Injection Of Lentiviral Vector Research Articles

BOP1 contributes to the activation of autophagy in polycystic ovary syndrome via nucleolar stress response

Abnormal autophagy is one of the vital features in polycystic ovary syndrome (PCOS). However, the underlying molecular mechanisms remain unelucidated. In this study, we aimed to investigate whether Block of Proliferation 1 (BOP1) is involved in the onset of autophagy activation of granulosa cells in PCOS. Firstly, we found that BOP1 expression was significantly down-regulated in the ovaries of PCOS mice, which was associated with the development of PCOS. Next, local injection of lentiviral vectors in the ovary for the overexpression of BOP1 significantly alleviated the phenotypes of elevated androgens, disturbed estrous cycle, and abnormal follicular development in PCOS mice. Subsequently, we found that knockdown of BOP1 activated autophagy of granulosa cells in the in vitro experiments, whereas overexpression of BOP1 inhibited autophagy in both in vivo and in vitro models. Mechanistically, BOP1 knockdown triggered the nucleolus stress response, which caused RPL11 to be released from the nucleolus into the nucleoplasm and inhibited the E3 ubiquitination ligase of MDM2, thereby enhancing the stability of p53. Subsequently, P53 inhibited mTOR, thereby activating autophagy in granulosa cells. In addition, the mRNA level of BOP1 was negatively correlated with antral follicle count (AFC), body–mass index (BMI), serum androgen levels, and anti-Mullerian hormone (AMH) in patients with PCOS. In summary, our study demonstrates that BOP1 downregulation inhibits mTOR phosphorylation through activation of the p53-dependent nucleolus stress response, which subsequently contributes to aberrant autophagy in granulosa cells, revealing that BOP1 may be a key target for probing the mechanisms of PCOS.

Improved intravenous lentiviral gene therapy based on endothelial-specific promoter-driven factor VIII expression for hemophilia A

BackgroundHemophilia A (HA) is an X-linked monogenic disorder caused by deficiency of the factor VIII (FVIII) gene in the intrinsic coagulation cascade. The current protein replacement therapy (PRT) of HA has many limitations including short term effectiveness, high cost, and life-time treatment requirement. Gene therapy has become a promising treatment for HA. Orthotopic functional FVIII biosynthesis is critical to its coagulation activities.MethodsTo investigate targeted FVIII expression, we developed a series of advanced lentiviral vectors (LVs) carrying either a universal promoter (EF1α) or a variety of tissue-specific promoters, including endothelial-specific (VEC), endothelial and epithelial-specific (KDR), and megakaryocyte-specific (Gp and ITGA) promoters.ResultsTo examine tissue specificity, the expression of a B-domain deleted human F8 (F8BDD) gene was tested in human endothelial and megakaryocytic cell lines. Functional assays demonstrated FVIII activities of LV-VEC-F8BDD and LV-ITGA-F8BDD in the therapeutic range in transduced endothelial and megakaryocytic cells, respectively. In F8 knockout mice (F8 KO mice, F8null mice), intravenous (iv) injection of LVs illustrated different degrees of phenotypic correction as well as anti-FVIII immune response for the different vectors. The iv delivery of LV-VEC-F8BDD and LV-Gp-F8BDD achieved 80% and 15% therapeutic FVIII activities over 180 days, respectively. Different from the other LV constructs, the LV-VEC-F8BDD displayed a low FVIII inhibitory response in the treated F8null mice.ConclusionsThe LV-VEC-F8BDD exhibited high LV packaging and delivery efficiencies, with endothelial specificity and low immunogenicity in the F8null mice, thus has a great potential for clinical applications.

Ataxin-2 in the hypothalamus at the crossroads between metabolism and clock genes.

ATXN2 gene, encoding for ataxin-2, is located in a trait locus for obesity. Atxn2 knockout (KO) mice are obese and insulin resistant; however, the cause for this phenotype is still unknown. Moreover, several findings suggest ataxin-2 as a metabolic regulator, but the role of this protein in the hypothalamus was never studied before. The aim of this work was to understand if ataxin-2 modulation in the hypothalamus could play a role in metabolic regulation. Ataxin-2 was overexpressed/re-established in the hypothalamus of C57Bl6/Atxn2 KO mice fed either a chow or a high-fat diet (HFD). This delivery was achieved through stereotaxic injection of lentiviral vectors encoding for ataxin-2. We show, for the first time, that HFD decreases ataxin-2 levels in mouse hypothalamus and liver. Specific hypothalamic ataxin-2 overexpression prevents HFD-induced obesity and insulin resistance. Ataxin-2 re-establishment in Atxn2 KO mice improved metabolic dysfunction without changing body weight. Furthermore, we observed altered clock gene expression in Atxn2 KO that might be causative of metabolic dysfunction. Interestingly, ataxin-2 hypothalamic re-establishment rescued these circadian alterations. Thus, ataxin-2 in the hypothalamus is a determinant for weight, insulin sensitivity and clock gene expression. Ataxin-2's potential role in the circadian clock, through the regulation of clock genes, might be a relevant mechanism to regulate metabolism. Overall, this work shows hypothalamic ataxin-2 as a new player in metabolism regulation, which might contribute to the development of new strategies for metabolic disorders.

Abstract PR003: Pioneering genetic rat models of Ductal Carcinoma in situ (DCIS)

Abstract DCIS has been virtually unknown before the introduction of breast cancer screening programs in the 1980s. Since then, its incidence has been rising steeply and it now accounts for about 25% of all newly diagnosed ‘breast cancer’ cases. Despite the increase in occurrence, the molecular underpinnings regulating DCIS initiation and progression remain elusive. In addition, almost all DCIS cases are hormone dependent, a characteristic which remains one of the biggest challenges to recapitulate in breast cancer research. We could successfully establish models for DCIS as well as estrogen receptor positive disease subtypes by somatically engineering the rat mammary gland via intraductal injection of lentiviral vectors encoding for the most commonly mutated genes (Ccnd1, Myc, Pik3ca and p53) in (pre-) breast cancer. Models with one perturbed gene remained mostly mammary tumor-free after an observation period of 18 months. However, when perturbed in two loci, animals showed mammary lesion formation within 5 and after up to 40 weeks. Histopathological analysis of these lesions characterized a broad spectrum of tumor types including ductal and lobular carcinomas, with both in situ and invasive phenotypes. Among these tumor types, DCIS lesions as well as invasive ductal carcinoma showed estrogen receptor positivity after immunohistochemical staining. Two distinct growth patterns were observed: a flat growing tumor, shaped by, and replacing the host rat mammary duct, and a round growing lesion, disregarding ductal structures. Interestingly, the flat growing tumor pattern evolved mostly in models expressing luminal drivers (Ccnd1 and Pik3ca) and strongly correlated with in situ growth. To increase luminal subtypes and to narrow down the broad tumor spectrum observed in these models, we generated a luminal cell type-specific promoter expressing our genes of interest. Pathological analysis of the arisen tumors indeed revealed a more homogeneous tumor subtype for all models, with an average tumor latency of 28 to 40 weeks. These genetically engineered models for DCIS and estrogen receptor positive breast cancer may aid in an improved treatment stratification by distinguishment between indolent and aggressive DCIS, a persisting challenge as of today. Additionally, these models broaden the scope of available hormone dependent breast cancer models, benefitting the search for novel treatment regiments for patients suffering from this breast cancer subtype. Citation Format: Catrin Lutz, Madelon Badoux, Timo Eijkman, Bim de Klein, Ji-Ying Song, Linda Henneman, Colinda Scheele, Jelle Wesseling, Jos Jonkers. Pioneering genetic rat models of Ductal Carcinoma in situ (DCIS) [abstract]. In: Proceedings of the AACR Special Conference on Rethinking DCIS: An Opportunity for Prevention?; 2022 Sep 8-11; Philadelphia, PA. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_1): Abstract nr PR003.

Improved Lentiviral In Vivo Gene Therapy for Chronic Granulomatous Disease

Background: Chronic granulomatous disease (CGD) is a congenital immunodeficiency characterized by severe life-threatening infections. CGD patients lack reactive oxygen species (ROS) in phagocytic leukocytes and often develop widespread tissue granulomas. The current treatment for CGD is lifelong antibiotic prophylaxis or hematopoietic stem cell transplantation (HSCT). However, most patients cannot find matched donors, and HSCT is known to have increased risk in CGD patients. Gene therapy has become a promising treatment for CGD. At present, "ex vivo" HSC gene therapy is the widely applied strategy. "In vivo" gene therapy is based on direct delivery of gene therapy vectors into patients, which could overcome the difficulties in HSCT. Methods: We have developed an in vivo CGD gene therapy strategy by intravenous (iv) injection of lentiviral vectors (LVs) carrying an universal EF1a promoter or a myeloid-specific miR223 promoter driving the expression of CYBB (cytochrome B-245 beta chain, or p91-phox) in X-CGD mice (X-CGD; B6.129S-Cybbtm1Din/J). Each mouse received 1x109 transduction units (TU) of LV-CYBB via iv injection after non-myeloablative conditioning. A second injection of 1x109 TU of LV-CYBB after re-conditioning was given 12 weeks later. Results: We developed a chemotherapy conditioning regimen to improve gene delivery efficiency by using busulfan, cyclophosphamide and dexamethasone. The X-CGD mice received two iv injections of LVs, and the expression and function of the LV-CYBB gene were analyzed by intracellular gp91-phox staining and DHR123 assay 4 weeks after each injection. Based on flow cytometry, we detected gp91-phox positive cells in the blood as the following, EF1a: 1.14-5.18% and 5.27-14.36%, and miR223: 1.65-5.17% and 4.59-17.89% after the first and the second injections, respectively, as compared with the WT mice: 79.9-87.6%. Functional analyses of ROS showed the following, EF1a: 1.93-5.2% and 2.39-18.51%, miR223: 2.65-6.67% and 3.75-19.8%, after the first and the second injections respectively, as compared with WT: 72.6-85.31% (Figure A). The LV vector copy number (VCN) in the peripheral blood mononuclear cells was detected by qPCR as the following, EF1a: 1.11-2.32% and 1.35-9.32%, and miR223: 1.01-2.39% and 2.71-8.51% after the first and the second injections, respectively. These results indicated that iv LV injection effectively delivered and expressed the LV-CYBB gene in vivo, and repeated injection of the LVs could increase the gene transfer efficiency in the CGD mice. Importantly, after the second injection, we found no antibody response to the LV particle-associated protein p24. However, after 3-12 months, markedly reduced VCN was found in the blood of the treated mice, suggesting exhaustion of the transgenes in the somatic cells. To improve the gene delivery into HSCs, we treated the mice with AMD3100 to mobilize HSCs into peripheral blood. The mice were treated with chemo-conditioning and AMD3100 prior to LV ivinjection. After four weeks, the gp91-phox positive HSCs of total Sca1+ HSCs in blood with or without (+/-) AMD3100 treatment were examined by flow cytometry, and the results showed the following, EF1a: (-) 9.52-15.43%, (+) 16.1-29.4%, and miR223: (-) 4.49-10.35% and (+) 7.06-16.84%. To confirm stem cell gene transfer, we transplanted the bone marrow cells from the LV-CYBB-treated CGD mice into untreated CGD mice. The VCN in blood was examined before (donor mice) and 4 weeks after (recipient mice) the HSCT, and the results showed the following, EF1a: before 4.49% (in the donor) and after 1.05-2.57% (in the recipients), and miR223: before 4.91% (in the donor) and after 1.04-2.69% (in the recipients) (Figure B). This transplantation study confirmed that the AMD3100 treatment effectively increased iv LV gene delivery into HSCs. Conclusion: We have developed an improved LV iv gene delivery strategy by applying chemo-conditioning and AMD3100 mobilization prior to the injection of the LVs into CGD mice. This in vivo LV gene therapy strategy could potentially be translated into future CGD gene therapy applications. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Intravenous Lentiviral Gene Therapy for Hemophilia a: Endothelial-Specific Expression and Increased Glycosylation for Improved FVIII Function

Background: Hemophilia A (HA) is an X-linked monogenic coagulation disorder in the intrinsic coagulation cascade resulting from deficiency of the factor VIII (FVIII, F8) gene. The current treatment of HA is based on protein replacement therapy (PRT) with limitations of short half-life, high cost, and life-time requirement of the treatment. Gene therapy has become a promising treatment option for HA. Methods: An advanced lentiviral vector (LV) system for intravenous (iv) F8 gene therapy has been established based on a novel B domain deleted (BDD) F8 gene, which restored all N-glycosylation sites (F8-299) in the B domain with increased coagulation function (DOI:10.1016/j.jbc.2021.101397). LV-F8-299 containing a universal (EF1a) or endothelial-specific (VEC) promoter was investigated in vitro using a human endothelial cell line, EA-hy926, and biologically and immunologically characterized in vivo in F8 knockout (KO) mice and in a non-human primate. Results: We found that LV-VEC-F8-299 (VEC-299) exhibited higher virus packaging (100 folds) and transduction efficiencies than EF1a-299. The F8 protein expression was verified in EA-hy926 by RT-PCR, Western blot and ELISA. We detected F8 activities close to 4 folds above the normal plasma level from the EF1a-299 transduced cells, whereas the VEC-299 expressed only 0.1 fold of the normal plasma F8 level. In vivo studies in F8 KO mice via iv injection of LVs after non-myeloablative radiation conditioning illustrated variable phenotypic corrections as well as anti-F8 immune responses from the two LVs. The iv gene therapy of EF1a-299 and VEC-299 LVs illustrated therapeutic F8 activities over time, in the range of 90% and 40%, respectively, in 60 days. Importantly, VEC-299 steadily reached a very high level (180%) of F8 activities, whereas EF-299 dropped to 3% after 120 days (Figure A). Kinetic analyses of anti-F8 IgG and inhibitor titer showed that the VEC-299 treated mice exhibited the lowest F8 inhibitory immune response over time. We next assessed the distribution of LV F8 gene in a macaque monkey (Macaca fascicularis). The monkey was chemo-conditioned with methylprednisolone (10 mg/kg) and prednisone (2 mg/kg) and iv injected with 1x109 TU/kg LV-VEC-299. The vector copy number (VCN) in body fluids was closely monitored by qPCR. From day 1 to week 14 after injection, the VCN in the blood remained stable (~1%), while in feces and saliva was high on day 1 (~3%), and in urine reached peak (~5%) on day 7, but all of which decreased with time. The VCN in body fluids (blood, urine, faces, saliva and tear) became nearly undetectable after 14 weeks. A second LV injection of 1x109TU/kg LV-VEC-299 was performed using a modified non-myeloablative conditioning with busulfan (2 mg/kg), cyclophosphamide (40 mg/kg) and dexamethasone (3 mg/kg). The VCN in the blood reached 20% from day 1 to day 7, then reduced to 5% and remained stable to week 6 (Figure B). The blood biochemical analyses including Alanine Transaminase (ALT), Aspartate Transaminase (AST), Total Protein (TP) and Albumin (ALB) remained in the normal range before and after treatment. Conclusion: These results demonstrated that the LV-VEC-299 exhibited delivery consistency, high coagulation function and low immunogenicity. The in vivo study supported that iv LV-VEC-299 gene therapy could be a safe, convenient and effective HA gene therapy strategy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Directly injected lentiviral vector-based T cell vaccine protects mice against acute and chronic viral infection.

Lentiviral vector-based dendritic cell vaccines induce protective T cell responses against viral infection and cancer in animal models. In this study, we tested whether preventative and therapeutic vaccination could be achieved by direct injection of antigen-expressing lentiviral vector, obviating the need for ex vivo transduction of dendritic cells. Injected lentiviral vector preferentially transduced splenic dendritic cells and resulted in long-term expression. Injection of a lentiviral vector encoding an MHC class I-restricted T cell epitope of lymphocytic choriomeningitis virus (LCMV) and CD40 ligand induced an antigen-specific cytolytic CD8+ T lymphocyte response that protected the mice from infection. The injection of chronically infected mice with a lentiviral vector encoding LCMV MHC class I and II T cell epitopes and a soluble programmed cell death 1 microbody rapidly cleared the virus. Vaccination by direct injection of lentiviral vector was more effective in sterile alpha motif and HD-domain containing protein 1-knockout (SAMHD1-knockout) mice, suggesting that lentiviral vectors containing Vpx, a lentiviral protein that increases the efficiency of dendritic cell transduction by inducing the degradation of SAMHD1, would be an effective strategy for the treatment of chronic disease in humans.

HIV-1 Tat drives the Fabp4/NF-κB feedback loop in microglia to mediate inflammatory response and neuronal apoptosis.

Fatty acid-binding proteins (FABPs) are relevant to multiple neurodegenerative diseases. However, the roles and mechanisms of FABPs in HIV-associated neurocognitive disorder (HAND) remain yet unclear. In this study, cultured BV-2 microglial cells and HT-22 neuronal cells were used for in vitro experiments and HAND mouse models were constructed through intracerebroventricular injection of lentiviral vectors for in vivo experiments. FABP expression was determined using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot. The interrelationship between Fabp4 and NF-κB signaling was investigated using chromatin immunoprecipitation, qRT-PCR, and Western blot. The role of Fabp4 in regulating inflammatory response was determined using qRT-PCR, enzyme-linked immunosorbent assay, Western blot, and immunofluorescence staining. Cell viability and apoptosis were analyzed using cell counting kit-8 assay and flow cytometry assay, respectively. Our results suggested an upregulation of Fabp4 expression in the presence of Tat. Tat-induced Fabp4 expression was directly regulated by NF-κB p65, followed by, Fabp4 facilitating Tat-activated NF-κB signaling pathway. We also observed that Fabp4 knockdown in microglial cells significantly suppressed inflammatory response and neuronal apoptosis both in vitro and in vivo. In conclusion, the presence of Tat in microglial cells results in Fabp4 and NF-κB to form a positive feedback loop leading to exacerbate inflammatory response and neuronal apoptosis.

PLK1 promotes cholesterol efflux and alleviates atherosclerosis by up-regulating ABCA1 and ABCG1 expression via the AMPK/PPARγ/LXRα pathway

Polo-like kinase 1 (PLK1) is a serine/threonine kinase involving lipid metabolism and cardiovascular disease. However, its role in atherogenesis has yet to be determined. The aim of this study was to observe the impact of PLK1 on macrophage lipid accumulation and atherosclerosis development and to explore the underlying mechanisms. We found a significant reduction of PLK1 expression in lipid-loaded macrophages and atherosclerosis model mice. Lentivirus-mediated overexpression of PLK1 promoted cholesterol efflux and inhibited lipid accumulation in THP-1 macrophage-derived foam cells. Mechanistic analysis revealed that PLK1 stimulated the phosphorylation of AMP-activated protein kinase (AMPK), leading to activation of the peroxisome proliferator-activated receptor γ (PPARγ)/liver X receptor α (LXRα) pathway and up-regulation of ATP binding cassette transporter A1 (ABCA1) and ABCG1 expression. Injection of lentiviral vector expressing PLK1 increased reverse cholesterol transport, improved plasma lipid profiles and decreased atherosclerotic lesion area in apoE-deficient mice fed a Western diet. PLK1 overexpression also facilitated AMPK and HSL phosphorylation and enhanced the expression of PPARγ, LXRα, ABCA1, ABCG1 and LPL in the aorta. In summary, these data suggest that PLK1 inhibits macrophage lipid accumulation and mitigates atherosclerosis by promoting ABCA1- and ABCG1-dependent cholesterol efflux via the AMPK/PPARγ/LXRα pathway.

Asprosin inhibits macrophage lipid accumulation and reduces atherosclerotic burden by up-regulating ABCA1 and ABCG1 expression via the p38/Elk-1 pathway

BackgroundAsprosin, a newly discovered adipokine, is a C-terminal cleavage product of profibrillin. Asprosin has been reported to participate in lipid metabolism and cardiovascular disease, but its role in atherogenesis remains elusive.MethodsAsprosin was overexpressed in THP-1 macrophage-derived foam cells and apoE−/− mice using the lentiviral vector. The expression of relevant molecules was determined by qRT-PCR and/or western blot. The intracellular lipid accumulation was evaluated by high-performance liquid chromatography and Oil red O staining. HE and Oil red O staining was employed to assess plaque burden in vivo. Reverse cholesterol transport (RCT) efficiency was measured using [3H]-labeled cholesterol.ResultsExposure of THP-1 macrophages to oxidized low-density lipoprotein down-regulated asprosin expression. Lentivirus-mediated overexpression of asprosin promoted cholesterol efflux and inhibited lipid accumulation in THP-1 macrophage-derived foam cells. Mechanistic analysis revealed that asprosin overexpression activated p38 and stimulated the phosphorylation of ETS-like transcription factor (Elk-1) at Ser383, leading to Elk-1 nuclear translocation and the transcriptional activation of ATP binding cassette transporters A1 (ABCA1) and ABCG1. Injection of lentiviral vector expressing asprosin diminished atherosclerotic lesion area, increased plaque stability, improved plasma lipid profiles and facilitated RCT in apoE−/− mice. Asprosin overexpression also increased the phosphorylation of p38 and Elk-1 as well as up-regulated the expression of ABCA1 and ABCG1 in the aortas.ConclusionAsprosin inhibits lipid accumulation in macrophages and decreases atherosclerotic burden in apoE−/− mice by up-regulating ABCA1 and ABCG1 expression via activation of the p38/Elk-1 signaling pathway.

Editing T cell repertoire by thymic epithelial cell-directed gene transfer abrogates risk of type 1 diabetes development

Insulin is the primary autoantigen (Ag) targeted by T cells in type 1 diabetes (T1D). Although biomarkers precisely identifying subjects at high risk of T1D are available, successful prophylaxis is still an unmet need. Leaky central tolerance to insulin may be partially ascribed to the instability of the MHC-InsB9-23 complex, which lowers TCR avidity, thus resulting in defective negative selection of autoreactive clones and inadequate insulin-specific T regulatory cell (Treg) induction. We developed a lentiviral vector (LV)-based strategy to engineer thymic epithelial cells (TECs) to correct diabetogenic T cell repertoire. Intrathymic (it) LV injection established stable transgene expression in EpCAM+ TECs, by virtue of transduction of TEC precursors. it-LV-driven presentation of the immunodominant portion of ovalbumin allowed persistent and complete negative selection of responsive T cells in OT-II chimeric mice. We successfully applied this strategy to correct the diabetogenic repertoire of young non-obese diabetic mice, imposing the presentation by TECs of the stronger agonist InsulinB9-23R22E and partially depleting the existing T cell compartment. We further circumscribed LV-driven presentation of InsulinB9-23R22E by micro-RNA regulation to CD45− TECs without loss of efficacy in protection from diabetes, associated with expanded insulin-specific Tregs. Overall, our gene transfer-based prophylaxis fine-tuned the central tolerance processes of negative selection and Treg induction, correcting an autoimmune prone T cell repertoire.

Lentiviral-Induced Spinal Cord Gliomas in Rat Model.

Intramedullary spinal cord tumors are a rare and understudied cancer with poor treatment options and prognosis. Our prior study used a combination of PDGF-B, HRAS, and p53 knockdown to induce the development of high-grade glioma in the spinal cords of minipigs. In this study, we evaluate the ability of each vector alone and combinations of vectors to produce high-grade spinal cord gliomas. Eight groups of rats (n = 8/group) underwent thoracolumbar laminectomy and injection of lentiviral vector in the lateral white matter of the spinal cord. Each group received a different combination of lentiviral vectors expressing PDGF-B, a constitutively active HRAS mutant, or shRNA targeting p53, or a control vector. All animals were monitored once per week for clinical deficits for 98 days. Tissues were harvested and analyzed using hematoxylin and eosin (H&E) and immunohistochemical (IHC) staining. Rats injected with PDGF-B+HRAS+sh-p53 (triple cocktail) exhibited statistically significant declines in all behavioral measures (Basso Beattie Bresnahan scoring, Tarlov scoring, weight, and survival rate) over time when compared to the control. Histologically, all groups except the control and those injected with sh-p53 displayed the development of tumors at the injection site, although there were differences in the rate of tumor growth and the histopathological features of the lesions between groups. Examination of immunohistochemistry revealed rats receiving triple cocktail displayed the largest and most significant increase in the Ki67 proliferation index and GFAP positivity than any other group. PDGF-B+HRAS also displayed a significant increase in the Ki67 proliferation index. Rats receiving PDGF-B alone and PDGF-B+ sh-p53 displayed more a significant increase in SOX2-positive staining than in any other group. We found that different vector combinations produced differing high-grade glioma models in rodents. The combination of all three vectors produced a model of high-grade glioma more efficiently and aggressively with respect to behavioral, physiological, and histological characteristics than the rest of the vector combinations. Thus, the present rat model of spinal cord glioma may potentially be used to evaluate therapeutic strategies in the future.

Intravenous Lentiviral Gene Therapy for Hemophilia a: In Vitro and In Vivo Assessment of Universal Versus Tissue-Specific Promoters

Background：Hemophilia A (HA) is an X-linked monogenic coagulation disorder resulting from deficiency of the factor VIII (FVIII, F8) gene in the intrinsic coagulation cascade. The current treatment of HA is based on protein replacement therapy (PRT) through plasma-derived coagulation factors or recombinant proteins with limitations of short half-life, high cost, and life-time requirement of the treatment. Gene therapy has become a promising treatment for HA.Methods and Materials: We developed an advanced lentiviral vector (LV) system for intravenous (iv) F8 gene therapy. A selective codon optimized and B-domain deleted human F8 (hF8BDD) gene was synthesized, sequenced and functionally verified. LVs carrying a universal EF1α promoter, or several modified tissue-specific promoters including endothelial- (VEC), endothelial and epithelial- (KDR), and two megakaryocyte-specific (ITGA and Gp) promoters, were biologically and immunologically characterized in vitro using human endothelial and megakaryocytic cell lines, EA-hy926 and DAMI, and in vivo using F8 knockout (KO) mice.Results: We investigated the different LV promoter constructs and found that LV-VEC-F8BDD exhibited the highest virus packaging (1.3x10 9 transduction units/ml) and transduction efficiencies compared with the other LV constructs. Tissue-specific expression of the VEC, KDR, ITGA and Gp promoters was confirmed in EA-hy926 and DAMI cells by RT-PCR, Western blot and ELISA analyses. We detected F8 activities close to 6 folds and 4.5 folds above the normal plasma level from the EF1a LV-F8BDD transduced EA-hy926 cells and DAMI cells, respectively, whereas the VEC LV in EA-hy926 cells and the ITGA-LV in DAMI cells exhibited F8 activities at 1.5 folds and 5 folds above the normal plasma level, respectively. In vivo studies in F8 KO mice via iv injection of LVs after reduced radiation conditioning illustrated preferential vector expression in different cell lineages, with high expression of EF1a vector in CD11b, F4/80 and Ly-6G positive immune cells, and preferential expression of VEC vector in CD31 positive endothelial cells, and ITGA and Gp vectors in CD41 positive megakaryocytes. In addition, we detected variable phenotypic corrections as well as anti-F8 immune responses in the F8 KO mice treated with the different LVs. The iv deliveries of VEC and Gp F8BDD vectors illustrated therapeutic F8 activities over time, around 25% and 8%, respectively, in 60 days, which increased to high levels (80% and 25%, respectively) after 120 days (Figure A). Kinetic analyses of anti-F8 IgG and inhibitor titers (Bethesda assay) of the treated mice showed that the VEC vector exhibited the lowest F8 inhibitory immune response over time (Figure B).Conclusion: Based on the in vitro and in vivo studies, our results suggest that for HA gene therapy, optimal rather than high F8 expression is critical, and tissue-specific expression but not universal expression can reduce adverse inhibitor effect. We demonstrated that the LV-VEC-F8BDD vector displayed high tissue specificity in vivo, and high transgene delivery efficiency, high coagulation function and low immunogenicity. In addition, iv LV gene therapy could be a safe, convenient and effective HA gene therapy strategy. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

CISD1 protects against atherosclerosis by suppressing lipid accumulation and inflammation via mediating Drp1

Atherosclerosis still remains the leading cause of morbidity and mortality worldwide, and deeper understanding of target signaling that protect from the atherosclerosis progression may provide novel therapeutic strategies. CDGSH iron-sulfur domain-containing protein 1 (CISD1) is a protein localized on the outer membrane of mitochondria, and plays key roles in regulating cell death and oxidative stress. However, its potential on atherosclerosis development and the underlying mechanisms are largely unknown. Here, in our study, we found markedly decreased CISD1 expression in lipid-laden THP1 macrophages. Notably, lentivirus (LV)-mediated CISD1 over-expression remarkably ameliorated lipid deposition in macrophages stimulated by ox-LDL. Furthermore, cellular total ROS and mitochondrial ROS generation, and impairment of mitochondrial membrane potential (MMP) were highly induced by ox-LDL in THP1 cells, while being considerably reversed upon CISD1 over-expression. Inflammatory response caused by ox-LDL was also significantly restrained in macrophages with CISD1 over-expression. Mechanistically, we found that CISD1 could interact with dynamin-related protein 1 (Drp1). Intriguingly, CISD1-improved mitochondrial dysfunction and inflammation in ox-LDL-treated macrophages were strongly abolished by Drp1 over-expression, indicating that Drp1 suppression might be necessary for CISD1 to perform its protective effects in vitro. In high fat diet (HFD)-fed apolipoprotein E-deficient (ApoE−/−) mice, tail vein injection of lentiviral vector expressing CISD1 remarkably decreased atherosclerotic lesion area, serum LDL cholesterol levels and triglyceride contents. Inflammatory response, cellular total and mitochondrial ROS production, and Drp1 expression levels in aorta tissues were also dramatically ameliorated in HFD-fed ApoE−/− mice, contributing to the inhibition of atherosclerosis in vivo. Therefore, improving CISD1 expression may be a novel therapeutic strategy for atherosclerosis treatment.

Identification of the transcription factor, AFF4, as a new target of miR-203 in CNS

MiR-203 was identified as a hub of a potential regulatory miRNA network in central nervous system. Overexpressing of miR-203 in the frontal cortex of C57BL/6J wild type mouse induced neurodegeneration by increasing the apoptotic pathway and neuron death. AFF4, a transcription factor, was identified as a new bona fida protein target of miR-203 in CNS. The miRNA:mRNA interaction of miR-203 and AFF4 was verified using Dural-luciferase assay. Down-regulated expression of AFF4 was induced by overexpressing miR-203 both in vitro and in vivo. Open field test, Y maze and Morris water maze test were conducted for the behavioral assessment of the mice with stereotactic injection of lentiviral vector overexpressing miR-203 in the hippocampus. No anxiety-like behavior or impaired cognition was noticed in these mice. Consistent with the results of the behavioral assessment, the electron micrograph and Nissl staining revealed no significant change in the synaptic density and no neuron injuries in the hippocampus of mice overexpressing miR-203, respectively. Our results indicated that instead of promoting neurodegenerative phenotype, a more profound function should be ascribed to miR-203 in regulating neuron behavioral activities and cognition. Neuron-type specific functions of miR-203 are likely to be executed via its various downstream protein interactors.

CTRP12 ameliorates atherosclerosis by promoting cholesterol efflux and inhibiting inflammatory response via the miR-155-5p/LXR\u03b1 pathway

C1q tumor necrosis factor-related protein 12 (CTRP12), a conserved paralog of adiponectin, is closely associated with cardiovascular disease. However, little is known about its role in atherogenesis. The aim of this study was to examine the influence of CTRP12 on atherosclerosis and explore the underlying mechanisms. Our results showed that lentivirus-mediated CTRP12 overexpression inhibited lipid accumulation and inflammatory response in lipid-laden macrophages. Mechanistically, CTRP12 decreased miR-155-5p levels and then increased its target gene liver X receptor α (LXRα) expression, which increased ATP binding cassette transporter A1 (ABCA1)- and ABCG1-dependent cholesterol efflux and promoted macrophage polarization to the M2 phenotype. Injection of lentiviral vector expressing CTRP12 decreased atherosclerotic lesion area, elevated plasma high-density lipoprotein cholesterol levels, promoted reverse cholesterol transport (RCT), and alleviated inflammatory response in apolipoprotein E-deficient (apoE−/−) mice fed a Western diet. Similar to the findings of in vitro experiments, CTRP12 overexpression diminished miR-155-5p levels but increased LXRα, ABCA1, and ABCG1 expression in the aortas of apoE−/− mice. Taken together, these results suggest that CTRP12 protects against atherosclerosis by enhancing RCT efficiency and mitigating vascular inflammation via the miR-155-5p/LXRα pathway. Stimulating CTRP12 production could be a novel approach for reducing atherosclerosis.

Neuroinflammation in the normal-appearing white matter (NAWM) of the multiple sclerosis brain causes abnormalities at the nodes of Ranvier.

Changes to the structure of nodes of Ranvier in the normal-appearing white matter (NAWM) of multiple sclerosis (MS) brains are associated with chronic inflammation. We show that the paranodal domains in MS NAWM are longer on average than control, with Kv1.2 channels dislocated into the paranode. These pathological features are reproduced in a model of chronic meningeal inflammation generated by the injection of lentiviral vectors for the lymphotoxin-α (LTα) and interferon-γ (IFNγ) genes. We show that tumour necrosis factor (TNF), IFNγ, and glutamate can provoke paranodal elongation in cerebellar slice cultures, which could be reversed by an N-methyl-D-aspartate (NMDA) receptor blocker. When these changes were inserted into a computational model to simulate axonal conduction, a rapid decrease in velocity was observed, reaching conduction failure in small diameter axons. We suggest that glial cells activated by pro-inflammatory cytokines can produce high levels of glutamate, which triggers paranodal pathology, contributing to axonal damage and conduction deficits.

Dexmedetomidine inhibits inflammatory response and autophagy through the circLrp1b/miR-27a-3p/Dram2 pathway in a rat model of traumatic brain injury.

Circular RNAs (circRNAs) have a regulatory function on inflammation and autophagy, of which rno-circRNA_010705 (circLrp1b) appears to be significantly up-regulated following traumatic brain injury (TBI). Dexmedetomidine (DEX) shows improvement effects in TBI by inhibiting NLRP3/caspase-1. However, whether circLrp1b plays critical roles in DEX-mediated TBI attenuation and the underlying mechanisms remain unclear. After TBI was established in rats by controlled cortical impact (CCI) to cause brain trauma, they received an intracerebroventricular injection of lentiviral vector, followed by intraperitoneal injection of DEX. Administration of DEX ameliorated autophagy in rats following TBI, accompanied by up-regulated circLrp1b and Dram2 and down-regulated miR-27a-3p. DEX promoted the effects of circLrp1b in attenuating TBI-induced neurologic impairment, autophagy, and inflammation, which was significantly reversed by inhibition of miR-27a-3p or Dram2 overexpression. Mechanistically, northern blot and luciferase reporter assays indicated that circLrp1b up-regulated Dram2 expression by functioning as a sponge for miR-27a-3p to promote autophagy involved in TBI, which was reversed by DEX treatment. Collectively, this study demonstrated that DEX inhibits inflammatory response and autophagy involved in TBI in vivo through inactivation of the circLrp1b/miR-27a-3p/Dram2 signaling pathway.

Mzb1 protects against myocardial infarction injury in mice via modulating mitochondrial function and alleviating inflammation.

Myocardial infarction (MI) leads to the loss of cardiomyocytes, left ventricle dilation and cardiac dysfunction, eventually developing into heart failure. Mzb1 (Marginal zone B and B1 cell specific protein 1) is a B-cell-specific and endoplasmic reticulum-localized protein. Mzb1 is an inflammation-associated factor that participates a series of inflammatory processes, including chronic periodontitis and several cancers. In this study we investigated the role of Mzb1 in experimental models of MI. MI was induced in mice by ligation of the left descending anterior coronary artery, and in neonatal mouse ventricular cardiomyocytes (NMVCs) by H2O2 treatment in vitro. We showed that Mzb1 expression was markedly reduced in the border zone of the infarct myocardium of MI mice and in H2O2-treated NMVCs. In H2O2-treated cardiomyocytes, knockdown of Mzb1 decreased mitochondrial membrane potential, impaired mitochondrial function and promoted apoptosis. On contrary, overexpression of Mzb1 improved mitochondrial membrane potential, ATP levels and mitochondrial oxygen consumption rate (OCR), and inhibited apoptosis. Direct injection of lentiviral vector carrying Len-Mzb1 into the myocardial tissue significantly improved cardiac function and alleviated apoptosis in MI mice. We showed that Mzb1 overexpression significantly decreased the levels of Bax/Bcl-2 and cytochrome c and improved mitochondrial function in MI mice via activating the AMPK-PGC1α pathway. In addition, we demonstrated that Mzb1 recruited the macrophages and alleviated inflammation in MI mice. We conclude that Mzb1 is a crucial regulator of cardiomyocytes after MI by improving mitochondrial function and reducinginflammatory signaling pathways, implying a promising therapeutic target in ischemic cardiomyopathy.

In vivo delivery of MMP3-shRNA and Sox9 lentivirus cocktail enhances matrix synthesis to prevent lumbar disc degeneration.

Intervertebral disc degeneration (IDD) is characterized by increased proteolytic degradation of the extracellular matrix (ECM), leading to a loss of collagen II and proteoglycan in the nucleus pulposus (NP). Although MMP3 has been reported to play a central role in disc degeneration, it is still unknown whether gene therapy targeting MMP3 can inhibit IDD. To investigate whether lentivirus-mediated MMP3 knockdown is capable of attenuating IDD. More importantly, we also explored whether combined gene therapy that simultaneously antagonizes MMP3 and overexpresses Sox9 can synergistically inhibit IDD and induce augmented matrix reconstitution in the degenerative NP. We performed direct injection of lentiviral vectors LV-MMP3-shRNA and/or LV-Sox9 into rabbit lumbar discs. The animals were scanned using magnetic resonance imaging (MRI) at 8, 12 and 24 weeks after the operation. We also evaluated the gene expression and synthesis of NP matrix components, including collagen II, aggrecan and proteoglycan. The MRI scans showed remarkable needle-puncture-induced progressive IDD in animals injected with PBS or 10^7 viral particles (VP) of the control virus. In contrast, injection of 10^7 VP of LV-MMP3-shRNA or LV-Sox9 substantially inhibited IDD. MMP3 knockdown or Sox9 overexpression stimulated collagen II and aggrecan expression, as well as proteoglycan synthesis. Notably, the injection of a cocktail of LV-MMP3-shRNA and LV-Sox9 (5 × 10^6 VP each) greatly delayed the development of IDD and induced the highest levels of collagen II and proteoglycan production, indicating a synergistic effect in ECM induction. Our results suggest that gene therapy targeting MMP3 is an efficient way to delay IDD. Combined gene therapy possesses a stronger capacity to induce matrix components in degenerative NP tissue than single-gene delivery.