Abstract 14720: Pre-clinical Safety Assessment of SLN360, a Novel Short Interfering Ribonucleic Acid Targeting LPA

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
David Rider ◽  
Daniel Swerdlow ◽  
Mona Eisermann ◽  
Kathrin Loeffler ◽  
Judith Hauptmann ◽  
...  
Keyword(s):  
Target Genes ◽  
Clinical Chemistry ◽  
Recovery Period ◽  
Human Hepatocytes ◽  
Saline Control ◽  
Mrna Levels ◽  
Clinical Safety ◽  
Primary Human Hepatocytes

Introduction: SLN360 is a liver-targeted N-acetyl galactosamine (GalNAc)-conjugated siRNA with a promising profile for addressing Lp(a)-related cardiovascular risk. We previously reported the pharmacodynamic effects of SLN360 in female (F) cynomolgus monkeys (cynos) 1 . Here we describe the findings from key pre-clinical safety studies. Methods: SLN360 was tested in vitro in primary human hepatocytes for LPA knockdown and effects on hypothetical off targets identified through in silico screening. An in vivo biodistribution study was performed in rats over 21 days (4 males [M]/4 F per timepoint; single 10 mg/kg s.c. injection). In a GLP safety study, healthy cynos received 5 once weekly s.c. injections of saline control or SLN360 (3 M / 3 F per group, up to 200 mg/kg) followed by an 8-week recovery period (2 M / 2 F in saline and top dose). A standard battery of safety assessments was performed. Results: In vitro, SLN360 reduced LPA expression in primary human hepatocytes with no effects on any hypothetical liver-expressed human off-target genes at concentrations >300-fold the human LPA IC 50 . SLN360 showed a typical rodent GalNAc distribution pattern, with significant levels in liver and kidney (peak 126 or 246 mg/g tissue at 6h respectively; estimated liver half-life of 56 hours). Levels in other organs (including reproductive organs) were <1% of peak liver levels. In cynos, no clinical observations were observed following 5 s.c. doses of SLN360. Liver LPA mRNA levels were significantly reduced by 98% at day 30 and 95% after the 8-week recovery period, while serum Lp(a) was undetectable at both timepoints. Findings were restricted to the liver (increased weight and diffuse hepatocyte hypertrophy) and lymph nodes (vacuolated macrophages) at day 30. These were considered non-adverse due to reversibility after recovery. No dose-related changes in clinical chemistry, hematology, circulatory and ECG parameters, respiratory rate, neurobehavior, plasma cytokines, complement activation or CRP were noted. The NOAEL was defined as 200 mg/kg, >60-fold the active dose in cynos. Conclusions: Overall, the off-target profile, biodistribution and NOAEL of SLN360 indicate suitability for entry into clinical studies. 1 Aleku et al., 2019. Circulation. 2019, 140:A9538.

scholarly journals Circular RNA CDR1as Exerts Oncogenic Properties Partially through Regulating MicroRNA 641 in Cholangiocarcinoma

2020 ◽  
Vol 40 (15) ◽  
Author(s):  
Dingyang Li ◽  
Zhe Tang ◽  
Zhiqiang Gao ◽  
Pengcheng Shen ◽  
Zhaochen Liu ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Target Genes ◽  
Circular Rna ◽  
Tumor Xenograft ◽  
Mrna Levels ◽  
Cell Behaviors ◽  
Xenograft Growth ◽  
Tumor Xenograft Growth

ABSTRACT It has been found that the circular RNA (circRNA) CDR1as is upregulated in cholangiocarcinoma (CCA) tissues. In this study, we tried to explore the roles of CDR1as in CCA. CDR1as was overexpressed or knocked down in human CCA cells to assess the effects of CDR1as on cell behaviors and tumor xenograft growth. In vitro, the CDR1as level was significantly increased in CCA cell lines. The results showed that CDR1as promoted the cell proliferation, migration, invasion, and activation of the AKT3/mTOR pathway in CCA cells. Moreover, miR-641, a predicted target microRNA (miRNA) of CDR1as, could partially reverse the effects of CDR1as on cell behaviors in CCA cells. Furthermore, CDR1as improved tumor xenograft growth, and it could be attenuated by miR-641 in vivo. Additionally, CDR1as expression was inversely correlated with miR-641 in CCA cells, and miR-641 could directly bind with CDR1as and its target genes, the AKT3 and mTOR genes. Mechanistically, CDR1as could bind with miR-641 and accelerate miR-641 degradation, which possibly leads to the upregulation of the relative mRNA levels of AKT3 and mTOR in RBE cells. In conclusion, our findings indicated that CDR1as might exert oncogenic properties, at least partially, by regulating miR-641 in CCA. CDR1as and miR-641 could be considered therapeutic targets for CCA.

scholarly journals Astaxanthin Treatment Induces Maturation and Functional Change of Myeloid-Derived Suppressor Cells in Tumor-Bearing Mice

Antioxidants ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 350
Author(s):  
Seong Mun Jeong ◽  
Yeon-Jeong Kim
Keyword(s):  
Mhc Class Ii ◽  
Target Genes ◽  
Suppressor Cells ◽  
Functional Change ◽  
Related Factor ◽  
Mrna Levels ◽  
Myeloid Derived Suppressor Cells ◽  
Antitumor Effects

Myeloid-derived suppressor cells (MDSCs) are immature myeloid cells which accumulate in stress conditions such as infection and tumor. Astaxanthin (ATX) is a well-known antioxidant agent and has a little toxicity. It has been reported that ATX treatment induces antitumor effects via regulation of cell signaling pathways, including nuclear factor erythroid-derived 2-related factor 2 (Nrf2) signaling. In the present study, we hypothesized that treatment with ATX might induce maturation of MDSCs and modulate their immunosuppressive activity. Both in vivo and in vitro treatment with ATX resulted in up-regulation of surface markers such as CD80, MHC class II, and CD11c on both polymorphonuclear (PMN)-MDSCs and mononuclear (Mo)-MDSCs. Expression levels of functional mediators involved in immune suppression were significantly reduced, whereas mRNA levels of Nrf2 target genes were increased in ATX-treated MDSCs. In addition, ATX was found to have antioxidant activity reducing reactive oxygen species level in MDSCs. Finally, ATX-treated MDSCs were immunogenic enough to induce cytotoxic T lymphocyte response and contributed to the inhibition of tumor growth. This demonstrates the role of ATX as a regulator of the immunosuppressive tumor environment through induction of differentiation and functional conversion of MDSCs.

MUSCLE-INDUCED PATELLOFEMORAL JOINT LOADING RAPIDLY AFFECTS CARTILAGE mRNA LEVELS IN A SITE SPECIFIC MANNER

2004 ◽  
Vol 08 (01) ◽  
pp. 1-12 ◽  
Author(s):  
Andrea L. Clark ◽  
Linda Mills ◽  
David A Hart ◽  
Walter Herzog
Keyword(s):  
Articular Cartilage ◽  
Mechanical Loading ◽  
Patellofemoral Joint ◽  
Mrna Level ◽  
Recovery Period ◽  
Cartilage Explants ◽  
Mrna Levels ◽  
Biological Responses

Mechanical loading of articular cartilage affects the synthesis and degradation of matrix macromolecules. Much of the work in this area has involved mechanical loading of articular cartilage explants or cells in vitro and assessing biological responses at the mRNA and protein levels. In this study, we developed a new experimental technique to load an intact patellofemoral joint in vivo using muscle stimulation. The articular cartilages were cyclically loaded for one hour in a repeatable and measurable manner. Cartilage was harvested from central and peripheral regions of the femoral groove and patella, either immediately after loading or after a three hour recovery period. Total RNA was isolated from the articular cartilage and biological responses were assessed on the mRNA level using the reverse transcriptase-polymerase chain reaction. Articular cartilage from intact patellofemoral joints demonstrated heterogeneity at the mRNA level for six of the genes assessed independent of the loading protocol. Cyclical loading of cartilage in its native environment led to alterations in mRNA levels for a subset of molecules when assessed immediately after the loading period. However, the increases in TIMP-1 and decreases in bFGF mRNA levels were transient; being present immediately after load application but not after a three hour recovery period.

Combinatorial epigenetic treatment approach for hepatocellular carcinoma

2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 10066-10066
Author(s):  
M. Bitzer ◽  
S. Venturelli ◽  
S. Armeanu ◽  
T. S. Weiss ◽  
M. Gregor ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Histone Acetylation ◽  
Treatment Approach ◽  
Methylation Status ◽  
Human Hepatocytes ◽  
Cellular Damage ◽  
Primary Human Hepatocytes ◽  
Hcc Cells

10066 Hepatocellular carcinoma (HCC) displays a striking resistance to chemotherapeutic drugs. Alternative approaches comprise employment of epigenetic compounds such as histone deacetylase inhibitors (HDAC-I) or demethylating agents (DA). Since we could recently demonstrate that HDAC-I exhibit inherent in vitro therapeutic activities against HCC cells, we now explored the potential of a HDAC-I / DA combined epigenetic treatment approach for the highly chemotherapy resistant tumor entity HCC. Methods: HCC cell lines (HepG2, Hep3B, HuH7) and primary human hepatocytes (PHH) were treated with a HDAC-I compound (Suberoylanilide hydroxamic acid - SAHA) together with or without a DA (5-aza-2dC) and examined for cellular damage (enzyme release), proliferation inhibition (SRB), apoptotic cells (FACS), histone acetylation pattern (Western blot) and methylation status (methylation specific PCR). The in vivo activity of our approach was investigated in a HuH7 xenograft mouse hepatoma model. Results: Both SAHA and 5-aza-2dC were found to induce substantial antiproliferative effects and apoptotic cell death in HCC cells; interestingly, combinatorial treatment (SAHA plus 5-aza-2dC) resulted in strongly enhanced anti-HCC effects. Interferences between DA and HDAC-I activities could be excluded by determining both (i) the methylation status of two model genes (p16, SOCS-1; known to be abberantly methylated in HCC) as well as (ii) patterns of histone acetylation. Most importantly, non-malignant primary human hepatocytes (5 different donors) did not exhibit any relevant cellular damage even when high doses of SAHA plus 5-aza-2dC were applied. Finally, in vivo testing of our combinatorial regimen (SAHA plus 5-aza-2dC) was found to be superior in suppression of tumor cell growth when compared to the application of single substances (SAHA or 5-aza-2dC) or placebo. Conclusion: Our combined epigenetic approach in chemotherapy resistant HCC not only has been found to be a highly active treatment option with profound anti-tumor effects both in vitro and in a small animal model in vivo, but did not impair primary human hepatocytes. Thus, this combination therapy now is considered for further investigation in clinical trials. No significant financial relationships to disclose.

A toxicogenomics-based parallelogram approach to evaluate the relevance of coumarin-induced responses in primary human hepatocytes in vitro for humans in vivo

2009 ◽  
Vol 23 (6) ◽  
pp. 1163-1169 ◽  
Author(s):  
Anne S. Kienhuis ◽  
Marcel C.G. van de Poll ◽  
Cornelis H.C. Dejong ◽  
Ralph Gottschalk ◽  
Marcel van Herwijnen ◽  
...  
Keyword(s):  
Human Hepatocytes ◽  
Induced Responses ◽  
Primary Human Hepatocytes

scholarly journals Novel use of a chemically modified siRNA for robust and sustainable in vivo gene silencing in the retina

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Takazumi Taniguchi ◽  
Ken-ichi Endo ◽  
Hidetoshi Tanioka ◽  
Masaaki Sasaoka ◽  
Kei Tashiro ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Intravitreal Injection ◽  
Target Genes ◽  
Vitreous Body ◽  
Cell Types ◽  
Retinal Cell ◽  
Mrna Levels ◽  
Chemically Modified

AbstractDespite efficient and specific in vitro knockdown, more reliable and convenient methods for in vivo knockdown of target genes remain to be developed particularly for retinal research. Using commercially available and chemically modified siRNA so-called Accell siRNA, we established a novel in vivo gene silencing approach in the rat retina. siRNA designed for knockdown of the house keeping gene Gapdh or four retinal cell type-specific genes (Nefl, Pvalb, Rho and Opn1sw) was injected into the vitreous body, and their retinal mRNA levels were quantified using real-time PCR. Intravitreal injection of siRNA for Gapdh resulted in approximately 40–70% reduction in its retinal mRNA levels, which lasted throughout a 9-day study period. Furthermore, all the selected retinal specific genes were efficiently down-regulated by 60–90% following intravitreal injection, suggesting injected siRNA penetrated into major retinal cell types. These findings were consistent with uniform distribution of a fluorescence-labeled siRNA injected into the vitreous body. Interestingly, gene silencing of Grin1, a core subunit of NMDA receptor, was accompanied by significant prevention from NMDA-induced retinal ganglion cell death. Thus, we provide single intravitreal injection of Accell siRNA as a versatile technique for robust and sustainable in vivo retinal gene silencing to characterize their biological functions under physiological and pathophysiological conditions.

scholarly journals Bacterial Nucleoid-Associated Protein Uncouples Transcription Levels from Transcription Timing

mBio ◽  
2014 ◽  
Vol 5 (5) ◽  
Author(s):  
Igor Zwir ◽  
Won-Sik Yeo ◽  
Dongwoo Shin ◽  
Tammy Latifi ◽  
Henry Huang ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Target Genes ◽  
Transcriptional Regulator ◽  
Mrna Levels ◽  
Content Type ◽  
Expression Of Genes ◽  
Serovar Typhimurium ◽  
Expression Behavior

ABSTRACTThe histone-like nucleoid-structuring (H-NS) protein binds to horizontally acquired genes in the bacteriumSalmonella entericaserovar Typhimurium, silencing their expression. We now report that overcoming the silencing effects of H-NS imposes a delay in the expression of genes activated by the transcriptional regulator PhoP. We determine that PhoP-activated genes ancestral toSalmonellaare expressed before those acquired horizontally. This expression timing reflects thein vivooccupancy of the corresponding promoters by the PhoP protein. These results are surprising because some of these horizontally acquired genes reached higher mRNA levels than ancestral genes expressed earlier and were transcribed from promoters harboring PhoP-binding sites with higherin vitroaffinity for the PhoP protein. Our findings challenge the often-made assumption that for genes coregulated by a given transcription factor, early genes are transcribed to higher mRNA levels than those transcribed at later times. Moreover, they provide a singular example of how gene ancestry can impact expression timing.IMPORTANCEWe report that gene ancestry dictates the expression behavior of genes under the direct control of theSalmonellatranscriptional regulator PhoP. That is, ancestral genes are transcribed before horizontally acquired genes. This reflects both the need to overcome silencing by the H-NS protein of the latter genes and the architecture of the corresponding promoters. Unexpectedly, transcription levels do not reflect transcription timing. Our results illustrate how a bacterium can exhibit an elaborate temporal expression behavior among genes coregulated by a transcription factor even though the products encoded by the target genes do not participate in a morphological or developmental pathway.

scholarly journals Visualizing Dynamic E2F-Mediated Repression In Vivo

2006 ◽  
Vol 26 (12) ◽  
pp. 4448-4461 ◽  
Author(s):  
Monica Agromayor ◽  
Elzbieta Wloga ◽  
Benedetta Naglieri ◽  
John Abrashkin ◽  
Kapil Verma ◽  
...  
Keyword(s):  
Target Gene ◽  
Target Genes ◽  
Suppressor Gene ◽  
Mrna Levels ◽  
Wild Type ◽  
Retinoblastoma Tumor Suppressor ◽  
Ets Family ◽  
Retinoblastoma Tumor

ABSTRACT Although many E2F target genes have been identified recently, very little is known about how any single E2F site controls the expression of an E2F target gene in vivo. To test the requirement for a single E2F site in vivo and to learn how E2F-mediated repression is regulated during development and tumorigenesis, we have constructed a novel series of wild-type and mutant Rb promoter-LacZ transgenic reporter lines that allow us to visualize the activity of a crucial E2F target in vivo, the retinoblastoma tumor suppressor gene (Rb). Two mutant Rb promoter-LacZ constructs were used to evaluate the importance of a single E2F site or a nearby activator (Sp1/Ets) site that is found mutated in low-penetrance retinoblastomas. The activity of the wild-type Rb promoter is dynamic, varying spatially and temporally within the developing nervous system. While loss of the activator site silences the Rb promoter, loss of the E2F site stimulates its activity in the neocortex, retina, and trigeminal ganglion. Surprisingly, E2F-mediated repression of Rb does not act globally or in a static manner but, instead, is a highly dynamic process in vivo. Using neocortical extracts, we detected GA-binding protein α (GABPα, an Ets family member) bound to the activator site and both E2F1 and E2F4 bound to the repressor site of the Rb promoter in vitro. Additionally, we detected binding of both E2F1 and E2F4 to the Rb promoter in vivo using chromatin immunoprecipitation analysis on embryonic day 13.5 brain. Unexpectedly, we detect no evidence for Rb promoter autoregulation in neuroendocrine tumors from Rb +/−; RbP-LacZ mice that undergo loss of heterozygosity at the Rb locus, in contrast to the situation in human retinoblastomas where high RB mRNA levels are found. In summary, this study provides the first demonstration that loss of an E2F site is critical for target gene repression in vivo and underscores the complexity of the Rb and E2F family network in vivo.

scholarly journals Coordinated Roles of Pregnane X Receptor and Constitutive Androstane Receptor in Autoinduction of Voriconazole Metabolism in Mice

2012 ◽  
Vol 57 (3) ◽  
pp. 1332-1338 ◽  
Author(s):  
Masato Ohbuchi ◽  
Kouichi Yoshinari ◽  
Hayato Kaneko ◽  
Satoru Matsumoto ◽  
Akiko Inoue ◽  
...  
Keyword(s):  
Target Genes ◽  
Mammalian Species ◽  
Constitutive Androstane Receptor ◽  
Pregnane X Receptor ◽  
Human Hepatocytes ◽  
Mrna Levels ◽  
Wild Type ◽  
Primary Human Hepatocytes ◽  
Metabolic Activities ◽  
Species Specific

ABSTRACTThe antifungal efficacy of voriconazole (VRC) differs among host species, with potent efficacy in humans but less in rodents. We investigated the possible involvement of pregnane X receptor (PXR) and constitutive androstane receptor (CAR) in the species-specific efficacy of VRC through pharmacokinetic analyses using genetically modified mice and primary human hepatocytes. VRC (30 mg/kg) was orally administered to wild-type,Pxr-null,Car-null, andPxr- andCar-null (Pxr/Car-null) mice for 7 days. Hepatic VRC metabolism was significantly increased by VRC administration, and the elimination rates of plasma VRC were much higher on day 7 than on day 1 in wild-type mice. This autoinduction was also observed inPxr-null andCar-null mice but not inPxr/Car-null mice, suggesting coordinated roles of PXR and CAR in the autoinduction of VRC metabolism in mice. HepaticCyp3a11mRNA levels were increased by VRC administration, hepatic metabolic activities for VRC were correlated with CYP3A activities, and the induced VRC metabolism was inhibited by ketoconazole (a CYP3A inhibitor). In primary human hepatocytes, VRC barely increased mRNA levels ofCYP3A4andCYP2B6(human PXR/CAR target genes) at its therapeutic concentrations. In conclusion, these results suggest that VRC is metabolized mainly by CYP3A11 in mouse livers and that PXR- and CAR-mediated CYP3A11 induction, namely, autoinduction of VRC metabolism, is a primary reason for the ineffectiveness of VRC in mice. A limited ability of VRC to activate human PXR/CAR at its clinical concentration might explain the VRC efficacy in humans. Therefore, the ability to activate PXR/CAR might determine the VRC efficacy in different mammalian species.

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