New insights and tools to study innovative delivery strategies of therapeutic peptides: Apolipoprotein A-I Milano, administered orally via genetically modified rice plants, exerts anti-inflammatory effects in vivo.

In the proof-of-concept study published by Romano and others in Int.Cardiology 2018, was shown a new approach for treatment of cardiovascular disease (CVDs) because, despite the high therapeutic potential of Apolipoprotein A-I protein, the practical application is hampered by the low efficiency of purification and delivery. A genetically modified rice plants expressing Apolipoprotein A-I variant Milano (AIM) were created. The product was tested in vitro, and in vivo mouse atherosclerotic model treated with genetically modified rice-milk (in the form of protein extract of rice seed proteins) or wild type rice-milk and it was found to be safe and effective. For subsequent aims, initially, in this context, the APO rice flour (RF) product was better characterized to further understand the potential of transgenic rice seeds as an efficient oral production and delivery system for recombinant AIM protein. WT and APO RF were analysed by ELISA and Western Blot (WB) and APO protein was detected in APO rice as expected. To better understand the biodistribution of the AIM protein administered orally in APO3 pre-clinical study and the molecular mechanisms underlying the athero-protective and anti-inflammatory effects, ELISA, WB and immunohistochemistry (IHC) assays were performed on mice liver and/or other organs. AIM in liver and intestine lysates was not detected by ELISA and WB. By increasing the spatial resolution by IHC, positive spots were detected in the liver sections of the treated mice. These investigations were fundamental to lay the foundations for the main purpose, namely the development of a new system of functional AIM product to improve both in quality and quantity of delivered protein to be able to extend the product for future use in humans. In this order, a set of vectors carrying the coding sequence of the AIM and the respective control vectors was constructed, to be tested at a functional level in vitro and subsequently to be used as source of the AIM sequence to engineer the new rice product.