Expression and characterization of a novel human recombinant factor IX molecule with enhanced in vitro and in vivo clotting activity

Perot, Eloïse; Enjolras, Nathalie; Quellec, Sandra Le; Indalecio, Alice; Girard, J.; Négrier, Claude; Dargaud, Yesim

doi:10.1016/j.thromres.2015.02.034

Cited by 15 publications

(23 citation statements)

References 37 publications

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“…The preclinical development of FIX-Padua may provide a roadmap for how the efficacy and safety concerns of immunogenicity and thrombogenicity could begin to be addressed for these variants 135, 136, 137, 138, 139, 140Table 3Hyperactive FIX VariantsAmino Acid SubstitutionsaFold Change in FIX Activity (Relative to FIX-WT)bReferencesG4Y1.2 161 V10K1.6161, 211V86A1.1 212 K265T1.9213, 214E277A1.3 212 R338L8.0 131 N346A1.2 215 S377W1.4 161 E410H4.6 216 R338L + S377W12 161 V10K + R338L + S337W19 161 R318Y + R338E + T343R17(S.-B. Hong et al., 2016, Am.…”

Section: Main Textmentioning

confidence: 99%

Protein-Engineered Coagulation Factors for Hemophilia Gene Therapy

Samelson-Jones

Arruda

2019

Molecular Therapy - Methods & Clinical Development

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Hemophilia A (HA) and hemophilia B (HB) are X-linked bleeding disorders due to inheritable deficiencies in either coagulation factor VIII (FVIII) or factor IX (FIX), respectively. Recently, gene therapy clinical trials with adeno-associated virus (AAV) vectors and protein-engineered transgenes, B-domain deleted (BDD) FVIII and FIX-Padua, have reported near-phenotypic cures in subjects with HA and HB, respectively. Here, we review the biology and the clinical development of FVIII-BDD and FIX-Padua as transgenes. We also examine alternative bioengineering strategies for FVIII and FIX, as well as the immunological challenges of these approaches. Other engineered proteins and their potential use in gene therapy for hemophilia with inhibitors are also discussed. Continued advancement of gene therapy for HA and HB using protein-engineered transgenes has the potential to alleviate the substantial medical and psychosocial burdens of the disease.

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Section: Main Textmentioning

confidence: 99%

Protein-Engineered Coagulation Factors for Hemophilia Gene Therapy

Samelson-Jones

Arruda

2019

Molecular Therapy - Methods & Clinical Development

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“…We were not only able to analyze the abundance and complexity of the proteome as it shifted during the bioprocess and after purification, but we were also able to monitor the abundance of diverse PTMs with site specificity, including the critically important g- Discussion rFIX is commercialized as a replacement therapy for Haemophilia B [8,10]. Considerable effort has been devoted to optimize rFIX bioprocess and expression systems [7,[10][11][12][109][110][111][112][113]. FIX is highly post-translationally modified with a variety of heterogeneous PTMs, and GLA domain gcarboxylation is the most functionally relevant PTM [14,[19][20][21].…”

Section: Predicting Purity Of Purified Rfix By Measuring Changes In Hmentioning

confidence: 99%

Analysis of coagulation factor IX in bioreactor cell culture medium predicts yield and quality of the purified product

Zacchi

Recinos

Pegg

et al. 2020

Preprint

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Coagulation factor IX (FIX) is a highly complex post-translationally modified human serum glycoprotein and a high-value biopharmaceutical. The quality of recombinant FIX (rFIX), especially complete g-carboxylation, is critical for rFIX clinical efficacy. Changes in bioreactor operating conditions can impact rFIX production and occupancy and structure of rFIX posttranslational modifications (PTMs). We hypothesized that monitoring the bioreactor cell culture supernatant with Data Independent Acquisition Mass Spectrometry (DIA-MS) proteomics would allow us to predict product yield and quality after purification. With the goal of optimizing rFIX production, we developed a suite of MS proteomics analytical methods and used these to investigate changes in rFIX yield, g-carboxylation, other PTMs, and host cell proteins during bioreactor culture and after purification. Our methods provided a detailed overview of the dynamics of site-specific PTM occupancy and abundance on rFIX during production, which accurately predicted the efficiency of purification and the quality of the purified product from different culture conditions. In addition, we identified new PTMs in rFIX, some of which were near the GLA domain and could impact rFIX GLA-dependent purification efficiency and protein function. The workflows presented here are applicable to other biologics and expression systems, and should aid in the optimization and quality control of upstream and downstream bioprocesses. Figure 1: Coagulation Factor IX (FIX) structural domains and post-translational modifications (PTMs). Schematic of mature FIX with previously described PTMs in plasma derived FIX and/or recombinant FIX (figure modified from [13]). The GLA domain (red) contains 12 potential sites of g-carboxylation on E 7-40 and one O-glycan at T 38/39 . The EGF-like 1 domain (pink) contains two O-glycans on S 53 and S 61 , b-hydroxylation of D 64 , and phosphorylation of S 68 . The EGF-like 2 domain (violet) has no known PTMs. The short domain (white) linking the EGFlike 2 domain with the activation peptide (AP, green) contains one O-glycan at S 141 . The AP contains two N-glycans at N 157 and N 167 , four O-glycans at T 159 , T 169 , T 172 , and T 179 , sulfation of Y 155 , and phosphorylation of S 158 and T 159 . The serine protease domain (orange) contains one Nlinked glycan at N 258 .FIX is a highly post-translationally modified glycoprotein [7,14] (Fig. 1). Immature FIX is composed of six structural domains: an N-terminal propeptide, the GLA domain, two consecutive EGF-like domains followed by a short linker, the activation peptide (AP), and the C-terminal serine protease domain (Fig. 1). Most described PTMs are on or near the GLA, the EGF-like 1, and the AP domains. The propeptide and the AP are removed through proteolysis events, and both proteolysis are required for function [1,9,15]. FIX's propeptide is cleaved by the trans-Golgi protease PACE/Furin during transit through the secretory pathway [16,17]. FIX's AP is removed in the blood by Factor XIa as part of the c...

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“…Despite some changes in morphology compared to the normal cells, NuH-7 have maintained liver cell functions as shown by the production of plasma proteins [ 89 ]. Expression of recombinant factor IX (for hemophilia treatment) [ 90 , 91 ] and human erythropoietin (EPO) [ 92 ] has been reported. This cell line has not been studied under the influence of the MF.…”

Section: Magnetic Field Influence On Mammalian Cellsmentioning

confidence: 99%