Enhanced gene expression from retroviral vectors

<p>Abstract</p> <p>Background</p> <p>Retroviruses are widely used to transfer genes to mammalian cells efficiently and stably. However, genetic elements required for high-level gene expression are incompatible with standard systems. The retroviral RNA genome is produced by cellular transcription and post-transcriptional processing within packaging cells: Introns present in the retroviral genomic transcript are removed by splicing, while polyadenylation signals lead to the production of ineffective truncated genomes. Furthermore strong enhancer/promoters within the retroviral payload lead to detrimental competition with the retroviral enhancer/promoter.</p> <p>Results</p> <p>By exploiting a new method of producing the retroviral genome <it>in vitro </it>it is possible to produce infectious retroviral particles carrying a high-level expression cassette that completely prohibits production of infectious retroviral particles by conventional methods.</p> <p>We produced an expression cassette comprising a strong enhancer/promoter, an optimised intron, the GFP open reading frame and a strong polyadenylation signal. This cassette was cloned into both a conventional MMLV retroviral vector and a vector designed to allow <it>in vitro </it>transcription of the retroviral genome by T7 RNA polymerase.</p> <p>When the conventional retroviral vector was transfected into packaging cells, the expression cassette drove strong GFP expression, but no infectious retrovirus was produced. Introduction of the <it>in vitro </it>produced uncapped retroviral genomic transcript into the packaging cells did not lead to any detectable GFP expression. However, infectious retrovirus was easily recovered, and when used to infect target primary human cells led to very high GFP expression – up to 3.5 times greater than conventional retroviral LTR-driven expression.</p> <p>Conclusion</p> <p>Retroviral vectors carrying an optimized high-level expression cassette do not produce infectious virions when introduced into packaging cells by transfection of DNA. Infectious retrovirus carrying the same cassette is readily produced when packaging cells are transfected with <it>in vitro </it>transcribed retroviral genomic RNA. The applications of this technique are not limited to producing the higher levels of transgene expression demonstrated here. For example, novel reporters with alternatively spliced exon-intron configurations could readily be transduced into virtually any cell. Furthermore, because the <it>in vitro </it>transcripts are not translated within the packaging cells, retroviruses carrying genes lethal to the packaging cells can also be produced.</p

Osteogenic stimulatory conditions enhance growth and maturation of endothelial cell microvascular networks in culture with mesenchymal stem cells

To optimize culture conditions for in vitro prevascularization of tissue-engineered bone constructs, the development of organotypic blood vessels under osteogenic stimulatory conditions (OM) was investigated. Coculture of endothelial cells and mesenchymal stem cells was used to assess proangiogenic effects of mesenchymal stem cells on endothelial cells. Four different culture conditions were evaluated for their effect on development of microvascular endothelial cell networks. Mineralization, deposition of extracellular matrix, and perivascular gene expression were studied in OM. After 3 days, endothelial cells established elongated capillary-like networks, and upregulated expression of vascular markers was seen. After 15 days, all parameters evaluated were significantly increased for cultures in OM. Mature networks developed in OM presented lumens enveloped by basement membrane-like collagen IV, with obvious mineralization and upregulated perivascular gene expression from mesenchymal stem cells. Our results suggest osteogenic stimulatory conditions to be appropriate for in vitro development of vascularized bone implants for tissue engineering

Mesenchymal stem cells induce endothelial cell quiescence and promote capillary formation

Introduction: Rapid establishment of functional blood vessels is a prerequisite for successful tissue engineering. During vascular development, endothelial cells (ECs) and perivascular cells assemble into a complex regulating proliferation of ECs, vessel diameter and production of extracellular matrix proteins. The aim of this study was to evaluate the ability of mesenchymal stem cells (MSCs) to establish an endothelial-perivascular complex in tissue-engineered constructs comprising ECs and MSCs. Methods: Primary human ECs and MSCs were seeded onto poly(L-lactide-co-1,5-dioxepan-2-one) (poly(LLA-co-DXO)) scaffolds and grown in dynamic culture before subcutaneous implantation in immunocompromised mice for 1 and 3 weeks. Cellular activity, angiogenic stimulation and vascular assembly in cell/scaffold constructs seeded with ECs or ECs/MSCs in a 5:1 ratio was monitored with real-time RT-PCR, ELISA and immunohistochemical microscopy analysis. Results: A quiescent phenotype of ECs was generated, by adding MSCs to the culture system. Decreased proliferation of ECs, in addition to up-regulation of selected markers for vascular maturation was demonstrated. Baseline expression of VEGFa was higher for MSCs compared with EC (P <0.001), with subsequent up-regulated VEGFa-expression for EC/MSC constructs before (P <0.05) and after implantation (P <0.01). Furthermore, an inflammatory response with CD11b + cells was generated from implantation of human cells. At the end of the 3 week experimental period, a higher vascular density was shown for both cellular constructs compared with empty control scaffolds (P <0.01), with the highest density of capillaries being generated in constructs comprising both ECs and MSCs. Conclusions: Induction of a quiescent phenotype of ECs associated with vascular maturation can be achieved by co-seeding with MSCs. Hence, MSCs can be appropriate perivascular cells for tissue-engineered constructs

AXL inhibition prevents NAFLD progression in mice with soluble AXL as marker of the NAFLD to NASH transition

Trabajo presentado en el The Digital International Liver Congress, celebrado del 27 al 29 de agosto de 2020Background and Aims: TYRO3, AXL and MERTK are receptor tyrosine kinases activated by the ligand GAS6. AXL signalling is increased in NASH patients, promotes fibrosis in hepatic stellate cells and inflammation in Kupffer cells, while GAS6 protects hepatocytes against lipotoxicity via MERTK. Recent data has shown that the AXL kinase inhibitor bemcentinib, by blocking AXL signalling and increasing GAS6 levels, reduces experimental NASH. However, AXL's role in the NAFLD/NASH transition has not been addressed. Identifying mechanisms responsible for NAFLD progression into NASH could provide early markers and novel therapeutic targets. Method: Mice were fed a high-fat methionine-restricted choline deficient (HFCD) diet for 2 and 4 weeks, and a high-fat diet with fructose (HFF) for 4 months to induce different degrees of NAFLD/NASH. Mouse Gas6, soluble levels of Axl (sAXL) and Mertk were measured by ELISA. Human GAS6, sAXL and MERTK were measured by ELISA in control and patient serum, and compared with biochemical and histological data. Transaminases and triglycerides were measured at the Hospital Clinic Core. The collagen content was measured by staining with Sirius Red and quantified by imaging software. H&E staining was performed and NAS score evaluated. Transcriptomic analysis of genes related to liver inflammation and fibrosis were measured in commercial microarrays and by qPCR. Results: After 4 weeks feeding with HFCD diet, early NASH was detected featuring liver steatosis, liver inflammation, hepatocellular ballooning and fibrosis. AXL inhibition with bemcentinib for 2 weeks reduced all these hepatic anomalies, including triglyceride serums levels and liver steatosis, preventing NAFLD progression. After 2 weeks of HFCD diet, mice presented fatty liver without fibrosis; however, transcriptomic analysis evidenced strong upregulation of pro-fibrotic and pro-inflammatory genes, while soluble Axl levels were already increased. Of note, one week bemcentinib administration not only reduced specific inflammatory and fibrotic genes such as Ccr2 or Col1a1, but also hepatic steatosis and NAS score. In contrast,in HFF-fed mice only liver steatosis was observed, without evidence of fibrosis or inflammation nor changes in sAxl levels. Interestingly, previous and on-going clinical data show significant sAXL increase in clinical patients with only diagnosed liver steatosis, showing no histological signs of inflammation or fibrosis. These results justify further transcriptomic patient evaluation and possible evolution depending on sAXL levels. Conclusion: sAXL levels reveals as a potential NAFLD-NASH transition marker, indicative of the initiation of liver inflammation and fibrosis before histological detection. Early treatment with bemcentinib prevented experimental NASH appearance, pointing to AXL antagonism as possible strategy for future clinical trials

Local Delivery of Interleukin 4 by Retrovirus-Transduced T Lymphocytes Ameliorates Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory autoimmune disease of the central nervous system which serves as a model for the human disease multiple sclerosis. We demonstrate here that encephalitogenic T cells, transduced with a retroviral gene, construct to express interleukin 4, and can delay the onset and reduce the severity of EAE when adoptively transferred to myelin basic protein–immunized mice. Thus, T lymphocytes transduced with retroviral vectors can deliver “regulatory cytokines” in a site-specific manner and may represent a viable therapeutic strategy for the treatment of autoimmune disease

Axl-inhibitor bemcentinib alleviates mitochondrial dysfunction in the unilateral ureter obstruction murine model

Renal fibrosis is a progressive histological manifestation leading to chronic kidney disease (CKD) and associated with mitochondrial dysfunction. In previous work, we showed that Bemcentinib, an Axl receptor tyrosine kinase inhibitor, reduced fibrosis development. In this study, to investigate its effects on mitochondrial dysfunction in renal fibrosis, we analysed genome-wide transcriptomics data from a unilateral ureter obstruction (UUO) murine model in the presence or absence of bemcentinib (n = 6 per group) and SHAM-operated (n = 4) mice. Kidney ligation resulted in dysregulation of mitochondria-related pathways, with a significant reduction in the expression of oxidative phosphorylation (OXPHOS), fatty acid oxidation (FAO), citric acid cycle (TCA), response to reactive oxygen species and amino acid metabolism-related genes. Bemcentinib treatment increased the expression of these genes. In contrast, AKT/PI3K signalling pathway genes were up-regulated upon UUO, but bemcentinib largely inhibited their expression. At the functional level, ligation reduced mitochondrial biomass, which was increased upon bemcentinib treatment. Serum metabolomics analysis also showed a normalizing amino acid profile in UUO, compared with SHAM-operated mice following bemcentinib treatment. Our data suggest that mitochondria and mitochondria-related pathways are dramatically affected by UUO surgery and treatment with Axl-inhibitor bemcentinib partially reverses these effects.publishedVersio

A rapid in vivo screen for pancreatic ductal adenocarcinoma therapeutics

Pancreatic ductal adenocarcinoma (PDA) is the fourth leading cause of cancer-related deaths in the United States, and is projected to be second by 2025. It has the worst survival rate among all major cancers. Two pressing needs for extending life expectancy of affected individuals are the development of new approaches to identify improved therapeutics, addressed herein, and the identification of early markers. PDA advances through a complex series of intercellular and physiological interactions that drive cancer progression in response to organ stress, organ failure, malnutrition, and infiltrating immune and stromal cells. Candidate drugs identified in organ culture or cell-based screens must be validated in preclinical models such as KIC (p48Cre;LSL-KrasG12D;Cdkn2af/f) mice, a genetically engineered model of PDA in which large aggressive tumors develop by 4 weeks of age. We report a rapid, systematic and robust in vivo screen for effective drug combinations to treat Kras-dependent PDA. Kras mutations occur early in tumor progression in over 90% of human PDA cases. Protein kinase and G-protein coupled receptor (GPCR) signaling activates Kras. Regulators of G-protein signaling (RGS) proteins are coincidence detectors that can be induced by multiple inputs to feedback-regulate GPCR signaling. We crossed Rgs16::GFP bacterial artificial chromosome (BAC) transgenic mice withKIC mice and show that the Rgs16::GFP transgene is a KrasG12D-dependent marker of all stages of PDA, and increases proportionally to tumor burden in KIC mice. RNA sequencing (RNA-Seq) analysis of cultured primary PDA cells reveals characteristics of embryonic progenitors of pancreatic ducts and endocrine cells, and extraordinarily high expression of the receptor tyrosine kinase Axl, an emerging cancer drug target. In proof-of-principle drug screens, we find that weanling KIC mice with PDA treated for 2 weeks with gemcitabine (with or without Abraxane) plus inhibitors of Axl signaling (warfarin and BGB324) have fewer tumor initiation sites and reduced tumor size compared with the standard-of-care treatment. Rgs16::GFP is therefore an in vivo reporter of PDA progression and sensitivity to new chemotherapeutic drug regimens such as Axl-targeted agents. This screening strategy can potentially be applied to identify improved therapeutics for other cancers

Mural Cell Associated VEGF Is Required for Organotypic Vessel Formation

Background: Blood vessels comprise endothelial cells, mural cells (pericytes/vascular smooth muscle cells) and basement membrane. During angiogenesis, mural cells are recruited to sprouting endothelial cells and define a stabilizing context, comprising cell-cell contacts, secreted growth factors and extracellular matrix components, that drives vessel maturation and resistance to anti-angiogenic therapeutics. Methods and Findings: To better understand the basis for mural cell regulation of angiogenesis, we conducted high content imaging analysis on a microtiter plate format in vitro organotypic blood vessel system comprising primary human endothelial cells co-cultured with primary human mural cells. We show that endothelial cells co-cultured with mural cells undergo an extensive series of phenotypic changes reflective of several facets of blood vessel formation and maturation: Loss of cell proliferation, pathfinding-like cell migration, branching morphogenesis, basement membrane extracellular matrix protein deposition, lumen formation, anastamosis and development of a stabilized capillary-like network. This phenotypic sequence required endothelial-mural cell-cell contact, mural cell-derived VEGF and endothelial VEGFR2 signaling. Inhibiting formation of adherens junctions or basement membrane structures abrogated network formation. Notably, inhibition of mural cell VEGF expression could not be rescued by exogenous VEGF. Conclusions: These results suggest a unique role for mural cell-associated VEGF in driving vessel formation and maturation

Lithium-to-calcium ratios in Modern, Cenozoic, and Paleozoic articulate brachiopod shells

Li/Ca ratios in modern brachiopod shells generally correlate inversely with growth temperature, ranging from ∼20 µmol/mol at 30°C to ∼50 µmol/mol at 0°C with no apparent interspecific offsets. Causes of the temperature effect on Li/Ca ratios are not yet understood. Cenozoic brachiopod Li/Ca ratios average ∼30 µmol/mol, similar to the average observed in modern brachiopods. Relatively constant Li/Ca ratios for Eocene to Pleistocene nonluminescent brachiopod shells, consistent with previous observations of Cenozoic planktonic foraminifera, support the conclusion of little variation in Cenozoic seawater Li/Ca. Nonluminescent portions of Permian and Carboniferous brachiopods have Li/Ca ratios substantially lower (generally <10 µmol/mol) than modern, Cenozoic, or Devonian samples. Mass balance considerations, constrained by δ18O of brachiopods, suggest that low Li concentrations in Permo-Carboniferous seawater could be the result of a lower flux of dissolved Li from the continents and/or a higher flux of Li from seawater to clastic marine sediments. Nonluminescent Devonian brachiopods from a single hand specimen have Li/Ca ratios around 70% of the modern average. These Li/Ca ratios can be explained by either somewhat higher temperature with constant seawater Li/Ca, somewhat lower seawater Li/Ca at constant temperature, or a combination of slightly elevated temperature and slightly lower seawater Li/Ca

AXL targeting reduces fibrosis development in experimental unilateral ureteral obstruction

The AXL receptor tyrosine kinase (RTK) is involved in partial epithelial-to-mesenchymal transition (EMT) and inflammation - both main promoters of renal fibrosis development. The study aim was to investigate the role of AXL inhibition in kidney fibrosis due to unilateral ureteral obstruction (UUO). Eight weeks old male C57BL/6 mice underwent UUO and were treated with oral AXL inhibitor bemcentinib (n = 22), Angiotensin-converting enzyme inhibitor (ACEI, n = 10), ACEI and bemcentinib (n = 10) or vehicle alone (n = 22). Mice were sacrificed after 7 or 15 days and kidney tissues were analyzed by immunohistochemistry (IHC), western blot, ELISA, Sirius Red (SR) staining, and hydroxyproline (Hyp) quantification. RNA was extracted from frozen kidney tissues and sequenced on an Illumina HiSeq4000 platform. After 15 days the ligated bemcentinib-treated kidneys showed less fibrosis compared to the ligated vehicle-treated kidneys in SR analyses and Hyp quantification. Reduced IHC staining for Vimentin (VIM) and alpha smooth muscle actin (alpha SMA), as well as reduced mRNA abundance of key regulators of fibrosis such as transforming growth factor (Tgf beta), matrix metalloproteinase 2 (Mmp2), Smad2, Smad4, myofibroblast activation (Aldh1a2, Crlf1), and EMT (Snai1,2, Twist), in ligated bemcentinib-treated kidneys was compatible with reduced (partial) EMT induction. Furthermore, less F4/80 positive cells, less activity of pathways related to the immune system and lower abundance of MCP1, MCP3, MCP5, and TARC in ligated bemcentinib-treated kidneys was compatible with reduction in inflammatory infiltrates by bemcentinib treatment. The AXL RTK pathway represents a promising target for pharmacologic therapy of kidney fibrosis.Peer reviewe