Substitution Therapy in Haemophilia B

1961 ◽  
Vol 6 (02) ◽  
pp. 391-410 ◽  
Author(s):  
E. A Loeliger ◽  
A Hensen ◽  
M. J Mattern ◽  
E. E. J Alsbach
Keyword(s):  
Maintenance Dose ◽  
Factor Ix ◽  
Major Surgery ◽  
Substitution Therapy ◽  
Rapid Infusion ◽  
Haemophilia B ◽  
Human Factor Ix ◽  
Daily Dose ◽  
Daily Maintenance Dose

Conclusions1. Substitution therapy in haemophilia B without daily control of the Factor IX level in vivo by means of a reliable Factor IX assay cannot be adequate.2. The level of the Factor IX activity which, in cases of haemophilia B, ensures safe haemostasis after major trauma or in major surgery is at least 25% of normal.3. Assuming a normal t½ for Factor IX of 30 hours and a distribution pool of 7 l (normal adult), in cases of severe haemophilia the minimum amount of Factor IX to be rapidly transfused at the beginning of substitution (loading up of the pool up to 25%) is the amount present in 1.75 l of freshly drawn normal net plasma. The minimum daily dose necessary to maintain the 25% level is contained in 1 l. Due to the influence of body temperature, the required daily maintenance dose increases probably up to 2.4 l.4. Since rapid infusion of more than 1 l net plasma is not tolerated and since a daily dose of 2.4 l net plasma can hardly be achieved by means of exchange transfusion, partially purified and concentrated human Factor IX must be available. The plasma product PPSB, prepared by the “Centre National de Transfusion Sanguine” in Paris, contains, according to in vivo assays, about 10 times the activity of normal plasma for comparable volumes. The t½ of the Factor IX from PPSB appeared to be the same as from fresh plasma. The disappearance rate of the concentrated Factor IX from serum (CSB), prepared by the same institute, has not been estimated experimentally. According to clinical experience, however, the activity of CSB seems to be of a similar magnitude as that of PPSB. Adequate substitution with PPSB alone would require as initial loading-up dose 175 ml and as daily maintenance dose 100—240 ml of a 10-fold concentrated product, to be given in a continuous drip-infusion.5. In a case of mild haemophilia B (Factor IX ≅ 20%), transfusion of 0.8 l freshly drawn net plasma daily resulted in Factor IX levels of between 23 and 28% instead of the expected 35% of normal. This observation suggests that, for substitution therapy, mild cases of haemophilia B must be considered as rather severely affected.

scholarly journals Novel Caprine Adeno-Associated Virus (AAV) Capsid (AAV-Go.1) Is Closely Related to the Primate AAV-5 and Has Unique Tropism and Neutralization Properties

2005 ◽  
Vol 79 (24) ◽  
pp. 15238-15245 ◽  
Author(s):  
Alejandra E. Arbetman ◽  
Michael Lochrie ◽  
Shangzhen Zhou ◽  
Jennifer Wellman ◽  
Ciaran Scallan ◽  
...  
Keyword(s):  
Protein Expression ◽  
Neutralization Assay ◽  
Biological Properties ◽  
Scid Mice ◽  
Factor Ix ◽  
In Vivo Model ◽  
Adeno Associated Virus ◽  
Human Factor Ix

ABSTRACT Preexisting humoral immunity to adeno-associated virus (AAV) vectors may limit their clinical utility in gene delivery. We describe a novel caprine AAV (AAV-Go.1) capsid with unique biological properties. AAV-Go.1 capsid was cloned from goat-derived adenovirus preparations. Surprisingly, AAV-Go.1 capsid was 94% identical to the human AAV-5, with differences predicted to be largely on the surface and on or under the spike-like protrusions. In an in vitro neutralization assay using human immunoglobulin G (IgG) (intravenous immune globulin [IVIG]), AAV-Go.1 had higher resistance than AAV-5 (100-fold) and resistance similar to that of AAV-4 or AAV-8. In an in vivo model, SCID mice were pretreated with IVIG to generate normal human IgG plasma levels prior to the administration of AAV human factor IX vectors. Protein expression after intramuscular administration of AAV-Go.1 was unaffected in IVIG-pretreated mice, while it was reduced 5- and 10-fold after administration of AAV-1 and AAV-8, respectively. In contrast, protein expression after intravenous administration of AAV-Go.1 was reduced 7.1-fold, similar to the 3.8-fold reduction observed after AAV-8administration in IVIG-pretreated mice, and protein expression was essentially extinguished after AAV-2 administration in mice pretreated with much less IVIG (15-fold). AAV-Go.1 vectors also demonstrated a marked tropism for lung when administered intravenously in SCID mice. The pulmonary tropism and high neutralization resistance to human preexisting antibodies suggest novel therapeutic uses for AAV-Go.1 vectors, including targeting diseases such as cystic fibrosis. Nonprimate sources of AAVs may be useful to identify additional capsids with distinct tropisms and high resistance to neutralization by human preexisting antibodies.

Successful in vivo propagation of factor IX-producing hepatocytes in mice: Potential for cell-based therapy in haemophilia B

2008 ◽  
Vol 99 (11) ◽  
pp. 883-891 ◽  
Author(s):  
Kohei Tatsumi ◽  
Miho Kataoka ◽  
Masaru Shibata ◽  
Hiroyuki Naka ◽  
Midori Shima ◽  
...  
Keyword(s):  
Coagulation Factor ◽  
Isolated Hepatocytes ◽  
Factor Ix ◽  
Blood Levels ◽  
Recipient Mouse ◽  
Cytoplasmic Staining ◽  
Haemophilia B ◽  
Cell Based Therapy ◽  
Coagulation Factor Ix

SummaryCell-based therapies using isolated hepatocytes have been proposed to be an attractive application in the treatment of haemophilia B due to the normal production of coagulation factor IX (FIX) in these particular cells. Current cell culture technologies have largely failed to provide adequate isolated hepatocytes, so the present studies were designed to examine a new approach to efficiently proliferate hepatocytes that can retain normal biological function, including the ability to synthesize coagulation factors like FIX. Canine or human primary hepatocytes were transplanted into urokinase-type plasminogen activatorsevere combined immunodeficiency (uPA/SCID) transgenic mice. Both donor hepatocytes from canines and humans were found to progressively proliferate in the recipient mouse livers as evidenced by a sharp increase in the circulating blood levels of species-specific albumin, which was correlated with the production and release of canine and human FIX antigen levels into the plasma. Histological examination confirmed that the transplanted canine and human hepatocytes were able to proliferate and occupy >80% of the host livers. In addition, the transplanted hepatocytes demonstrated strong cytoplasmic staining for human FIX, and the secreted coagulation factor IX was found to be haemostatically competent using specific procoagulant assays. In all, the results from the present study indicated that developments based on this technology could provide sufficient FIX-producing hepatocytes for cell-based therapy for haemophilia B.

scholarly journals Electroimmunoassay of Factor IX in the Detection of Haemophilia B Carriers

1977 ◽  
Author(s):  
F. Panicucci ◽  
U. Baicchi ◽  
A. Sagripanti ◽  
E. Pinori
Keyword(s):  
Genetic Variants ◽  
Human Factor ◽  
Factor Ix ◽  
Agarose Gel ◽  
Haemophilia B ◽  
Method Factor ◽  
Human Factor Ix ◽  
Monospecific Antiserum ◽  
Normal Women ◽  
Almost All

Parallel determinations of factor IX activity and factor IX antigen were carried out on 28 haemophilia B carriers and on 20 normal women. Factor IX activity was measured by a one stage method. Factor IX antigen was quantified by electroimmuno assay in agarose gel containing heterologous monospecific antiserum against human factor IX.The activity was observed to be at the same level as the antigen in normal women. Discrepancy was not found between the antigen and the activity in almost all of carriers: only in the mothers of haemophiliacs Bor B+ or BM the factor IX antigen resulted greater than that activity. Our results show that electroimmuno-assay may be used to study carriers of haemophilia B genetic variants and confirm that in the majority of cases they can probably be diagnosed on the basis of factor IX activity alone.

scholarly journals Proteolytic inactivation of blood coagulation factor IX by thrombin

Blood ◽  
1985 ◽  
Vol 66 (6) ◽  
pp. 1302-1308 ◽  
Author(s):  
W Kisiel ◽  
KJ Smith ◽  
BA McMullen
Keyword(s):  
Human Factor ◽  
Coagulation Factor ◽  
Factor Ix ◽  
Light Chains ◽  
Factor X ◽  
Amino Terminal ◽  
Coagulation Factor Ix ◽  
Human Factor Ix ◽  
Coagulant Activity

Coagulation factor IX is a vitamin K-dependent glycoprotein that circulates in blood as a precursor of a serine protease. Incubation of human factor IX with human alpha-thrombin resulted in a time and enzyme concentration-dependent cleavage of factor IX yielding a molecule composed of a heavy chain (mol wt 50,000) and a doublet light chain (mol wt 10,000). The proteolysis of factor IX by thrombin was significantly inhibited by physiological levels of calcium ions. Under nondenaturing conditions, the heavy and light chains of thrombin- cleaved factor IX remained strongly associated, but these chains were readily separated by gel filtration in the presence of denaturants. Amino-terminal sequence analyses of the isolated heavy and light chains of thrombin-cleaved human factor IX indicated that thrombin cleaved peptide bonds at Arg327-Val328 and Arg338-Ser339 in this molecule. Comparable cleavages were observed in bovine factor IX by bovine thrombin and occurred at Arg319-Ser320 and Arg339-Ser340. Essentially, a complete loss of factor IX procoagulant activity was associated with its cleavage by thrombin. Furthermore, thrombin-cleaved factor IX neither developed coagulant activity after treatment with factor XIa nor inhibited the coagulant activity of native factor IX. These data indicate that thrombin cleaves factor IX near its active site serine residue, rendering it incapable of activating factor X. Whether or not this reaction occurs in vivo is unknown.

Adenovirus-mediated regulatable Expression of human Factor IX in vitro and in vivo

2003 ◽  
pp. 72-80
Author(s):  
M. A. Srour ◽  
H. Fechner ◽  
X. Wang ◽  
U. Siemetzki ◽  
T. Albert ◽  
...  
Keyword(s):  
Human Factor ◽  
Factor Ix ◽  
Human Factor Ix

scholarly journals 860. Sustained Long_Term In Vivo Expression of Human Factor IX from Keratinocytes Transduced with HIV_Based Vectors

2006 ◽  
Vol 13 ◽  
pp. S331-S332
Author(s):  
Angeles Escarti-Nebot ◽  
Fernando Serrano ◽  
Marcela delRio ◽  
Fernando Larcher ◽  
Antonio Bernad
Keyword(s):  
Human Factor ◽  
Factor Ix ◽  
Human Factor Ix ◽  
In Vivo Expression

scholarly journals Delivery of human factor IX in mice by encapsulated recombinant myoblasts: a novel approach towards allogeneic gene therapy of hemophilia B

Blood ◽  
1996 ◽  
Vol 87 (12) ◽  
pp. 5095-5103 ◽  
Author(s):  
G Hortelano ◽  
A Al-Hendy ◽  
FA Ofosu ◽  
PL Chang
Keyword(s):  
Gene Therapy ◽  
Human Factor ◽  
Ex Vivo ◽  
Cost Effective ◽  
Factor Ix ◽  
Hemophilia B ◽  
Therapy Strategy ◽  
Human Factor Ix

A potentially cost-effective strategy for gene therapy of hemophilia B is to create universal factor IX-secreting cell lines suitable for implantation into different patients. To avoid graft rejection, the implanted cells are enclosed in alginate-polylysine-alginate microcapsules that are permeable to factor IX diffusion, but impermeable to the hosts' immune mediators. This nonautologous approach was assessed by implanting encapsulated mouse myoblasts secreting human factor IX into allogeneic mice. Human factor IX was detected in the mouse plasma for up to 14 days maximally at approximately 4 ng/mL. Antibodies to human factor IX were detected after 3 weeks at escalating levels, which were sustained throughout the entire experiment (213 days). The antibodies accelerated the clearance of human factor IX from the circulation of the implanted mice and inhibited the detection of human factor IX in the mice plasma in vitro. The encapsulated myoblasts retrieved periodically from the implanted mice up to 213 days postimplantation were viable and continued to secrete human factor IX ex vivo at undiminished rates, hence suggesting continued factor IX gene expression in vivo. Thus, this allogeneic gene therapy strategy represents a potentially feasible alternative to autologous approaches for the treatment of hemophilia B.

A prospective registry of European haemophilia B patients receiving nonacog alfa, recombinant human factor IX, for usual use

Haemophilia ◽  
2011 ◽  
Vol 18 (4) ◽  
pp. 503-509 ◽  
Author(s):  
E. BERNTORP ◽  
D. KEELING ◽  
M. MAKRIS ◽  
A. TAGLIAFERRI ◽  
C. MALE ◽  
...  
Keyword(s):  
Human Factor ◽  
Factor Ix ◽  
Haemophilia B ◽  
Human Factor Ix ◽  
Recombinant Human Factor Ix

355 COMPARISON OF Tn5 AND SLEEPING BEAUTY SYSTEMS IN BOVINE EMBRYOS AND IN OVINE OFFSPRING

2015 ◽  
Vol 27 (1) ◽  
pp. 265
Author(s):  
R. J. Bevacqua ◽  
R. Fernandéz Martín ◽  
A. Gibbons ◽  
D. Teixeira ◽  
N. G. Canel ◽  
...  
Keyword(s):  
Sleeping Beauty ◽  
Factor Ix ◽  
Bovine Embryos ◽  
Embryo Viability ◽  
Fluorescent Reporter ◽  
Human Factor Ix ◽  
Recombinant Human Factor Ix ◽  
Beta Lactoglobulin

Current techniques for the production of transgenic domestic animals remain inefficient. Only recently, DNA transposons resulted in improved efficiencies for mouse and pig transgenesis. In this work, we evaluated Tn5 and Sleeping Beauty systems for transgenesis in bovine and ovine species. First, both transposon systems were assessed in vitro in bovine embryos employing transposons carrying fluorescent reporter genes. In vitro-produced bovine zygotes were microinjected with either 1) a complex of Tn5:egfp transposon (20 ng μL–1) (protein: transgene with mosaic ends recognised by Tn5, in Mg+2 free medium), or 2) two plasmids carrying Sleeping Beauty 100X (pSB100X, 5 ng μL–1) and pT2/Venus transposon (10 ng μL–1). In vitro results for Tn5 transgenesis in bovine showed that blastocysts, Day 4 egfp embryos and egfp blastocysts rates for the group injected with Tn5:egfp did not differ from the group injected with the egfp transposon alone (73/145, 50%; 86/145, 59%; and 65/145, 45% v. 65/129, 50%; 87/129, 67%; and 57/129, 44%, respectively). For SB transgenesis, blastocysts, D4 Venus embryos, and Venus blastocysts rates did not differ between co-injection of pSB100X and pT2/Venus or injection with pT2/Venus alone (46/99, 46.5%; 64/99, 64.6%; and 33/99, 33.3% v. 41/83, 49.4%; 52/83, 62.7%; and 26/83, 31.3%, respectively). However, Venus intensity in blastocysts was markedly higher for the group co-injected with pSB100X and pT2/Venus respective to pT2/Venus alone. Both systems were assessed in vivo for the production of transgenic lambs employing a functional transposon (hrFIX, recombinant human factor IX driven by a Beta-lactoglobulin promoter). Laparoscopic artificial insemination of donor sheep was performed, and presumptive zygotes were flushed from the oviducts. The microinjections were done identically as described for the bovine embryos. A total of 24 presumptive zygotes were recovered and injected with the Tn5:hrFIX complex. Then, 21 zygotes were transferred to 5 synchronized ewes; one pregnancy of siblings was obtained, and one animal was born. Genomic DNA from skin, placenta, and blood was genotyped by PCR, but the hrFIX gene could not be detected. For the SB approach, 64 presumptive zygotes were recovered from 4 superovulated ewes, microinjected with the SB plasmids, and 21 of them were transferred to 7 oestrous synchronized recipients. The remaining zygotes were cultured in vitro and blastocysts (n = 7) were vitrified. Currently, 3 donor ewes are pregnant, one with siblings (4 total fetuses). Deliveries are expected by the end of August of this year. Our results indicate that both Tn5 and SB systems are capable of resulting in the production of transgene expressing embryos, and the presence of the transposases does not affect embryo viability. However, phenotyping of blastocyst stages does not seem to be predictive for stable transgene integration. The in vivo results will help to better address the suitability of Tn5 and SB approaches for the production of transgenic sheep.

scholarly journals Proteolytic inactivation of blood coagulation factor IX by thrombin

Blood ◽  
1985 ◽  
Vol 66 (6) ◽  
pp. 1302-1308 ◽  
Author(s):  
W Kisiel ◽  
KJ Smith ◽  
BA McMullen
Keyword(s):  
Human Factor ◽  
Coagulation Factor ◽  
Factor Ix ◽  
Light Chains ◽  
Factor X ◽  
Amino Terminal ◽  
Coagulation Factor Ix ◽  
Human Factor Ix ◽  
Coagulant Activity

Abstract Coagulation factor IX is a vitamin K-dependent glycoprotein that circulates in blood as a precursor of a serine protease. Incubation of human factor IX with human alpha-thrombin resulted in a time and enzyme concentration-dependent cleavage of factor IX yielding a molecule composed of a heavy chain (mol wt 50,000) and a doublet light chain (mol wt 10,000). The proteolysis of factor IX by thrombin was significantly inhibited by physiological levels of calcium ions. Under nondenaturing conditions, the heavy and light chains of thrombin- cleaved factor IX remained strongly associated, but these chains were readily separated by gel filtration in the presence of denaturants. Amino-terminal sequence analyses of the isolated heavy and light chains of thrombin-cleaved human factor IX indicated that thrombin cleaved peptide bonds at Arg327-Val328 and Arg338-Ser339 in this molecule. Comparable cleavages were observed in bovine factor IX by bovine thrombin and occurred at Arg319-Ser320 and Arg339-Ser340. Essentially, a complete loss of factor IX procoagulant activity was associated with its cleavage by thrombin. Furthermore, thrombin-cleaved factor IX neither developed coagulant activity after treatment with factor XIa nor inhibited the coagulant activity of native factor IX. These data indicate that thrombin cleaves factor IX near its active site serine residue, rendering it incapable of activating factor X. Whether or not this reaction occurs in vivo is unknown.

Sign in / Sign up

Export Citation Format

Share Document