scholarly journals Targeting Protein S Using Small Interfering RNA Is Well Tolerated and Protects Mice with Hemophilia a from Acute Hemarthrosis

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 20-21 ◽  
Author(s):  
Raja Eladnani Prince ◽  
Ute Schaeper ◽  
Sibylle Dames ◽  
Sara Calzavarini ◽  
Claudia Quarroz ◽  
...  
Keyword(s):  
Gene Expression ◽  
Prothrombin Time ◽  
Small Interfering Rna ◽  
Hemophilia A ◽  
Alpha Angle ◽  
Coagulation Factor ◽  
Protein S ◽  
Mrna Levels ◽  
Spontaneous Bleeding ◽  
Interfering Rna

Introduction & Aim: Hemophilia A (HA) is an X-linked disorder caused by an absence or a reduction of coagulation factor VIII. Patients with HA often suffer from spontaneous bleeding within the musculoskeletal system, such as hemarthrosis. Hemarthrosis is caused by bleeding into joint spaces affecting the synovium, synovial blood vessels as well as cartilage and bone tissues. Current prophylactic treatments are not always effective and hemophilia patients can experience breakthrough bleeds. Recently, we demonstrated that inhibition of protein S (PS), a natural anticoagulant, controls coagulation and constitutes a potential therapeutic target in hemophilia (Blood 2018, 131:1360-1371). Here, we aim to translate our findings using small interfering RNA conjugated to an N-acetylgalactosamine (GalNAc) cluster to target Pros1 gene expression (GalNAc-PS siRNA) in vivo and exclusively in hepatocytes. siRNAs conjugated to a GalNAc cluster bind to asialoglycoprotein receptors expressed predominantly by hepatocytes thereby providing a potentially safe, specific and efficient delivery technology for therapeutic molecules. Methods & Results: Forty-two days after subcutaneous (s.c.) injection of GalNAc-PS siRNA (3mg/kg), wild-type (WT) mice were alive and did not display overt disseminated intravascular coagulation (DIC). In a second study in WT mice, DIC parameters assessed fourteen days after treatment with either 5mg/kg GalNAc-PS siRNA or with vehicle were also comparable between the two groups (platelet count: 578±284 vs 725±186 G/L, p>0.9, n=4-6; prothrombin time: 9.0±0.4 vs 8.9±0.3 seconds, p>0.9, n=4-6; fibrinogen: 1.5±0.5 vs 1.8±0.4 g/L, p>0.85, n=5-6; thrombin anti-thrombin complexes, TAT: 71±63 vs 115±34 μg/L, p>0.9, n=2-4) supporting that GalNAc-PS siRNA treatment can be safe. At the same time, mice treated with GalNAc-PS siRNA displayed lower plasma PS level compared to mice receiving the vehicle (52±12 vs 100±11 %, p<0.001, n=6). In the liver, PS mRNA levels were reduced by 69% compared to mice treated just with the vehicle (31±10 vs 100±24 %, p<0.0001, n=6). Importantly, in a murine model for hemophilia A (F8-/- mice) the intrinsically-activated test using ellagic acid (INTEM) assessed by rotative thromboelastometry (ROTEM®) was improved by treatment with GalNAc-PS siRNA (5mg/kg s.c) as compared to F8-/- mice treated with the vehicle (clotting time: 281±193 vs 802±330 seconds, p<0.01, n=6-11; clot formation time: 109±80 vs 657±466 seconds, p<0.05, n=6-11; alpha angle: 70±13 vs 35±24 mm, p<0.1, n=6-11). To assess if targeting PS using GalNAc-siRNA-PS protects mice from acute hemarthrosis (AH), we applied an AH model to F8-/- mice. Five days after injecting a single dose of 5mg/kg GalNAc-PS s.c., right knees were injured using a 30 gauge-needle and knee diameters were measured 72 hours later. Macroscopically, vehicle treated F8-/- mice developed extensive bleeding in injured knees as compared to GalNAc-siRNA-PS treated mice. Scores for intra-articular bleeding (2.4±0.9 vs 1.0±0.7, p=0.035, n=5-9) and synovial hyperplasia (2.4±0.9 vs 0.6±0.9, p=0.027, n=5-9) were higher in F8-/- mice treated by vehicle than in those treated by GalNAc-PS-siRNA. Moreover, knee joint swelling was reduced in GalNAc-siRNA-PS treated mice compared to those treated by vehicle (0.14±0.15 vs 0.78±0.50 mm, p=0.025, n=7-10). As expected, PS plasma levels were lower in GalNAc-PS siRNA treated mice compared to those which received vehicle (63±9 vs 101±19% of WT PS antigen level, p<0.0001, n=7-13) with no overt DIC (platelets count: 711±149 vs 681±189 G/L, p>0.9, n=7-12; prothrombin time: 8.5±0.3 vs 8.4±0.3 seconds, p>0.9, n=4-13; fibrinogen: 2.7±0.6 vs 3.0±0.8 g/L, p=0.73, n=7-12 and TAT: 33±42 vs 45±66 μg/L, p>0.9, n=6-10). Conclusion: These data provide the first evidence that using a GalNAc-siRNA conjugate to modulate Pros1 gene expression is well tolerated and has the ability to reduce plasma PS level and protect F8-/- mice from AH pointing to PS targeting using GalNAc-siRNA-PS as a new valuable therapeutic approach for hemophilia. Further analysis to understand if the inhibition of PS influences also the inflammatory processes causing the hemophilic arthropathy is ongoing. Disclosures Schaeper: Silence Therapeutics GmbH: Current Employment. Dames:Silence Therapeutics: Current Employment. Eisermann:Silence Therapeutics: Current Employment.

Low Prevalence of the Factor V Leiden Mutation Among “Severe” Hemophiliacs with a “Milder” Bleeding Diathesis

1995 ◽  
Vol 74 (05) ◽  
pp. 1255-1258 ◽  
Author(s):  
Arnaldo A Arbini ◽  
Pier Mannuccio Mannucci ◽  
Kenneth A Bauer
Keyword(s):  
Hemophilia A ◽  
Factor V Leiden ◽  
Protein S ◽  
Factor Ix ◽  
Hemophilia B ◽  
Factor V ◽  
Bleeding Diathesis ◽  
Mutation Site ◽  
Spontaneous Bleeding ◽  
Factor V Leiden Mutation

SummaryPatients with hemophilia A and B and factor levels less than 1 percent of normal bleed frequently with an average number of spontaneous bleeding episodes of 20–30 or more. However there are patients with equally low levels of factor VIII or factor IX who bleed once or twice per year or not at all. To examine whether the presence of a hereditary defect predisposing to hypercoagulability might play a role in amelio rating the hemorrhagic tendency in these so-called “mild severe” hemophiliacs, we determined the prevalence of prothrombotic defects in 17 patients with hemophilia A and four patients with hemophilia B selected from 295 and 76 individuals with these disorders, respectively, followed at a large Italian hemophilia center. We tested for the presence of the Factor V Leiden mutation by PCR-amplifying a fragment of the factor V gene which contains the mutation site and then digesting the product with the restriction enzyme Mnll. None of the patients with hemophilia A and only one patient with hemophilia B was heterozygous for Factor V Leiden. None of the 21 patients had hereditary deficiencies of antithrombin III, protein C, or protein S. Our results indicate that the milder bleeding diathesis that is occasionally seen among Italian hemophiliacs with factor levels that are less than 1 percent cannot be explained by the concomitant expression of a known prothrombotic defect.

scholarly journals Specific inhibition of gene expression using a stably integrated, inducible small‐interfering‐RNA vector

EMBO Reports ◽  
2003 ◽  
Vol 4 (6) ◽  
pp. 609-615 ◽  
Author(s):  
Marc van de Wetering ◽  
Irma Oving ◽  
Vanesa Muncan ◽  
Menno Tjon Pon Fong ◽  
Helen Brantjes ◽  
...  
Keyword(s):  
Gene Expression ◽  
Small Interfering Rna ◽  
Specific Inhibition ◽  
Interfering Rna

scholarly journals Optimization Procedure for Small Interfering RNA Transfection in a 384-Well Format

2007 ◽  
Vol 12 (4) ◽  
pp. 546-559 ◽  
Author(s):  
Jason Borawski ◽  
Alicia Lindeman ◽  
Frank Buxton ◽  
Mark Labow ◽  
L. Alex Gaither
Keyword(s):  
High Throughput Screening ◽  
Mammalian Cells ◽  
Small Interfering Rna ◽  
Dna Analysis ◽  
Lentiviral Vector ◽  
Mrna Levels ◽  
Sirna Transfection ◽  
Rna Transfection ◽  
Well Assay ◽  
Interfering Rna

High-throughput screening of RNAi libraries has become an essential part of functional analysis in academic and industrial settings. The transition of a cell-based RNAi assay into a 384-well format requires several optimization steps to ensure the phenotype being screened is appropriately measured and that the signal-to-background ratio is above a certain quantifiable threshold. Methods currently used to assess small interfering RNA (siRNA) efficacy after transfection, including quantitative PCR or branch DNA analysis, face several technical limitations preventing the accurate measurement of mRNA levels in a 384-well format. To overcome these difficulties, the authors developed an approach using a viral-based transfection system that measures siRNA efficacy in a standardized 384-well assay. This method allows measurement of siRNA activity in a phenotypically neutral manner by quantifying the knockdown of an exogenous luciferase gene delivered by a lentiviral vector. In this assay, the efficacy of a luciferase siRNA is compared to a negative control siRNA across many distinct assay parameters including cell type, cell number, lipid type, lipid volume, time of the assay, and concentration of siRNA. Once the siRNA transfection is optimized as a 384-well luciferase knockdown, the biologically relevant phenotypic analysis can proceed using the best siRNA transfection conditions. This approach provides a key technology for 384-well assay development when direct measurement of mRNA knockdown is not possible. It also allows for direct comparison of siRNA activity across cell lines from almost any mammalian species. Defining optimal conditions for siRNA delivery into mammalian cells will greatly increase the speed and quality of large-scale siRNA screening campaigns. ( Journal of Biomolecular Screening 2007:546-559)

scholarly journals Efficacy and safety of the drug Octofactor in prophylactic treatment in patients with severe haemophilia A

2018 ◽  
Vol 5 (3) ◽  
pp. 60-73 ◽  
Author(s):  
T. A. Andreeva ◽  
V. Yu. Zorenko ◽  
I. L. Davydkin ◽  
V. N. Konstantinova ◽  
O. E. Zalepukhina ◽  
...  
Keyword(s):  
Hemophilia A ◽  
Bleeding Episode ◽  
Coagulation Factor ◽  
Preventive Treatment ◽  
Haemophilia A ◽  
Severe Haemophilia ◽  
Efficacy And Safety ◽  
Spontaneous Bleeding ◽  
Bleeding Episodes ◽  
Blood Coagulation Factor Viii

Relevance.The development of a new recombinant blood coagulation factor VIII preparation is a promising step towards optimizing the treatment of hemophilia A. An introduction of a new medication into clinical practice precedes a clinical trials to evaluate the efficacy and safety.Materials and methods.The efficacy and safety of the domestic recombinant B-domain deleted blood coagulation factor VIII (FVIII) (moroctocog alfa, Octofactor®, JSC “GENERIUM”) were studied in the preventive treatment of 31 patients aged 21 to 52 years with severe haemophilia A. The Octofactor was administered in doses of 40 ± 5 IU/kg 3 times per week at intervals of at least 48 hours for 21 ± 1 weeks.Results.The efficacy of therapy was evaluated in 30 patients, since 1 patient refused to participate in the trial after the first injection of the study medication. There were registered 43 episodes of bleeding among 11 patients in the course of the preventive treatment with Octofactor. The average number of bleeding episodes was 1.4 ± 2.58. There were 43 bleeding episodes, 9 (20.9 %) of them were posttraumatic, 34 (79.1 %) of them were spontaneous. The average number of the spontaneous bleeding episodes (a major criterion of the efficacy) was 1.13 ± 2.19, which showed a low incidence of exacerbations of the hemorrhagic syndrome in the course of preventive treatment with Octofactor. Among all registered bleeding episodes there were 6 (14 %) mild episodes, 37 (86 %) moderate episodes. Among all spontaneous bleedings there were 6 mild episodes (17.6 %), 28 (82.4 %) moderate episodes. All posttraumatic bleedings were moderate. The vast majority (36, or 83.7 %) of bleeding episodes were stopped with administration of the Octofactor. The average number of administrations of the Octofactor for arresting 1 bleeding episode was 1.2 ± 0.56, for 1 spontaneous bleeding episode – 1.2 ± 0.59. On average, it was required to administer 3534.9 ± 2329.02 IU of the Octofactor to stop 1 episode of bleeding. In the vast majority of patients with severe hemophilia A (83.3–86.7 %),  the remaining activity FVIII was 1 % or more after the administration of the Octofactor in 48 hours. The total amount of the Octofactor, introduced for the prevention of bleeding, was 6,107,000 IU, to stop bleeding – 152,000 IU. The safety of therapy was evaluated in 31 patients. There were recorded 25 adverse events (AE) in 17 patients. Among them the laboratory ones prevailed in 23 (92 %) cases, which is not associated with the use of the trial medication. There were noted nausea and an unpleasant aftertaste in the mouth in 1 patient during the first administration of the Octofactor, and therefore he refused to continue to participate in the trial. Causality 2 AE with the study drug was regarded as definite. Such AE are expected and described in the instructions to the preparation. All AE were not serious and mild and resolved without outcomes. There were no presented thromboembolic events and immunogenic reactions.Conclusions.The obtained data testify to the efficacy and safety of the Octofactor both for preventive measures and for stopping bleeding in adult patients with severe hemophilia A.

Comparative study of specific inhibition of bcr-abl gene expression by chemically small interfering RNA and vector expression SiRNA

2008 ◽  
Vol 180 ◽  
pp. S245-S246
Author(s):  
Ali Zaree Mahmodabady ◽  
Ali Najafi ◽  
Mehdi Kamali ◽  
Hamid Javadi ◽  
Ali Khoshbaten
Keyword(s):  
Gene Expression ◽  
Comparative Study ◽  
Small Interfering Rna ◽  
Specific Inhibition ◽  
Interfering Rna

326 MICROINJECTIONS OF SMALL INTERFERING RNA AND COMPLEMENTARY RNA TO ELUCIDATE THE INVOLVEMENT OF ENDOGENOUS PHOSPHOLIPASE C ISOFORMS IN BOVINE OOCYTE ACTIVATION DURING FERTILIZATION

2010 ◽  
Vol 22 (1) ◽  
pp. 319
Author(s):  
B. R. Sessions ◽  
A. H. Bayles ◽  
J. Collier ◽  
K. Perry ◽  
L. S. Whitaker ◽  
...  
Keyword(s):  
Phospholipase C ◽  
Small Interfering Rna ◽  
Calcium Oscillations ◽  
Activation Process ◽  
Mrna Levels ◽  
Post Injection ◽  
Oocyte Activation ◽  
Bovine Oocyte ◽  
Interfering Rna ◽  
Bovine Oocytes

Phospholipase C (PLC) isoforms stimulate the hydrolysis of phosphatidyl inositol (4,5)-bisphosphate (PIP2) to produce diacylglycerol (DAG) and 1,4,5 inositol trisphosphate (IP3), with IP3 regulating the release of calcium (Ca2+) from the endoplasmic reticulum. This release of calcium is essential for oocyte activation, and a sperm-specific PLC isoform, PLCγ;, has been proposed as the primary agent that initiates the activation process. However, the oocyte contains many endogenous PLC isoforms (PLC β, γ, and δ) that could also be involved in regulating or initiating these calcium oscillations downstream of other initiating events. In order to better elucidate the involvement of endogenous PLC isoforms as well as the specific role of the sperm-specific form, small interfering RNA (siRNA) directed against the specific bovine PLC isoforms (PLCζ;, PLCγ1, PLCγ2, PLCδ1, PLCδ3, PLCδ4, PLCβ1, PLCβ3) were microinjected into bovine oocytes, and the subsequent effects on PLC mRNA levels and bovine fertilization were evaluated. Real-time PCR (qPCR) was used to quantify the levels of PLC message present in bovine oocytes at the time of injection (15 h post-maturation) and 6, 10, and 14 h post-injection. The qPCR results indicated a near-complete knockdown of mRNA levels in bovine oocytes 10 h post-injection for the isotypes PLCγ1, PLCγ2, PLCδ3, PLCδ4, PLCβ1, PLCβ3, but only partial knockdown of PLCS 1 mRNA. Oocytes microinjected with PLC siRNA were also fertilized and cultured in vitro according to our standard laboratory procedures (Reed et al. 1996 Theriogenology 45, 439-449). The oocytes microinjected with PLCζ;, PLCδ1, PLCδ3, PLCδ4, PLCβ1, PLCβ3 siRNA resulted in cleavage rates similar to the negative control siRNA, non-injected, and sham-injected treatment groups, whereas bovine oocytes microinjected with PLCγ1 and PLCγ2 siRNA had significantly lower cleavage rates compared with the controls. Additionally, complementary cRNA for each specific PLC isoform was microinjected into bovine oocytes to ascertain each isoform’s ability to induce parthenogenetic activation. Development was observed in oocytes microinjected with a variety of cRNAs, and the activating effects of the cRNA were negligible if the oocytes were microinjected with the corresponding siRNA before microinjection with cRNA. Interestingly, siRNA specific for PLCζ; failed to reduce cleavage when treated bovine oocytes were fertilized. These data illustrate the potential involvement of multiple endogenous PLC isoforms and not just the sperm-specific PLCζ; isoform in bovine oocyte activation during fertilization.

scholarly journals PP2B-mediated Dephosphorylation of c-Jun C Terminus Regulates Phorbol Ester-induced c-Jun/Sp1 Interaction in A431 Cells

2007 ◽  
Vol 18 (3) ◽  
pp. 1118-1127 ◽  
Author(s):  
Ben-Kuen Chen ◽  
Chi-Chen Huang ◽  
Wei-Chiao Chang ◽  
Yun-Ju Chen ◽  
Ushio Kikkawa ◽  
...  
Keyword(s):  
Gene Expression ◽  
Small Interfering Rna ◽  
Neuronal Nicotinic Acetylcholine Receptor ◽  
A431 Cells ◽  
Keratin 16 ◽  
Cytosolic Phospholipase ◽  
C Terminus ◽  
Genes Expression ◽  
Induced Genes ◽  
Interfering Rna

The c-Jun/Sp1 interaction is essential for growth factor- and phorbol 12-myristate 13-acetate (PMA)-induced genes expression, including human 12(S)-lipoxygenase, keratin 16, cytosolic phospholipase A2, p21WAF1/CIP1, and neuronal nicotinic acetylcholine receptor β4. Here, we examined the mechanism underlying the PMA-induced regulation on the interaction between c-Jun and Sp1. We found that treatment of cells with PMA induced a dephosphorylation at the C terminus of c-Jun at Ser-243 and a concomitant inhibition of PP2B by using PP2B small interfering RNA, resulting in reduction of PMA-induced gene expression as well as the c-Jun/Sp1 interaction. The c-Jun mutant TAM-67-3A, which contains three substitute alanines at Thr-231, Ser-243, and Ser-249 compared with TAM-67, binds more efficaciously with Sp1 and is about twice as efficacious as TAM-67 in inhibiting the PMA-induced activation of the 12(S)-lipoxygenase promoter. Importantly, PP2B not only dephosphorylates the c-Jun at Ser-243 but also interacts with c-Jun in PMA-treated cells. PMA stimulates the association of the PP2B/c-Jun/Sp1 complex with the promoter. These findings indicate the dephosphorylation of c-Jun C terminus is required for the c-Jun/Sp1 interaction and reveal that PP2B plays an important role in regulating c-Jun/Sp1 interaction in PMA-induced gene expression.

Common SNPs within or near Three Immune Response Genes Implicated in the Risk of FVIII Immunogenicity in Hemophilia A Do Not Influence Steady-State Levels of Their Encoded mRNAs

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3366-3366
Author(s):  
Tom E. Howard ◽  
Eugene Drigalenko ◽  
Matthew P. Johnson ◽  
Shelley S. Cole ◽  
Benjamin Kim ◽  
...  
Keyword(s):  
Gene Expression ◽  
Immune Response ◽  
Steady State ◽  
Hemophilia A ◽  
Minority Population ◽  
Mrna Levels ◽  
Promoter Polymorphisms ◽  
Inhibitor Development ◽  
Cis Acting ◽  
Immune Response Genes

Abstract Abstract 3366 Background/Aims: The hemophilia A (HA)-causing Factor (F)VIII gene (F8) mutation type is a well-established determinant of risk for the development of alloimmune inhibitors that neutralize replacement FVIII proteins in ∼20% of all HA patients. Studies have investigated variants of immune response genes to determine if they may account for the inter-individual variability in FVIII immunogenicity observed in patients with the same F8 abnormality, e.g., the intron-22 inversion. While some studies have found associations between inhibitor status and promoter polymorphisms in CTLA4, TNFA and/or IL10, others have not. If these promoter polymorphisms are indeed functional and truly influence inhibitor development, their alleles could modulate transcription initiation rates. The goal of this study was to investigate the possibility of cis-acting genotype-specific differences in mean steady-state mRNA levels encoded by CTLA4, TNFA and IL10. Methods: We examined the relationship of lymphocyte CTLA4, TNFA and IL10 mRNA levels with the genotypes of 265 SNPs located across their structural loci in 1189 Mexican American subjects in the San Antonio Family Heart Study. Expression profiles were generated using Illumina's HWG-6 BeadChips and genotypes came from the Illumina OmniExpress-12 BeadChip. Measured genotype association analyses that accounted for non-independence of family members and employed an additive model (in which testing to determine whether gene expression varies by genotype, with the model constrained so that each “dose” of the minor allele raises or lowers gene expression by an equal amount “beta”) were performed using the software package SOLAR. P-values were calculated using a 1 degree-of-freedom chi-square test comparing the likelihood of a model where the change in expression levels by genotype is estimated to the likelihood of a model where beta is constrained to zero. Results: None of the 265 genotyped SNPs within or near these three genes (i.e., 49 SNPs in IL10, 35 SNPs in CTLA4 & 181 SNPs in TNFA) function as cis-acting regulatory variants, as no significant genotype-specific associations with these genes' transcript levels were identified. Conclusions: We observed no evidence for cis-regulation of CTLA4, TNFA or IL10 in Mexican Americans, the largest and most rapidly growing minority population in the United States, despite having genotyped directly the previously implicated promoter polymorphisms in the current analysis (e.g., see Figure). Although Hispanic American HA patients were recently found to have a significantly higher risk for inhibitor development than White HA patients, it is possible that cis-acting functional variants in this minority population are rare and not well-represented by the common GWAS SNPs used for these analyses. Since linkage disequilibrium patterns between markers are population-specific, we are also currently genotyping these SNPs in a large cohort of African and Caucasian American HA patients. Disclosures: No relevant conflicts of interest to declare.

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