Novel therapeutic approach for hemophilia using gene delivery of an engineered secreted activated Factor VII

Margaritis, Paris; Arruda, Valder R.; Aljamali, Majed N.; Camire, Rodney M.; Schlachterman, Alexander; High, Katherine A.

doi:10.1172/jci20106

Cited by 56 publications

(71 citation statements)

References 44 publications

(24 reference statements)

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“…In an effort to bypass these drawbacks, we have previously demonstrated that a factor VII transgene engineered to be secreted in its activated form (FVIIa) can correct the hemostatic parameters in mouse models of hemophilia A (HA) and B (HB), after either adeno-associated viral (AAV) vector delivery 9 or in transgenic animals. 10 However, a necessary step preceding human application is the demonstration of safety and efficacy in a large animal (canine) model.…”

Section: Introductionmentioning

confidence: 99%

Successful treatment of canine hemophilia by continuous expression of canine FVIIa

Margaritis

Roy

Aljamali

et al. 2009

Blood

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103

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Section: Introductionmentioning

confidence: 99%

Successful treatment of canine hemophilia by continuous expression of canine FVIIa

Margaritis

Roy

Aljamali

et al. 2009

Blood

Self Cite

103

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“…Expression levels of the transgene reached up to 1000 ng/ml with the highest vector dose injected and didn't induce any thrombotic complications but still corrected phenotype [31,32]. In contrast, the overexpression at levels greater than 2 µg/ml caused mortality and pathological changes in heart and lung [33].…”

Section: Preclinical Fviia Gene Therapy Studiesmentioning

confidence: 99%

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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“…The enhanced GFP and the ATP7B molecule, identical to the coding sequence of human isoform a (GenBank reference sequence NM_000053.2), were inserted into the HIV-1-based transfer vector, a modified FG12 plasmid, 32 under the transcriptional control of a LSP, which contained elements of apolipoprotein E and alpha-1 antitrypsin, making it hepatocyte specific. 33 The transgene cassette contained the enhanced green fluorescent protein, eGFP reporter (Clontech) or both the reporter and the full-length ATP7B cDNA molecule. Bicistronic expression was achieved by inserting the therapeutic gene in-frame with the eGFP cDNA and the TaV sequence, a cis-acting hydrolase element derived from Thosea asigna virus.…”

Section: Lentiviral Vector Designmentioning

confidence: 99%

Early gestational gene transfer with targeted ATP7B expression in the liver improves phenotype in a murine model of Wilson's disease

et al. 2011

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The ideal gene therapy for metabolical liver disorders would target hepatocytes before the onset of disease and be durable, non-toxic and non-immunogenic. Early gestational gene transfer can achieve such goals. Here, we demonstrate that prenatal gene transfer of human Atp7b reduces liver pathology and improves biochemical markers in Atp7b À/À mice, a murine model of Wilson's disease (WD). Following prenatal injection of lentivirus vector containing the human Atp7b gene under the transcriptional control of a liver-specific promoter, the full-length ATP7B was detectable in mouse livers for the entire duration of experiments (20 weeks after birth). In contrast to a marked pathology in non-injected animals, livers from age-matched treated mice consistently demonstrated normal gross and histological morphology. Hepatic copper content was decreased in the majority of treated mice, although remaining copper levels varied. Improvement of hepatic copper metabolism was further apparent from the presence of copper-bound ceruloplasmin in the sera and normalization of the mRNA levels for HMG CoAreductase. With this approach, the complete loss of copper transport function can be ameliorated, as evident from phenotypical improvement in treated Atp7b À/À mice. This study provides proof of principle for in utero gene therapy in WD and other liver-based enzyme deficiencies.

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Novel therapeutic approach for hemophilia using gene delivery of an engineered secreted activated Factor VII

Cited by 56 publications

References 44 publications

Successful treatment of canine hemophilia by continuous expression of canine FVIIa

Successful treatment of canine hemophilia by continuous expression of canine FVIIa

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

Early gestational gene transfer with targeted ATP7B expression in the liver improves phenotype in a murine model of Wilson's disease

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