs on early-stage SONFH. This study provides new ideas and methods for resisting the stress-induced apoptosis of BMSCs and improving the transplantation effect of BMSCs on early-stage SONFH., (© 2021. The Author(s).)

Although many studies have explored the mechanism of epilepsy, it remains unclear and deserves further investigation. Vezatin has been reported to be a synaptic regulatory protein involved in regulating neuronal synaptic transmission (NST). However, the role of vezatin in epilepsy remains unknown. Therefore, the aims of this study are to investigate the underlying roles of vezatin in epilepsy. In this study, vezatin expression was increased in hippocampal tissues from pilocarpine (PILO)-induced epileptic mice and a Mg 2+ -free medium-induced in vitro seizure-like model. Vezatin knockdown suppressed seizure activity in PILO-induced epileptic mice. Mechanistically, vezatin knockdown suppressed AMPAR-mediated synaptic events in epileptic mice and downregulated the surface expression of the AMPAR GluA1 subunit (GluA1). Interestingly, vezatin knockdown decreased the phosphorylation of GluA1 at serine 845 and reduced protein kinase A (PKA) phosphorylation; when PKA phosphorylation was suppressed by H-89 (a selective inhibitor of PKA phosphorylation) in vitro, the effects of vezatin knockdown on reducing the phosphorylation of GluA1 at serine 845 and the surface expression of GluA1 were blocked. Finally, we investigated the pattern of vezatin in brain tissues from patients with temporal lobe epilepsy (TLE), and we found that vezatin expression was also increased in patients with TLE. In summary, the vezatin expression pattern is abnormal in individuals with epilepsy, and vezatin regulates seizure activity by affecting AMPAR-mediated NST and the surface expression of GluA1, which is involved in PKA-mediated phosphorylation of GluA1 at serine 845, indicating that vezatin-mediated regulation of epileptic seizures represents a novel target for epilepsy., (© 2021. The Author(s).)

Background: Long noncoding RNAs (lncRNAs) play important roles in many physiological and pathological processes, this indicates that lncRNAs can serve as potential targets for gene therapy. Stable expression is a fundamental technology in the study of lncRNAs. The lentivirus is one of the most widely used delivery systems for stable expression. However, it was initially designed for mRNAs, and the applicability of lentiviral vectors for lncRNAs is largely unknown., Results: We found that the lentiviral vector produces lncRNAs with improper termination, appending an extra fragment of ~ 2 kb to the 3'-end. Consequently, the secondary structures were changed, the RNA-protein interactions were blocked, and the functions were impaired in certain lncRNAs, which indicated that lentiviral vectors are not ideal delivery systems of lncRNAs. Here, we developed a novel lncRNA delivery method called the Expression of LncRNAs with Endogenous Characteristics using the Transposon System (ELECTS). By inserting a termination signal after the lncRNA sequence, ELECTS produces transcripts without 3'-flanking sequences and retains the native features and function of lncRNAs, which cannot be achieved by lentiviral vectors. Moreover, ELECTS presents no potential risk of infection for the operators and it takes much less time. ELECTS provides a reliable, convenient, safe, and efficient delivery method for stable expression of lncRNAs., Conclusions: Our study demonstrated that improper transcriptional termination from lentiviral vectors have fundamental effects on molecular action and cellular function of lncRNAs. The ELECTS system developed in this study will provide a convenient and reliable method for the lncRNA study., (© 2021. The Author(s).)

Rheumatoid arthritis (RA) is an erosive polyarthritis that can lead to severe joint destruction and painful disability if left untreated. Angiogenesis, a critical pathogenic mechanism in RA, attracts inflammatory leukocytes into the synovium, which promotes production of proinflammatory cytokines and destructive proteases. Adipokines, inflammatory mediators secreted by adipose tissue, also contribute to the pathophysiology of RA. The most abundant serum adipokine is adiponectin, which demonstrates proinflammatory effects in RA, although the mechanisms linking adiponectin and angiogenic manifestations of RA are not well understood. Our investigations with the human MH7A synovial cell line have revealed that adiponectin dose- and time-dependently increases vascular endothelial growth factor (VEGF) expression, stimulating endothelial progenitor cell (EPC) tube formation and migration. These adiponectin-induced angiogenic activities were facilitated by MEK/ERK signaling. In vivo experiments confirmed adiponectin-induced downregulation of microRNA-106a-5p (miR-106a-5p). Inhibiting adiponectin reduced joint swelling, bone destruction, and angiogenic marker expression in collagen-induced arthritis (CIA) mice. Our evidence suggests that targeting adiponectin has therapeutic potential for patients with RA. Clinical investigations are needed.

Human Immunodeficiency Virus (HIV-1) produces a persistent latent infection. Control of HIV-1 using combination antiretroviral therapy (cART) comes at the cost of life-shortening side effects and development of drug-resistant HIV-1. An ideal and safer therapy should be deliverable in vivo and target the stable epigenetic repression of the virus, inducing a stable "block and lock" of virus expression. Towards this goal, we developed an HIV-1 promoter-targeting Zinc Finger Protein (ZFP-362) fused to active domains of DNA methyltransferase 3 A to induce long-term stable epigenetic repression of HIV-1. Cells were engineered to produce exosomes packaged with RNAs encoding this HIV-1 repressor protein. We find here that the repressor loaded anti-HIV-1 exosomes suppress virus expression and that this suppression is mechanistically driven by DNA methylation of HIV-1 in humanized NSG mouse models. The observations presented here pave the way for an exosome-mediated systemic delivery platform of therapeutic cargo to epigenetically repress HIV-1 infection., (© 2021. The Author(s).)

We investigated the influence of signal transducer and activator of transcription-3 (STAT3) on the spinal cord tissue grafts of rat fetuses with spina bifida aperta. In particular, we hoped to identify whether transfection of the STAT3 overexpression plasmid increases the survival of spinal cord transplantation in order to improve therapeutic efficacy. The fetal rat model of spina bifida aperta was established using retinoic acid and treated with a microsurgical injection of bone marrow mesenchymal stem cells (BMSCs). The animals were divided into either the blank control group, negative control group or the experimental group. The optical density (OD) value of BMSCs viability was determined using the Cell Counting Kit-8 (CCK-8). The expression of STAT3, phosphorylated STAT3 (pSTAT3), neural markers and apoptosis-related factors were evaluated using real-time PCR and Western blot. The OD value in the experimental group was highest at eight hours after transplantation using CCK-8. The expression of pSTAT3, glial fibrillary acidic protein, neuron-specific enolase, neurofilament and nestin in the experimental group was significantly higher compared to the blank control group and negative control group ( P <0.05). However, STAT3 expression in the experimental group was statistically significantly decreased ( P< 0.05). The relative expression of caspase-8 and bcl-2 in the experimental group were significantly lower compared to the blank control group and negative control group ( P <0.05). Transfection of the recombinant lentivirus-mediated STAT3 overexpression plasmid with BMSCs can help improve the efficiency of transforming into neural cells and provide new seed cells for the treatment of congenital spina bifida aperta.