In vitro and in vivo transient human blood clotting factor IX (hFIX) production for characterization, evaluation of activity and functional screening via mammary tissues as a step for the production of hFIX in transgenic bovine milk

Abstract

This research project aims to integrate advanced biotechnology tools for the establishment of an efficient system of production of blood coagulation factor IX recombinant human (rhFIX) for the treatment of Hemophilia B, a hereditary disease linked to the X chromosome which is caused by deficiency of this functional protein in the blood coagulation cascade. The synthesis of hFIX in the liver occurs in the form of inactive serine protease, where it undergoes several post-translational modifications (MPT) before discharge to the plasma. The activated FIX (FIXa) activates Factor X coagulation and the formation of thrombin from prothrombin and the formation of fibrinogen, fibrin which, when polymerized, consolidates the platelet buffer or clot. The mutation in the hFIX fails coagulation and the Hemophilia B. The treatment of Hemophilia B is based on the replacement of the deficient hFIX based on hFIX intravenous infusion concentrate. Studies for producing recombinant therapeutic products have been developed in the world. In Brazil, the treatment is fully realized with FIX derived from human plasma, which is processed out of the country and sent back on lyophilized form, with high costs to SUS (R $ 52 million / year), as well as susceptibility to disease. RhFIX These production systems may be replaced by more productive methods, such as animal platform for protein production in the mammary gland, the main long-term goal of this proposal. Transgenic animal milk is considered one of the best sources for obtaining recombinant proteins for biomedical use, being highly competitive, with low implementation costs and high productivity. This project is aimed at the use of milk secretory capacity for the efficient production of complex proteins such as rhFIX, which is centered in the mammary gland which as the capacity to perform co- and post-translational modifications (cleavage of the pro-peptide, ³-carboxilic) demonstrably more efficiently than in cultured cells. In general, we aim at the production of recombinant human blood coagulation Factor IX (rhFIX) in its active form in large scale and efficiently in bovine milk using two excellent biological systems for screening and production of rhFIX-scale in an animal platform, seeking modern alternatives that guarantee the production of functional proteins in transgenic animal model. In view of the efficiency and speed in obtaining recombinant proteins, one system for obtaining and screening of rhFIX is the transient expression of the protein of interest (rhFIX) using vectors recombinant adenovirus (rAAV). It is proposed transient expression of recombinant proteins in vitro, in mammary gland cells in culture and in vivo, in the mammary gland of lactating goats and leporinas females by methods already well established by our employees in Brazil. They will be produced by particles of recombinant adenovirus in vitro for the purpose of injection into the mammary glands of cows and leporinas. Thus, the rFIX and rFurina be produced transiently in the milk, allowing test bicistronic construct and characterize the rhFIX produced in different systems. Once purified from the culture medium or milk, recombinant rhFIX be assessed for structure and activity, then to establish transgenic lines somatic bovine cells for the future (Phase II) are used for the development of transgenic cattle by cloning animal. The bicistronic gene construction of hFIX and hFurina genes will be inserted into a vector containing a mammary gland specific promoter. The efficient establishment of this transgenic animal model supports other technologies are used in combination the two expression systems, such as the use of safe harbor loci, and DNA editing system CRISPR / Cas9. Thus, the transgene will be used in co-transfection of bovine fetal fibroblasts with CRISPR system / Cas9 ROSA26 locus for the bovine genome, with subsequent selection and molecular diagnosis of transgenic cells with a view to future use in cloning SCNT. (AU)