Factor IX: Insights from knock-out and genetically engineered mice

2008 ◽  
Vol 100 (10) ◽  
pp. 563-575 ◽  
Author(s):  
Paul E. Monahan
Keyword(s):  
Mouse Models ◽  
Positional Cloning ◽  
Coagulation Factors ◽  
Genetically Engineered ◽  
Factor Ix ◽  
Mouse Strains ◽  
Knock Out ◽  
Haemophilia B ◽  
Genetically Engineered Mice ◽  
Knock Out Mice

SummaryThe study of coagulation factors has been rapidly advanced by studies performed in genetically engineered mouse strains. Investigation of factor IX (FIX) has benefited from excellent genedeleted mouse models that recapitulate many of the features of human haemophilia B. Moreover, advanced positional cloning techniques and availability of technology to allow not only knock-out mice, but also knock-in and knock-down mice, provide new opportunities to observe genotype-phenotype and structure-function correlations regarding FIX, as well as the interaction of FIX with inflammatory, immune, and tissue repair systems. In this paper, available FIX knock-out mice and additional haemophilia B mouse models are reviewed specifically in regards to observations these models have facilitated concerning: factor IX gene expression and factor IX protein pharmacokinetics; the role of FIX in haemostasis, thrombosis and wound healing; insights into coagulation FIX arising out of gene therapy applications in haemophilia mouse models; immunology of tolerance or loss of tolerance of FIX and inhibitor antibody formation.

scholarly journals Characterization and visualization of murine coagulation factor VIII-producing cells in vivo

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Morisada Hayakawa ◽  
Asuka Sakata ◽  
Hiroko Hayakawa ◽  
Hikari Matsumoto ◽  
Takafumi Hiramoto ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Factor Viii ◽  
Fluorescent Protein ◽  
Coagulation Factor ◽  
Coagulation Factors ◽  
Genetically Engineered ◽  
Coagulation Factor Viii ◽  
Liver Sinusoidal Endothelial Cells ◽  
Genetically Engineered Mice

AbstractCoagulation factors are produced from hepatocytes, whereas production of coagulation factor VIII (FVIII) from primary tissues and cell species is still controversial. Here, we tried to characterize primary FVIII-producing organ and cell species using genetically engineered mice, in which enhanced green fluorescent protein (EGFP) was expressed instead of the F8 gene. EGFP-positive FVIII-producing cells existed only in thin sinusoidal layer of the liver and characterized as CD31high, CD146high, and lymphatic vascular endothelial hyaluronan receptor 1 (Lyve1)+. EGFP-positive cells can be clearly distinguished from lymphatic endothelial cells in the expression profile of the podoplanin− and C-type lectin-like receptor-2 (CLEC-2)+. In embryogenesis, EGFP-positive cells began to emerge at E14.5 and subsequently increased according to liver maturation. Furthermore, plasma FVIII could be abolished by crossing F8 conditional deficient mice with Lyve1-Cre mice. In conclusion, in mice, FVIII is only produced from endothelial cells exhibiting CD31high, CD146high, Lyve1+, CLEC-2+, and podoplanin− in liver sinusoidal endothelial cells.

scholarly journals Deletion of Kif5c Does Not Alter Prion Disease Tempo or Spread in Mouse Brain

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1391
Author(s):  
Brent Race ◽  
Katie Williams ◽  
Chase Baune ◽  
James F. Striebel ◽  
Clayton W. Winkler ◽  
...  
Keyword(s):  
Prion Disease ◽  
Molecular Motors ◽  
Prion Diseases ◽  
Mouse Strains ◽  
Survival Times ◽  
Knock Out ◽  
Transport Vesicles ◽  
The Central Nervous System ◽  
Knock Out Mice ◽  
Scrapie Strains

In prion diseases, the spread of infectious prions (PrPSc) is thought to occur within nerves and across synapses of the central nervous system (CNS). However, the mechanisms by which PrPSc moves within axons and across nerve synapses remain undetermined. Molecular motors, including kinesins and dyneins, transport many types of intracellular cargo. Kinesin-1C (KIF5C) has been shown to transport vesicles carrying the normal prion protein (PrPC) within axons, but whether KIF5C is involved in PrPSc axonal transport is unknown. The current study tested whether stereotactic inoculation in the striatum of KIF5C knock-out mice (Kif5c−/−) with 0.5 µL volumes of mouse-adapted scrapie strains 22 L or ME7 would result in an altered rate of prion spreading and/or disease timing. Groups of mice injected with each strain were euthanized at either pre-clinical time points or following the development of prion disease. Immunohistochemistry for PrP was performed on brain sections and PrPSc distribution and tempo of spread were compared between mouse strains. In these experiments, no differences in PrPSc spread, distribution or survival times were observed between C57BL/6 and Kif5c−/− mice.

Home treatment for haemophilia

1978 ◽  
Vol 16 (13) ◽  
pp. 49-50
Keyword(s):  
Factor Viii ◽  
Coagulation Factors ◽  
Home Treatment ◽  
Factor Ix ◽  
Coagulation Cascade ◽  
Haemophilia A ◽  
Haemophilia B ◽  
Prolonged Bleeding ◽  
Factor Deficiency ◽  
Spontaneous Haemorrhage

Haemophilia A is caused by faulty synthesis of Factor VIII of the coagulation cascade. Haemophilia B (Christmas disease) is caused by a deficiency of Factor IX. The two conditions are clinically similar; all patients suffer from prolonged bleeding after trauma and in the more severely affected there is also spontaneous haemorrhage, particularly into joints and muscles. Correction of factor deficiency by plasma concentrates restores haemostasis but the intermittent nature of the haemorrhage, the scarcity of the transfused coagulation factors and their short plasma half-lives in most cases limit treatment to episodes of bleeding.

scholarly journals Rapid Evaluation of CRISPR Guides and Donors for Engineering Mice

Genes ◽  
2020 ◽  
Vol 11 (6) ◽  
pp. 628
Author(s):  
Elena McBeath ◽  
Jan Parker-Thornburg ◽  
Yuka Fujii ◽  
Neeraj Aryal ◽  
Chad Smith ◽  
...  
Keyword(s):  
Genomic Dna ◽  
Sanger Sequencing ◽  
High Efficiency ◽  
Genetically Engineered ◽  
Rapid Evaluation ◽  
Knock Out ◽  
Guide Rna ◽  
Guide Rnas ◽  
Genetically Engineered Mice ◽  
The Cost

Although the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/ CRISPR associated protein 9 (Cas9) technique has dramatically lowered the cost and increased the speed of generating genetically engineered mice, success depends on using guide RNAs and donor DNAs which direct efficient knock-out (KO) or knock-in (KI). By Sanger sequencing DNA from blastocysts previously injected with the same CRISPR components intended to produce the engineered mice, one can test the effectiveness of different guide RNAs and donor DNAs. We describe in detail here a simple, rapid (three days), inexpensive protocol, for amplifying DNA from blastocysts to determine the results of CRISPR point mutation KIs. Using it, we show that (1) the rate of KI seen in blastocysts is similar to that seen in mice for a given guide RNA/donor DNA pair, (2) a donor complementary to the variable portion of a guide integrated in a more all-or-none fashion, (3) donor DNAs can be used simultaneously to integrate two different mutations into the same locus, and (4) by placing silent mutations about every 6 to 10 bp between the Cas9 cut site and the desired mutation(s), the desired mutation(s) can be incorporated into genomic DNA over 30 bp away from the cut at the same high efficiency as close to the cut.

scholarly journals Cryopreservation of mouse resources

2020 ◽  
Vol 36 (1) ◽  
Author(s):  
Toru Takeo ◽  
Satohiro Nakao ◽  
Yoshiko Nakagawa ◽  
Jorge M. Sztein ◽  
Naomi Nakagata
Keyword(s):  
Research Team ◽  
Animal Experiments ◽  
Reproductive Technology ◽  
Genetically Engineered ◽  
Research Infrastructure ◽  
Mouse Strains ◽  
Research Projects ◽  
New Techniques ◽  
Genetically Engineered Mice ◽  
Animal Resources

Abstract The cryopreservation of sperm and embryos is useful to efficiently archive valuable resources of genetically engineered mice. Till date, more than 60,000 strains of genetically engineered mice have been archived in mouse banks worldwide. Researchers can request for the archived mouse strains for their research projects. The research infrastructure of mouse banks improves the availability of mouse resources, the productivity of research projects, and the reproducibility of animal experiments. Our research team manages the mouse bank at the Center for Animal Resources and Development in Kumamoto University and continuously develops new techniques in mouse reproductive technology to efficiently improve the system of mouse banking. In this review, we introduce the activities of mouse banks and the latest techniques used in mouse reproductive technology.

FIX-Triple, a gain-of-function factor IX variant, improves haemostasis in mouse models without increased risk of thrombosis

2010 ◽  
Vol 104 (08) ◽  
pp. 355-365 ◽  
Author(s):  
Chung-Yang Kao ◽  
Chia-Ni Lin ◽  
I-Shing Yu ◽  
Mi-Hua Tao ◽  
Hua-Lin Wu ◽  
...  
Keyword(s):  
Mouse Models ◽  
Specific Activity ◽  
Factor Ix ◽  
Wild Type ◽  
Haemophilia B ◽  
Virus Particles ◽  
Thrombosis Risk ◽  
Increased Risk ◽  
Recombinant Factor Ix ◽  
Fold Excess

SummaryEngineered recombinant factor IX (FIX) with augmented clotting activity may prove useful for replacement therapy, but it has not been studied for risk of thrombosis. We used three mouse models to evaluate thrombosis risk associated with the FIX variant FIX-Triple, which has a 13-fold higher specific activity than wild-type FIX (FIX-WT). Protein infusion of FIX-Triple into haemophilia B mice was not thrombogenic, even at a dose of 13-fold higher than FIX-WT. Gene knock-in to generate mice that constitutively produce FIX-WT or FIX-Triple protein revealed that all mice expressed equal antigen levels. FIX-Triple knock-in mice that exhibited 10-fold higher FIX clotting activity did not show hypercoagulation. Adeno-associated viral (AAV) delivery of the FIX gene into mice was used to mimic gene therapy. Haemophilia B and inbred C57Bl/6 mice injected with different doses of virus particles carrying FIX-WT or FIX-Triple and expressing up to a nearly 13-fold excess (1289% of normal) of FIX clotting activity did not show increased risk of thrombosis compared with untreated wild-type mice in a normal haemostatic state. When challenged with ferric chloride (FeCl3), the mesenteric venules of AAV-treated C57Bl/6 mice that gave a nearly five-fold excess (474%) of FIX clotting activity were not thrombotic; however, thrombosis became obvious in FeCl3-challenged mice expressing extremely high FIX clotting activities (976–1289%) achieved by AAV delivery of FIX-Triple. These studies suggest that FIX-Triple is not thrombogenic at therapeutic levels and is a potential therapeutic substitute for FIX-WT.

Recombinant Human Coagulation Factor IX Expressed in HEK293 Cells: Influence of Serin Phosphorylation and Tyrosine Sulfation on Pharmacokinetic Properties in FIX-Knock-out Mice

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4098-4098
Author(s):  
Ernst Boehm ◽  
Michael Dockal ◽  
Meinhard Hasslacher ◽  
Artur Mitterer ◽  
Eva M Muchitsch ◽  
...  
Keyword(s):  
Factor Ix ◽  
Hek293 Cells ◽  
High Yield ◽  
High Similarity ◽  
Tyrosine Sulfation ◽  
Knock Out ◽  
Pharmacokinetic Properties ◽  
Knock Out Mice ◽  
In Vivo Recovery

Abstract Recombinant factor IX (rFIX) expressed in Chinese hamster ovary (CHO) cells has been shown to be safe and effective in clinical studies, but differs in pharmacokinetics from plasma-derived FIX (pdFIX). In clinical studies, CHO-derived rFIX had a 30–50 % lower in-vivo recovery when compared to pdFIX, whereas mean residence time and terminal half-life did not differ between preparations. Although rFIX shows high similarity to pdFIX in structure and function, differences in glycosylation and gamma-carboxylation degree can be detected. Moreover, although experimental proof has yet to be published, the lower degree of phosphorylation of amino acid serine 155, and the lower degree of sulfation of tyrosine 158 have been hypothesized to be causative for the lower in-vivo recovery of rFIX. These two modifications occur at less than 20 % for the tyrosine-sulfation and at less than 1 % for the serine phosphorylation in rFIX, whereas pdFIX has both modifications to more than 90 % completed. We identified human HEK293 cells to perform rFIX phosphorylation and sulfation to a higher extent than CHO cells. A rFIX-producing cell line derived from HEK293 cells was generated by stable transfection, and was adapted to suspension culture conditions to allow lab-scale fermentation. rFIX was produced and purified from a single fermentation run using two different down-stream process schemes: the first was able to enrich high-phosphorylated and -sulfated rFIX; the second to purify total rFIX from the supernatant at high yield. For pharmacokinetic comparison, these HEK293 materials, CHO-derived rFIX, and a pdFIX preparation were formulated in the same buffer. Determination of phosphorylation and sulfation by mass spectrometry showed a phosphorylation and sulfation degree of 50 % plus a 20 % single modification (phosphorylation or sulfation) for the HEK293-material purified by the modification enrichment method versus 15 % for both modifications plus a 15 % single modification for the material purified by the high-yield protocol. The values for CHO-derived rFIX and pdFIX were similar to those in the literature. Oligosaccharide mapping revealed glycosylation differences among CHO-, HEK293-, and pdFIX preparations, but high similarity between both HEK293-derived materials. We compared the pharmacokinetics of the various FIX preparations in FIX-knock-out mice. In-vivo recovery and area under the curve were statistically significantly higher for the high phosphorylated and sulfated HEK293-material than for total rFIX derived from HEK293 cells. However, these two parameters were lower for both HEK293-derived rFIX preparations than for CHO-derived rFIX, and lower for CHO-derived rFIX than for pdFIX. This may be due to glycosylation differences between these FIX preparations. Mean residence times and terminal half-lives were similar for all preparations. In summary, these findings emphasize that the degree of rFIX-sulfation and -phosphorylation influences the pharmacokinetic properties of rFIX.

scholarly journals Biochemical and Functional Comparisons ofmdxandSgcg−/−Muscular Dystrophy Mouse Models

2015 ◽  
Vol 2015 ◽  
pp. 1-11 ◽  
Author(s):  
Nathan W. Roberts ◽  
Jenan Holley-Cuthrell ◽  
Magdalis Gonzalez-Vega ◽  
Aaron J. Mull ◽  
Ahlke Heydemann
Keyword(s):  
Muscular Dystrophy ◽  
Mouse Models ◽  
Time Course ◽  
Genetically Engineered ◽  
Blue Dye ◽  
Null Mouse ◽  
Human Genetic Disease ◽  
Mild Disease ◽  
Diagnosis And Prognosis ◽  
Genetically Engineered Mice

Mouse models have provided an essential platform to investigate facets of human diseases, from etiology, diagnosis, and prognosis, to potential treatments. Muscular dystrophy (MD) is the most common human genetic disease occurring in approximately 1 in 2500 births. Themdxmouse, which is dystrophin-deficient, has long been used to model this disease. However, this mouse strain displays a rather mild disease course compared to human patients. Themdxmice have been bred to additional genetically engineered mice to worsen the disease. Alternatively, other genes which cause human MD have been genetically disrupted in mice. We are now comparing disease progression from one of these alternative gene disruptions, theγ-sarcoglycan null mouseSgcg−/−on the DBA2/J background, to themdxmouse line. This paper aims to assess the time-course severity of the disease in the mouse models and determine which is best for MD research. TheSgcg−/−mice have a more severe phenotype than themdxmice. Muscle function was assessed by plethysmography and echocardiography. Histologically theSgcg−/−mice displayed increased fibrosis and variable fiber size. By quantitative Evan’s blue dye uptake and hydroxyproline content two key disease determinants, membrane permeability and fibrosis respectively, were also proven worse in theSgcg−/−mice.

scholarly journals A Novel Lysine to Arginine Substitution at Position 301 Enhances Activity of Factor IX

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3772-3772
Author(s):  
Daniel Verhoef ◽  
Jonathan H. Foley ◽  
Andrew Goodale ◽  
Emma Macrae ◽  
Jenny McIntosh ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Specific Activity ◽  
Factor Ix ◽  
Wild Type ◽  
Gain Of Function ◽  
Employment Equity ◽  
Knock Out ◽  
Genome Integration ◽  
Equity Ownership ◽  
Knock Out Mice

Abstract Introduction: AAV-mediated gene transfer of blood coagulation Factor IX (FIX) has been established as a safe and long-term treatment for patients suffering from severe hereditary Haemophilia B. A gain-of-function F9 transgene (F9-R338L; Padua) has recently been used to achieve higher functional levels of FIX, effectively eliminating the need for regular prophylaxis. The naturally-occurring R338L Padua mutation is situated in the catalytic domain of FIX on a helical side loop (region 332-339) that is involved in FVIIIa-mediated stimulation of substrate turnover. Here, we examined if a single amino acid substitution of a lysine at position 301 leads to gain of function. This basic residue sits adjacent to the 332-339 loop on an exposed helical segment (292-303) that has been implicated to interact with the FVIIIa A2 domain in the FIXa-FVIIIa tenase complex. Methods: We examined the lysine at position 301 (numbering based on mature polypeptide chain) in more detail by conservative mutation to arginine (K301R) and non-conservative mutation to leucine (K301L). To assess specific FIX activity, F9-K301 variants were transiently expressed in HEK293T cells and tested for antigenic FIX levels and chromogenic activity 48 hours post transfection. To assess specific activity in plasma, AAV-mediated gene transfer (1x1010vg/mouse) of F9-K301 variants in hemophilia B knock-out mice (CL57B6) was carried out. In addition, we investigated whether the F9-K301R mutation enhances specific activity in combination with the F9-R338L Padua mutation via site-specific genome integration. Results: Transient transfection of F9-K301 variants in HEK293T cells showed a 25% increase in specific activity with F9-K301R but a 50% reduction in activity with F9-K301L as compared to wild type F9 (WT-F9). Validation of gain-of-function was done by AAV-mediated gene transfer in hemophilia B knock-out mice. Four weeks post injection, plasma FIX antigen levels were similar in mice transduced with either F9-K301R (0.91±0.3 U/ml; N=3), F9-K301L (0.93±0.0 U/ml; N=2) or WT-F9 (0.94±0.19 U/ml; N=4) constructs. Interestingly, specific chromogenic activity in plasma from F9-K301R mice (2.71±0.66 U/ml) was more than 2-fold higher compared to plasma from mice in the WT-F9 cohort (1.25±0.2 U/ml). On the other hand, specific activity in the F9-K301L cohort (0.37±0.07 U/ml) was reduced compared to wild type F9, consistent with a haemophilic phenotype. Next, we investigated whether the F9-K301R mutation enhances activity in combination with the F9-R338L Padua mutation. To do so, we stably expressed wild type FIX (WT-FIX) and three FIX gain-of-function variants (FIX-K301R, FIX-R338L and FIX-K301R/R338L) in HEK293 cells via site-specific genome integration. Interestingly, higher FIX antigen levels were observed in conditioned media from cells (1.5x106) stably expressing FIX-K301R (0.14±0.01 U/ml) FIX-R338L (0.11±0.01 U/ml) and FIX-K301R/R338L (0.10±0.01 U/ml) relative to cells expressing WT-FIX (0.08±0.01 U/ml). Similar to previous results, specific chromogenic activity was more than 2-fold higher in FIX-K301R (1.25±0.08 U/ml) compared to WT-FIX (0.54±0.06 U/ml). In addition, specific activity was higher in FIX-K301R/R338L (7.71±0.35 U/ml) compared to FIX-R338L (6.69±0.32 U/ml), suggesting molecular synergism between both gain-of-function mutations. Ongoing studies are focused on characterizing these recombinant FIX variants in purified and plasma-based activity assays and unraveling the mechanism(s) leading to increased expression/secretion of these gain-of-function variants. Conclusion: In summary, these results show that the K301R mutation enhances catalytic activity of FIX in vitro and in vivo and synergistically enhances activity in combination with the R338L Padua mutation. As such, this gain-of-function mutation could potentially serve to facilitate higher levels of FIX activity in the plasma of Haemophilia B patients following AAV-mediated gene transfer. Disclosures Verhoef: Freeline: Employment, Equity Ownership. Foley:Freeline: Employment, Equity Ownership. Goodale:Freeline: Employment, Equity Ownership. Macrae:Freeline: Employment, Equity Ownership. McIntosh:BioMarin: Patents & Royalties; Freeline: Consultancy, Equity Ownership. Corbau:Freeline: Employment, Equity Ownership. Nathwani:Freeline: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

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