Generation of a novel factor IX with augmented clotting activities in vitro and in vivo

Lin, Chien Nan; Kao, Chung‐Yao; Miao, Carol H.; Hamaguchi, Nobuko; Wu, Hua; Shi, Guey Yueh; Liu, Y. L.; High, Katherine A.; Lin, Shu‐Wha

doi:10.1111/j.1538-7836.2010.03913.x

Cited by 27 publications

(34 citation statements)

References 34 publications

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“…Hence the observed improvement in catalytic activity was almost certainly not caused through elimination of a putative thrombin cleavage site at amino acid 338 [37]. The observed increase in catalytic efficiency was rather the outcome of the greater binding affinity of FIXa-R338A to FVIIIa [40]. Since those reports several independent studies were implemented in the bioengineering of new combined variants with the substitution R338A in order to J Genet Syndr Gene Ther Gene Therapy for Hemophilia ISSN:2157-7412 JGSGT an open access journal obtain an additive effect in catalytic activity [40,41] and thereby to succeed more efficacious gene therapy approaches by means of novel FIX variants with enhanced procoagulant potential for treatment of hemophilia B [40,42].…”

Section: Engineering Fix Variants With Enhanced Specific Activitymentioning

confidence: 86%

“…Another group generated a similar increase in specific clotting activity (12.6 fold higher than wt-FIX) through replacement of the FIX-EGF-1 domain by the corresponding sequence of FVII supplementary to the R338A substitution [41]. Moreover, expression of FIX-R338A variants following liver-directed gene transfer led to an improvement in specific clotting activity and hemostasis [42] in hemophilia B mice following delivery of both adeno-associated viral (AAV) vectors [40,42] as well as helper-dependent adenoviral (HDAd) vectors [41]. Lastly, even one more efficacious variant with a single substitution at position 338 from arginine to leucine was identified in a patient with juvenile thrombophilia in the Padua University Hospital [43].…”

Section: Engineering Fix Variants With Enhanced Specific Activitymentioning

confidence: 96%

“…Since those reports several independent studies were implemented in the bioengineering of new combined variants with the substitution R338A in order to J Genet Syndr Gene Ther Gene Therapy for Hemophilia ISSN:2157-7412 JGSGT an open access journal obtain an additive effect in catalytic activity [40,41] and thereby to succeed more efficacious gene therapy approaches by means of novel FIX variants with enhanced procoagulant potential for treatment of hemophilia B [40,42]. A further increase in FIX activity could be achieved by combining R338A with two other mutations (V86A and E277A) resulting in a FIX triple variant which exhibited 13-fold higher specific coagulation activity and 10-fold higher affinity for human FVIIIa [40]. Another group generated a similar increase in specific clotting activity (12.6 fold higher than wt-FIX) through replacement of the FIX-EGF-1 domain by the corresponding sequence of FVII supplementary to the R338A substitution [41].…”

Section: Engineering Fix Variants With Enhanced Specific Activitymentioning

confidence: 99%

See 2 more Smart Citations

Engineered Factor VII, Factor IX, and Factor X Variants for Hemophilia Gene Therapy

2012

J Genet Syndr Gene Ther

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FVII/FVIIaPatients with hemophilia A and B have deficient protein concentrations of FVIII and FIX, respectively and therefore are treated by protein substitution with plasma-derived or recombinant factor FVIII or FIX concentrates. In about 20-30% of the patients with hemophilia A and in 3-5% of those with hemophilia B this can lead to the development of neutralizing antibodies against the infused proteins which makes therapy inefficient [1,2]. Recombinant activated factor VII (rFVIIa) protein is currently an alternative therapy available for inhibitor patients to treat excessive bleeding. However, supraphysiological concentrations and repeated administration are required to induce hemostasis [3].Consequently, FVIIa analogs were created for future protein replacement therapy or gene delivery approaches based on crystalline structure analysis of free and TF-bound FVIIa or on sequence homology of other more potent proteases [4,5].Like the other vitamin K dependent coagulation factors, FVII is physiologically synthesized in the liver. FVII is secreted into the circulation as a 406-residue single-chain polypeptide which contains an N-terminal γ-carboxyglutamic acid (Gla) domain in which all ten Glu residues are post-translationally carboxylated followed by two regions homologous to epidermal growth factor domains and a serine protease domain [3,6,7]. Upon vascular injury, the cofactor tissue factor (TF) is exposed to FVII and triggers the extrinsic pathway of the coagulation cascade by activation of FIX, factor X and FVII auto-activation on the TF-bearing cells resulting in large-scale thrombin generation and a fibrin clot. FVII is activated to FVIIa by internal proteolysis due to a cleavage of a single Arg152-Ile153 peptide bond. The physiological plasma concentration of FVII is around 10 nM, however, only about 1% of FVII is circulating in its free and active form. FVIIa has a plasma halflife of approximately 2.5 hours [8]. FVII activation results in connected FVII light and heavy chains which form a one-to-one complex with TF in the presence of Ca 2+ ions. The complex formation is an essential process in which TF allosterically leads to a fully active FVIIa [9]. The cleavage alone leaves FVIIa in a zymogen-like conformation of quite low specific activity [3,10]. Several residues in the first EGF-like domain of FVII and in the protease domain, especially methionine at position 306, seem to be pivotal for the cofactor-mediated allosteric stimulation [11]. TF stabilizes the active conformation of FVIIa for accelerated activation of its substrates FIX and FX [12,13] by inducing a conformational change and establishing a stable salt bridge between the residues Ile153 and Asp343 [14] and stabilization of the interaction between the residues Leu305 and Phe374 which in turn stabilizes S 1 and S 3 substrate pocket and the activation pocket [15]. These findings contributed to the development of FVIIa variants with enhanced TFindependent (intrinsic) specific activity by mimicking the effect of TF binding [16]. Additional fin...

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Section: Engineering Fix Variants With Enhanced Specific Activitymentioning

confidence: 86%

Section: Engineering Fix Variants With Enhanced Specific Activitymentioning

confidence: 96%

Section: Engineering Fix Variants With Enhanced Specific Activitymentioning

confidence: 99%

See 1 more Smart Citation

Engineered Factor VII, Factor IX, and Factor X Variants for Hemophilia Gene Therapy

2012

J Genet Syndr Gene Ther

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“…Another possibility would be to replace the wild-type FIX cDNA with a hyperactive form of FIX. Researchers have described that the R338L mutation resulted in 8-fold higher biological activity, whereas V86A, E277A, and R338A resulted in 13-fold higher biological activity than wild-type FIX (Simioni et al, 2009;Lin et al, 2010).…”

Section: Discussionmentioning

confidence: 99%

Genetically Modified Adipose Tissue-Derived Stem/Stromal Cells, Using Simian Immunodeficiency Virus-Based Lentiviral Vectors, in the Treatment of Hemophilia B

et al. 2013

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Hemophilia is an X-linked bleeding disorder, and patients with hemophilia are deficient in a biologically active coagulation factor. This study was designed to combine the efficiency of lentiviral vector transduction techniques with murine adipose tissue-derived stem/stromal cells (mADSCs) as a new method to produce secreted human coagulation factor IX (hFIX) and to treat hemophilia B. mADSCs were transduced with simian immunodeficiency virus (SIV)-hFIX lentiviral vector at multiplicities of infection (MOIs) from 1 to 60, and the most effective dose was at an MOI of 10, as determined by hFIX production. hFIX protein secretion persisted over the 28-day experimental period. Cell sheets composed of lentiviral vector-transduced mADSCs were engineered to further enhance the usefulness of these cells for future therapeutic applications in transplantation modalities. These experiments demonstrated that genetically transduced ADSCs may become a valuable cell source for establishing cell-based gene therapies for plasma protein deficiencies, such as hemophilia.

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“…This could be achieved through the use of hepatocyte-specific promoter/enhancers in conjunction with an additional layer of miR-regulation. Additionally, to further improve the clotting activities of FVIII and FIX, codonoptimization and protein engineering technologies can be applied [66,70,101,102]. LVs are well suited to achieve sustained hemostatic correction of hemophilia after genetic modification of HSC, MSC or BOEC.…”

Section: Conclusion and Future Perspectivesmentioning

confidence: 99%

Recent Advances and Future Perspectives in Lentiviral Gene Therapy for Hemophilia A and B

Mátrai¹,

Chuah²,

VandenDriessche³

2012

J Genet Syndr Gene Ther

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Hemophilia A and B are rare incurable hereditary diseases due to deficiencies in clotting factor VIII (FVIII) and factor IX (FIX), respectively. These genetic defects result in potentially life-threatening, uncontrolled bleeding episodes. Current treatment by protein substitution therapy does not constitute a cure making gene therapy an attractive alternative. Lentiviral vectors (LVs) have many distinctive features that make them especially well suited for FVIII or FIX gene delivery. This includes the lack of vector-specific pre-existing immunity, their ability to permanently transduce both dividing and non-dividing cells and their capacity to readily accommodate FIX and FVIII expression cassettes, consistent with their packaging capacity of 10 kb. LVs have been used to achieve sustained therapeutic clotting factor expression levels and hemostatic correction in preclinical hemophilic mouse models. The liver has been the target organ of choice for direct in vivo LV transduction of FVIII or FIX genes, resulting in sustained therapeutic effects. Nevertheless, the potential development of neutralizing antibodies to the clotting factors following ex vivo or in vivo gene therapy with LVs can preclude long-term phenotypic correction. These risks can be minimized by preventing ectopic expression in antigen-presenting cells. LVs are well suited to deliver the clotting factor genes into hematopoietic stem/progenitor cells, allowing for stable FVIII or FIX expression upon hematopoietic reconstitution. In addition, therapeutic FVIII and FIX expression levels have been achieved in vivo after transplantation of lentivirally transduced endothelial and mesenchymal stem/progenitor cells. Current challenges relate primarily to the translation of these findings to larger preclinical animal models and ultimately to patients suffering from hemophilia.

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Generation of a novel factor IX with augmented clotting activities in vitro and in vivo

Cited by 27 publications

References 34 publications

Engineered Factor VII, Factor IX, and Factor X Variants for Hemophilia Gene Therapy

Engineered Factor VII, Factor IX, and Factor X Variants for Hemophilia Gene Therapy

Genetically Modified Adipose Tissue-Derived Stem/Stromal Cells, Using Simian Immunodeficiency Virus-Based Lentiviral Vectors, in the Treatment of Hemophilia B

Recent Advances and Future Perspectives in Lentiviral Gene Therapy for Hemophilia A and B

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