PRODUCTION AND CONTROLLED RELEASE OF A NOVEL THERAPEUTIC PROTEIN FOR THE TREATMENT OF MUSCULAR DYSTROPHIES

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 death (1). Cripto, an extracellular protein, has been recently found by our collaborator, Prof. Minchiotti (IGB Naples, Italy), to have great potential therapeutic value in alleviating muscle injury and diseases. Cripto was found to regulate muscle regeneration and satellite cell progression toward the myogenic lineage (2). We propose to improve the production capacity of exogenous therapeutic Cripto by using suspension bioreactors in combination with a microgel 3D cultivation system. Simultaneously, we are developing a controlled release delivery system for the purified Cripto protein. PEG-fibrinogen (PF) hydrogels provide the comprehensive solution for both needs. For the efficient of production of Cripto, a PF microcarrier was designed which allows mammalian cells to survive, proliferate, and secrete amounts of large 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. Additionally, a PF micro-scale delivery system was developed for intramuscular injection to provide stability during and after the procedure, as well as immunoprotection for the Cripto. 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.

1.Mercuri E, Muntoni F, Lancet 2013; 381:845-860

2.Guardiola O et al., Proc Natl Acad Sci USA 2012; 109:E3231-E32