Production and controlled release of a novel therapeutic protein for the treatment of muscular dystrophies

Rachel Lev, Olga Kossover, Yonatan Lati, Dror Seliktar
Muscular dystrophies (MDs) are genetic disorders characterized by progressive muscle wasting, leading to limitations in motor capacity and in many cases to progressive paralysis and death1. Cripto, an extracellular protein, has been recently found by our collaborator, Prof. Minchiotti (IGB Naples, Italy), to have therapeutic value in alleviating muscle injury and diseases by acting as a local recruitment factor for stem cells. The local recruitment of progenitor cells facilities muscle regeneration and promotes satellite cell progression toward the myogenic lineage2. A wide range of bio-therapeutic recombinant proteins are currently synthesized using mammalian cell culture methods. However, since protein-based therapeutics may require large doses over a long period of time, manufacturing capacity becomes a critical issue as the drug substance must be produced effectively in large scale for both commercial and clinical requirements. In this study, we improve upon the production capacity of exogenous therapeutic Cripto by using suspension bioreactors in combination with a microgel 3D cultivation system. Subsequently, we have been developing a controlled release delivery system for the recombinantly expressed Cripto protein. Semi-synthetic hydrogels made from PEG-fibrinogen (PF) provide the substrate for protein production as well as the controlled release in situ delivery vehicle. For efficient production of Cripto, PF microcarriers were designed to enable mammalian cell survival, proliferation, and secretion of large amounts of therapeutic proteins. Cripto overexpressing HEK 293T cell lines were encapsulated in the PF microcarriers and cultivated in stirred suspension bioreactors. PF microcarriers were prepared to exhibit high mechanical strength in order to resist shear forces and biodegradation associated with long–term suspension culture in the bioreactors. In addition, a PF micro-carrier delivery system was developed for intramuscular injection of the purified Cripto protein to provide stability and protection during and after the in vivo procedure. Preliminary studies of the in vitro controlled release of Cripto from the micro-scale delivery system shows a biphasic release profile, characterized by an initial burst release followed by a sustained release phase. In vivo experiments will soon be performed to examine and characterize the essential properties of our therapeutic protein release system.
In summary, PEG-Fibrinogen microcarriers hold great promise for both large scale protein production and for controlled release of therapeutic proteins.
1. Mercuri E, Muntoni F., Lancet 2013; 381:845-860.
2. Guardiola O et al., Proc Natl Acad Sci USA 2012; 109:E3231-E32