Bioengineering of coagulation factor VIII for improved secretion

Blood ◽  
2004 ◽  
Vol 103 (9) ◽  
pp. 3412-3419 ◽  
Author(s):  
Hongzhi Z. Miao ◽  
Nongnuch Sirachainan ◽  
Lisa Palmer ◽  
Phillip Kucab ◽  
Michael A. Cunningham ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Factor Viii ◽  
Hemophilia A ◽  
Coagulation Factor ◽  
Mrna Levels ◽  
Golgi Transport ◽  
Recombinant Fviii ◽  
Therapy Strategies

Abstract Factor VIII (FVIII) functions as a cofactor within the intrinsic pathway of blood coagulation. Quantitative or qualitative deficiencies of FVIII result in the inherited bleeding disorder hemophilia A. Expression of FVIII (domain structure A1-A2-B-A3-C1-C2) in heterologous mammalian systems is 2 to 3 orders of magnitude less efficient compared with other proteins of similar size compromising recombinant FVIII production and gene therapy strategies. FVIII expression is limited by unstable mRNA, interaction with endoplasmic reticulum (ER) chaperones, and a requirement for facilitated ER to Golgi transport through interaction with the mannose-binding lectin LMAN1. Bioengineering strategies can overcome each of these limitations. B-domain-deleted (BDD)-FVIII yields higher mRNA levels, and targeted point mutations within the A1 domain reduce interaction with the ER chaperone immunoglobulin-binding protein. In order to increase ER to Golgi transport we engineered several asparagine-linked oligosaccharides within a short B-domain spacer within BDD-FVIII. A bioengineered FVIII incorporating all of these elements was secreted 15- to 25-fold more efficiently than full-length FVIII both in vitro and in vivo. FVIII bioengineered for improved secretion will significantly increase potential for success in gene therapy strategies for hemophilia A as well as improve recombinant FVIII production in cell culture manufacturing or transgenic animals. (Blood. 2004;103: 3412-3419)

scholarly journals Biochemical, immunological, and in vivo functional characterization of B-domain-deleted factor VIII

Blood ◽  
1993 ◽  
Vol 81 (11) ◽  
pp. 2925-2935 ◽  
Author(s):  
DD Pittman ◽  
EM Alderman ◽  
KN Tomkinson ◽  
JH Wang ◽  
AR Giles ◽  
...  
Keyword(s):  
Factor Viii ◽  
Hemophilia A ◽  
Specific Activity ◽  
Functional Characterization ◽  
Chinese Hamster ◽  
Coagulation Factor ◽  
Thrombin Activation ◽  
Recombinant Fviii

Abstract Coagulation factor VIII (FVIII) is a cofactor in the intrinsic pathway of blood coagulation for which deficiency results in the bleeding disorder hemophilia A. FVIII contains a domain structure of A1-A2-B-A3- C1-C2 of which the B domain is dispensable for procoagulant activity in vitro. In this report, we compare the properties of B-domain-deleted FVIII (residues 760 through 1639, designated LA-VIII) to wildtype recombinant FVIII. In transfected Chinese hamster ovary (CHO) cells, LA- VIII was expressed at a 10- to 20-fold greater level compared with wildtype FVIII. The specific activity of purified LA-VIII was indistinguishable from wild-type recombinant FVIII and both exhibited similar thrombin activation coefficients. Wildtype recombinant-derived FVIII and LA-VIII also displayed similar timecourses of thrombin activation and heavy chain cleavage. However, compared with wildtype recombinant-derived FVIII, the light chain of LA-VIII was cleaved fivefold more rapidly by thrombin. Addition of purified von Willebrand factor (vWF) did not alter the kinetics of thrombin cleavage or activation of either wildtype recombinant-derived FVIII or LA-VIII. The immunogenicity of LA-VIII was compared with wildtype FVIII in a novel model of neonatal tolerance induction in mice. The results did not detect any immunologic differences between wildtype FVIII and LA-VIII, suggesting that LA-VIII does not contain significant new epitopes that are absent in wildtype FVIII. LA-VIII was tolerated well on infusion into FVIII-deficient dogs and was able to correct the cuticle bleeding time similar to wildtype recombinant factor VIII. In vivo, LA-VIII was bound to canine vWF and exhibited a half-life similar to wildtype recombinant FVIII. These studies support that B-domain-deleted FVIII may be efficacious in treatment of hemophilia A in humans.

scholarly journals Biochemical, immunological, and in vivo functional characterization of B-domain-deleted factor VIII

Blood ◽  
1993 ◽  
Vol 81 (11) ◽  
pp. 2925-2935 ◽  
Author(s):  
DD Pittman ◽  
EM Alderman ◽  
KN Tomkinson ◽  
JH Wang ◽  
AR Giles ◽  
...  
Keyword(s):  
Factor Viii ◽  
Hemophilia A ◽  
Specific Activity ◽  
Functional Characterization ◽  
Chinese Hamster ◽  
Coagulation Factor ◽  
Thrombin Activation ◽  
Recombinant Fviii

Coagulation factor VIII (FVIII) is a cofactor in the intrinsic pathway of blood coagulation for which deficiency results in the bleeding disorder hemophilia A. FVIII contains a domain structure of A1-A2-B-A3- C1-C2 of which the B domain is dispensable for procoagulant activity in vitro. In this report, we compare the properties of B-domain-deleted FVIII (residues 760 through 1639, designated LA-VIII) to wildtype recombinant FVIII. In transfected Chinese hamster ovary (CHO) cells, LA- VIII was expressed at a 10- to 20-fold greater level compared with wildtype FVIII. The specific activity of purified LA-VIII was indistinguishable from wild-type recombinant FVIII and both exhibited similar thrombin activation coefficients. Wildtype recombinant-derived FVIII and LA-VIII also displayed similar timecourses of thrombin activation and heavy chain cleavage. However, compared with wildtype recombinant-derived FVIII, the light chain of LA-VIII was cleaved fivefold more rapidly by thrombin. Addition of purified von Willebrand factor (vWF) did not alter the kinetics of thrombin cleavage or activation of either wildtype recombinant-derived FVIII or LA-VIII. The immunogenicity of LA-VIII was compared with wildtype FVIII in a novel model of neonatal tolerance induction in mice. The results did not detect any immunologic differences between wildtype FVIII and LA-VIII, suggesting that LA-VIII does not contain significant new epitopes that are absent in wildtype FVIII. LA-VIII was tolerated well on infusion into FVIII-deficient dogs and was able to correct the cuticle bleeding time similar to wildtype recombinant factor VIII. In vivo, LA-VIII was bound to canine vWF and exhibited a half-life similar to wildtype recombinant FVIII. These studies support that B-domain-deleted FVIII may be efficacious in treatment of hemophilia A in humans.

Codon optimization of human factor VIII cDNAs leads to high-level expression

Blood ◽  
2011 ◽  
Vol 117 (3) ◽  
pp. 798-807 ◽  
Author(s):  
Natalie J. Ward ◽  
Suzanne M. K. Buckley ◽  
Simon N. Waddington ◽  
Thierry VandenDriessche ◽  
Marinee K. L. Chuah ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Factor Viii ◽  
Viral Vectors ◽  
Hemophilia A ◽  
Lentiviral Vector ◽  
Wild Type ◽  
Fviii Activity ◽  
Human Factor Viii

Abstract Gene therapy for hemophilia A would be facilitated by development of smaller expression cassettes encoding factor VIII (FVIII), which demonstrate improved biosynthesis and/or enhanced biologic properties. B domain deleted (BDD) FVIII retains full procoagulant function and is expressed at higher levels than wild-type FVIII. However, a partial BDD FVIII, leaving an N-terminal 226 amino acid stretch (N6), increases in vitro secretion of FVIII tenfold compared with BDD-FVIII. In this study, we tested various BDD constructs in the context of either wild-type or codon-optimized cDNA sequences expressed under control of the strong, ubiquitous Spleen Focus Forming Virus promoter within a self-inactivating HIV-based lentiviral vector. Transduced 293T cells in vitro demonstrated detectable FVIII activity. Hemophilic mice treated with lentiviral vectors showed expression of FVIII activity and phenotypic correction sustained over 250 days. Importantly, codon-optimized constructs achieved an unprecedented 29- to 44-fold increase in expression, yielding more than 200% normal human FVIII levels. Addition of B domain sequences to BDD-FVIII did not significantly increase in vivo expression. These significant findings demonstrate that shorter FVIII constructs that can be more easily accommodated in viral vectors can result in increased therapeutic efficacy and may deliver effective gene therapy for hemophilia A.

scholarly journals A mutated factor X activatable by thrombin corrects bleedings in vivo in a rabbit model of antibody-induced hemophilia A

Haematologica ◽  
2019 ◽  
Vol 105 (9) ◽  
pp. 2335-2340
Author(s):  
Toufik Abache ◽  
Alexandre Fontayne ◽  
Dominique Grenier ◽  
Emilie Jacque ◽  
Alain Longue ◽  
...  
Keyword(s):  
Factor Viii ◽  
Hemophilia A ◽  
Factor Viia ◽  
Rabbit Model ◽  
Coagulation Factor ◽  
Factor Ix ◽  
Factor X ◽  
Factor Viiia

Rendering coagulation factor X sensitive to thrombin was proposed as a strategy that can bypass the need for factor VIII. In this paper, this non-replacement strategy was evaluated in vitro and in vivo in its ability to correct factor VIII but also factor IX, X and XI deficiencies. A novel modified factor X, named Actiten, was generated and produced in the HEK293F cell line. The molecule possesses the required post-translational modifications, partially keeps its ability to be activated by RVV-X, factor VIIa/tissue factor, factor VIIIa/factor IXa and acquires the ability to be activated by thrombin. The potency of the molecule was evaluated in respective deficient plasmas or hemophilia A plasmas, for some with inhibitors. Actiten corrects dose dependently all the assayed deficient plasmas. It is able to normalize the thrombin generation at 20 μg/mL showing however an increased lagtime. It was then assayed in a rabbit antibody-induced model of hemophilia A where, in contrast to recombinant factor X wild-type, it normalized the bleeding time and the loss of hemoglobin. No sign of thrombogenicity was observed and the generation of activated factor X was controlled by the anticoagulation pathway in all performed coagulation assays. This data indicates that Actiten may be considered as a possible non replacement factor to treat hemophilia's with the advantage of being a zymogen correcting bleedings only when needed.

scholarly journals Recombinant VWF Fragments Improve Bioavailability of Subcutaneous Factor VIII in Hemophilia A Mice

Blood ◽  
2020 ◽  
Author(s):  
Nadine Vollack-Hesse ◽  
Olga Oleshko ◽  
Sonja Werwitzke ◽  
Barbara Solecka-Witulska ◽  
Christoph Kannicht ◽  
...  
Keyword(s):  
Factor Viii ◽  
Half Life ◽  
Hemophilia A ◽  
Coagulation Factor ◽  
Dependent Manner ◽  
High Concentrations ◽  
Von Willebrand ◽  
Willebrand Factor

Conventional treatment of hemophilia A (HA) requires repetitive intravenous (IV) injection of coagulation factor VIII (FVIII). Subcutaneous (SC) administration of FVIII is inefficient because of binding to the extravascular matrix, in particular to phospholipids (PL), and subsequent proteolysis. To overcome this, recombinant dimeric fragments of von Willebrand factor (VWF) containing the FVIII stabilizing D3 domain were engineered. Two fragments, called VWF-12 and VWF-13, demonstrated high binding affinity to recombinant human FVIII (rhFVIII) and suppressed PL-binding in a dose-dependent manner. High concentrations of VWF fragments did not interfere with the functional properties of full-length VWF in vitro. The HA mouse model was used to study the effects of VWF-12 or VWF-13 on the in vivo pharmacokinetics of rhFVIII, demonstrating (i) no significant impact on rhFVIII recovery or half-life after a single IV administration; (ii) enhanced bioavailability (up to 18.5 %) of rhFVIII after SC administration; (iii) slow absorption (cmax 6h) and prolonged half-life (up to 2.5-fold) of rhFVIII after SC administration. Formation of anti-FVIII antibodies was not increased after administration of rhFVIII/VWF-12 SC compared to rhFVIII IV. A single SC dose of rhFVIII/VWF-12 provided protection in the HA tail bleeding model for up to 24h. In conclusion, recombinant VWF fragments support FVIII delivery through the SC space into vascular circulation without interfering with VWF or FVIII function. Slow resorption and excretion of FVIII after SC administration highlight the potential application of VWF fragments for SC FVIII prophylaxis in HA.

DDAVP-Induced Increase of Factor VIII Activity in Blood Is Likely Due To Release of Factor VIII That Is Synthesized by Endothelial Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 692-692 ◽  
Author(s):  
Lingfei Xu ◽  
Timothy C. Nichols ◽  
Stephanie McCorquodale ◽  
Aaron Dillow ◽  
Elizabeth Merricks ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Endothelial Cells ◽  
Factor Viii ◽  
Hemophilia A ◽  
Fold Increase ◽  
Von Willebrand Disease ◽  
Major Site ◽  
Von Willebrand

Abstract Desmopressin (1-deamino-8-D-arginine vasopressin, DDAVP) is commonly used as a nonreplacement therapy for mild von Willebrand disease (VWD) and hemophilia A. In humans, IV injection of 0.3 μg/kg of DDAVP induces a rapid 2 to 5-fold increase in plasma levels of both von Willebrand factor (VWF) and Factor VIII (FVIII) within 30–60 minutes, which is due to release from Wiebel-Palade bodies (WPBs) in endothelial cells. The stored FVIII may be synthesized by endothelial cells, which express FVIII in vitro. However, hepatoma cells can also express FVIII in vitro, and liver transplantation can correct hemophilia A. Thus, the liver may be the major site of production of FVIII in vivo, thus, an alternative explanation is that endothelial cells take up FVIII from blood and store it in WPBs with VWF, which can be released after DDAVP. DDAVP is effective in humans and dogs, but not in mice. In this study, we tested the effect of DDAVP on hemophilia A dogs after neonatal hepatic gene therapy with a retroviral vector (RV) expressing canine FVIII (cFVIII). With this gene therapy approach, canine hepatocytes express high levels of a reporter gene from an RV, but no expression is observed in endothelial cells. Thus, the major site of FVIII synthesis is the hepatocyte in this model. Our hypothesis is that if DDAVP increases FVIII levels in this dog model, it would indicate that the FVIII increase is due to uptake from blood by endothelial cells. Alternatively, if no increase in FVIII occurs after DDAVP stimulation, it would suggest that the increase in normal dogs is due to synthesis of FVIII by endothelial cells. An RV that contains the liver-specific human α1-antitrypsin promoter and the canine B-domain deleted FVIII cDNA was generated. RV was given IV to two hemophilia A dogs at 8x109 transducing units (TU)/kg at 3 days after birth. The whole blood clotting time (WBCT) and APTT time in both dogs have been normalized, and the plasma cFVIII COATEST activity has been maintained at 100–200% of normal for 11 months to date. DDAVP was injected IV at 0.5 μg/kg into RV-treated hemophilia A dogs at 7 months of age. Two separate doses of DDAVP were given with an interval of one week. The same dose of DDAVP was given to normal dogs as controls (N=4). In normal dogs, both VWF and FVIII levels increased 40% and 50% between 15 to 60 minutes after DDAVP, respectively. However, FVIII levels were not changed in RV-treated dogs, although VWF levels increased 150% or 60%. Thus, our data suggest that the normal FVIII increase after DDAVP administration is due to release of FVIII that is synthesized by endothelial cells. These data also demonstrate that DDAVP will not be effective at increasing FVIII activity in patients that receive liver-directed gene therapy and only achieve partial correction. Such patients would need to be treated with factor replacement if bleeding episodes occur.

Development of neutralizing antibodies in application of various concentrates of blood coagulation factor VIII in the treatment of hemophilia A

2021 ◽  
Author(s):  
Н.И. Зозуля
Keyword(s):  
Factor Viii ◽  
Cell Line ◽  
Neutralizing Antibodies ◽  
Hemophilia A ◽  
Chinese Hamster ◽  
Coagulation Factor ◽  
Human Cell Line ◽  
Chinese Hamster Cell ◽  
Coagulation Factor Viii ◽  
Recombinant Fviii

Серьезным осложнением, связанным с лечением гемофилии А, является развитие ингибиторов. В последние годы был проведён ряд исследований, посвящённых данной проблеме: RODIN, INSIGHT, FranceCoag, SIPPET и NuProtect. В данном обзоре суммируются основные результаты этих исследований. Согласно результатам рандомизированного исследования SIPPET, препараты плазматического фактора свертывания крови VIII (FVIII) обладают меньшей иммуногенностью, чем препараты рекомбинантного FVIII, синтезированного из клеточной линии китайских хомячков, что следует учитывать при выборе стратегии лечения. Согласно результатам исследования NuProtect, опубликованным в 2019 г., концентрат рекомбинантного FVIII, полученный из клеточной линии человека, демонстрирует профиль иммуногенности, сходный с таковым у препаратов плазматического FVIII. У ранее нелеченых пациентов с ненулевыми мутациями при применении симоктоког альфа не наблюдалось образования ингибиторов, также как и в случае применения препаратов плазматического FVIII в исследовании SIPPET. Inhibitor development is a serious complication associated with hemophilia A therapy. A number of studies have been carried out of this issue — RODIN, INSIGHT, FranceCoag, SIPPET, and NuProtect. This review summarizes the main results of these studies. According to the results of the SIPPET randomized trial, plasma-derived coagulation factor VIII (FVIII) products are less immunogenic than recombinant FVIII products synthesized from a Chinese hamster cell line; this fact should be taken into account in choosing a treatment strategy. According to the results of NuProtect study published in 2019, the concentrate of human cell line-derived recombinant FVIII demonstrates immunogenicity profi le similar to the one in plasma-derived FVIII products. Previously untreated patients with non-zero mutations receiving simoctocog alfa did not show development of inhibitors as well as in case of administration of plasma-derived FVIII products in SIPPET study.

IN VIVO STUDIES ON RED CELLS AND PLATELETS STORED IN HALFSTRENGTH CITRATE

1987 ◽  
Author(s):  
C Prewse ◽  
K Bell ◽  
B Griffin
Keyword(s):  
Factor Viii ◽  
Coagulation Factor ◽  
Red Cells ◽  
In Vivo Studies ◽  
Dramatic Improvement ◽  
Half Strength ◽  
Survival Studies ◽  
Cellular Components

We have previously shown that donation of blood into anticoagulants containing half the normal amount of citrate results in a dramatic improvement in the stability of coagulation factor VIII and has no adverse effect on the in vitro qualities of red cells or platelets during storage. To confirm the viability of stored cellular components we are now performing autologous survival studies in healthy volunteers using radiolabelled cells from red cells and platelets stored for 35 and 5 days respectively. Results to date indicate a 24 hour survival of 80% for red cells stored at a haematocrit of 0.70 for 35 days. Infusion of Ill-In oxine labelled platelets after storage for 5 days in full or half-strength citrate gave recoveries of 40% and survivals of 7 days. These encouraging results suggest use of halfstrength citrate may be a route to increasing factor VIII supply without any additional donor recruitment. Further in vitro studies have also been performed on cellular components and reveal adequate in vitro quality for half-strength citrate blood held at room temperature for 20 hours prior to component preparation.

Animal Testing of Retroviral-Mediated Gene Therapy for Factor VIII Deficiency

1999 ◽  
Vol 82 (08) ◽  
pp. 555-561 ◽  
Author(s):  
Douglas Jolly ◽  
Judith Greengard
Keyword(s):  
Gene Therapy ◽  
Factor Viii ◽  
Hemophilia A ◽  
Joint Damage ◽  
Coagulation Factor ◽  
The United States ◽  
Severe Hemophilia ◽  
Factor Viii Gene ◽  
Bleeding Episodes

IntroductionHemophilia A results from the plasma deficiency of factor VIII, a gene carried on the X chromosome. Bleeding results from a lack of coagulation factor VIII, a large and complex protein that circulates in complex with its carrier, von Willebrand factor (vWF).1 Severe hemophilia A (<1% of normal circulating levels) is associated with a high degree of mortality, due to spontaneous and trauma-induced, life-threatening and crippling bleeding episodes.2 Current treatment in the United States consists of infusion of plasma-derived or recombinant factor VIII in response to bleeding episodes.3 Such treatment fails to prevent cumulative joint damage, a major cause of hemophilia-associated morbidity.4 Availability of prophylactic treatment, which would reduce the number and severity of bleeding episodes and, consequently, would limit such joint damage, is limited by cost and the problems associated with repeated venous access. Other problems are associated with frequent replacement treatment, including the dangers of transmission of blood-borne infections derived from plasma used as a source of factor VIII or tissue culture or formulation components. These dangers are reduced, but not eliminated, by current manufacturing techniques. Furthermore, approximately 1 in 5 patients with severe hemophilia treated with recombinant or plasma-derived factor VIII develop inhibitory humoral immune responses. In some cases, new inhibitors have developed, apparently in response to unnatural modifications introduced during manufacture or purification.5 Gene therapy could circumvent most of these difficulties. In theory, a single injection of a vector encoding the factor VIII gene could provide constant plasma levels of factor in the long term. However, long-term expression after gene transfer of a systemically expressed protein in higher mammals has seldom been described. In some cases, a vector that appeared promising in a rodent model has not worked well in larger animals, for example, due to a massive immune response not seen in the rodent.6 An excellent review of early efforts at factor VIII gene therapy appeared in an earlier volume of this series.7 A summary of results from various in vivo experiments is shown in Table 1. This chapter will focus on results pertaining to studies using vectors based on murine retroviruses, including our own work.

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