Development and characterization of recombinant ovine coagulation factor VIII.

Animal models of the bleeding disorder, hemophilia A, have been an integral component of the biopharmaceutical development process and have facilitated the development of recombinant coagulation factor VIII (fVIII) products capable of restoring median survival of persons with hemophilia A to that of the general population. However, there remain several limitations to recombinant fVIII as a biotherapeutic, including invasiveness of intravenous infusion, short half-life, immunogenicity, and lack of availability to the majority of the world's population. The recently described ovine model of hemophilia A is the largest and most accurate phenocopy. Affected sheep die prematurely due to bleeding-related pathogenesis and display robust adaptive humoral immunity to non-ovine fVIII. Herein, we describe the development and characterization of recombinant ovine fVIII (ofVIII) to support further the utility of the ovine hemophilia A model. Full-length and B-domain deleted (BDD) ofVIII cDNAs were generated and demonstrated to facilitate greater biosynthetic rates than their human fVIII counterparts while both BDD constructs showed greater expression rates than the same-species full-length versions. A top recombinant BDD ofVIII producing baby hamster kidney clone was identified and used to biosynthesize raw material for purification and biochemical characterization. Highly purified recombinant BDD ofVIII preparations possess a specific activity nearly 2-fold higher than recombinant BDD human fVIII and display a differential glycosylation pattern. However, binding to the carrier protein, von Willebrand factor, which is critical for stability of fVIII in circulation, is indistinguishable. Decay of thrombin-activated ofVIIIa is 2-fold slower than human fVIII indicating greater intrinsic stability. Furthermore, intravenous administration of ofVIII effectively reverses the bleeding phenotype in the murine model of hemophilia A. Recombinant ofVIII should facilitate the maintenance of the ovine hemophilia A herd and their utilization as a relevant large animal model for the research and development of novel nucleic acid and protein-based therapies for hemophilia A

Target-Cell-Directed Bioengineering Approaches for Gene Therapy of Hemophilia A

Potency is a key optimization parameter for hemophilia A gene therapy product candidates. Optimization strategies include promoter engineering to increase transcription, codon optimization of mRNA to improve translation, and amino-acid substitution to promote secretion. Herein, we describe both rational and empirical design approaches to the development of a minimally sized, highly potent AAV-fVIII vector that incorporates three unique elements: a liver-directed 146-nt transcription regulatory module, a target-cell-specific codon optimization algorithm, and a high-expression bioengineered fVIII variant. The minimal synthetic promoter allows for the smallest AAV-fVIII vector genome known at 4,832 nt, while the tissue-directed codon optimization strategy facilitates increased fVIII transgene product expression in target cell types, e.g., hepatocytes, over traditional genome-level codon optimization strategies. As a tertiary approach, we incorporated ancient and orthologous fVIII sequence elements previously shown to facilitate improved biosynthesis through post-translational mechanisms. Together, these technologies contribute to an AAV-fVIII vector that confers sustained, curative levels of fVIII at a minimal dose in hemophilia A mice. Moreover, the first two technologies should be generalizable to all liver-directed gene therapy vector designs. Keywords: vector optimization, AAV, hemophilia, factor VIII, codon optimization, promoter desig

Discrimination of the BDD OfVIII Heavy and Light Chains.

<p>Immunoprecipitation using domain specific MAbs was performed by incubation with ofVIII in the presence and absence of thrombin. Heavy and light chains were dissociated using M-PER lysis buffer supplemented with 150 mM NaCl prior to MAb addition. FVIII heavy chain was precipitated with 4F4 1B, an A2 domain-specific mAb, and light chain was precipitated with I14 1B, a C2 domain-specific mAb. MAbs incubated with vehicle served as negative controls.</p

Thrombin-Activated Decay Rate of OfVIIIa.

<p>Human (closed circle) and ovine (open circle) fVIIIa decay was measured by chromogenic Xase assay in which 20 nM fVIII was activated with thrombin and then stopped with desulfatohirudin. Activated fVIIIa in complex with phospholipid vesicles, activated factor IXa, and factor X was measured at 0.5, 3, 5, 8, 15, and 30 minutes to determine residual fVIIIa activity. Half-lives of 1.8±0.09 and 3.5±0.37 minutes were calculated for human and ovine fVIIIa, respectively. Data shown represents the percent of initial activity by semi-log extrapolation to time  = 0. Regression analysis revealed Pearson correlation coefficients of 0.999 for both treatments.</p

Biochemical Analysis of BDD OfVIII.

<p>Recombinant ofVIII (2 µg) ± thrombin and PNGase treatment was resolved by SDS-PAGE and visualized by Coomassie blue staining. A molecular weight ladder was used to determine the relative mobility of the polypeptides.</p

BDD OfVIII Binding to VWF.

<p>Kinetic ELISA was conducted using human VWF to capture human (closed circles) or ovine (open circles) fVIII. Plates were coated with 50 µl of 6 mg/ml human VWF and blocked with 2% BSA. Monoclonal A2 domain fVIII antibody 4F4 1B was added to each well and colorimetric transmission was activated with <i>para</i>-nitrophenylphosphate substrate following streptavidin alkaline phosphatase. Data shown are the mean of three independent experiments ± sample standard deviation.</p

In Vivo Efficacy of OfVIII in Hemophilia A Mice.

<p>Hemophilia A mice were injected with either 100 µl saline or 300 U/kg ofVIII in 100 µl sterile saline via tail vein injection (n = 8). After 15 min, bleeding challenge was induced via tail transaction at 2 mm diameter. Blood was collected in pre-weighed vials of 13 ml sterile saline at 37°C. Blood loss was calculated and displayed as mg/g body weight. Mean blood loss for saline and ofVIII treatments were 32.2±9.37 and 1.15±2.57 mg/g body weight, respectively (<i>P</i><0.001; Mann-Whitney <i>U</i> test).</p

Expression of Recombinant Ovine and Human fVIII.

<p>BHK-M cells were stably transfected with full-length and BDD fVIII constructs and selected with geneticin. Individual colonies were expanded in 6-well plates and fVIII activity was measured by one-stage coagulation assay in serum-free media after 24 hr culture. Cell numbers were determined at the time of activity measurement and data was normalized to 10⁶ cells. The horizontal lines depict the mean values for each data set.</p

Mechanochemical evolution of the giant muscle protein titin as inferred from resurrected proteins

The sarcomere-based structure of muscles is conserved among vertebrates; however, vertebrate muscle physiology is extremely diverse. A molecular explanation for this diversity and its evolution has not been proposed. We use phylogenetic analyses and single-molecule force spectroscopy (smFS) to investigate the mechanochemical evolution of titin, a giant protein responsible for the elasticity of muscle filaments. We resurrect eight-domain fragments of titin corresponding to the common ancestors to mammals, sauropsids, and tetrapods, which lived 105-356 Myr ago, and compare them with titin fragments from some of their modern descendants. We demonstrate that the resurrected titin molecules are rich in disulfide bonds and display high mechanical stability. These mechanochemical elements have changed over time, creating a paleomechanical trend that seems to correlate with animal body size, allowing us to estimate the sizes of extinct species. We hypothesize that mechanical adjustments in titin contributed to physiological changes that allowed the muscular development and diversity of modern tetrapods.Research has been supported by the Ministry of Economy and Competitiveness (MINECO) grant BIO2016-77390-R, BFU2015-71964 to R.P.-J., BIO2014-54768-P and RYC-2014-16604 to J.A-C., and CTQ2015-65320-R to D.D.S., and the European Commission grant CIG Marie Curie Reintegration program FP7-PEOPLE-2014 to R.P.-J. A.A.-C. is funded by the predoctoral program of the Basque Government. R.P.-J. and D.D.S., thank CIC nanoGUNE and the Ikerbasque Foundation for Science for financial support. CNIC is supported by the Spanish Ministry of Economy and Competitiveness (MINECO) and the Pro-CNIC Foundation and is a Severo Ochoa Center of Excellence (MINECO award SEV-2015-0505). Plasmid pQE80-(I91-32/75)8 was a kind gift from J. Fernandez (Columbia University). We thank R. Zardoya (National Museum of Natural Sciences, Madrid) for helpful discussions and comments. The authors acknowledge technical support provided by IZO-SGI SGIker of UPV/EHU and European funding (ERDF and ESF) for the use of the Arina HPC cluster and the assistance provided by T. Mercero and E. Ogando.S