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Title: “Genetically engineered apolipoproteins immobilized on nanoparticles: a Molecular Trojan horse targeting atherosclerotic plaque” (APGEN)

Title (in Romanian language): Apolipoproteine generate prin inginerie genetica imobilizate pe nanoparticule: un cal troian molecular pentru tintirea plăcii aterosclerotice (APGEN)

Abstract: Apolipoprotein E (apoE) plays a pivotal role against atherosclerosis. This role is exerted only by apoE delivered at the lesion level. The main aim of this project is to obtain apolipoprotein-fullerene based nano-systems (AFN) targeted toward the atherosclerotic plaque through transferrin receptor. The transferrin or anti-transferrin receptor monoclonal antibody included in AFN will facilitate their transcytosis through the endothelial layer and will enhance their binding on the macrophage-foam cells. We plan to achieve the following objectives: (1) Setting up a series of AFNs using recombinant apoE3 or apoE3-HRP chimera alone or together with transferrin or with anti-transferrin receptor monoclonal antibodies; (2) Biochemical characterization of the obtained AFNs by size, apolipoprotein/Tf or TfRMoAb ratio, lipid binding capacity; (3). Functional characterization: (i) In vitro tests of the capacity of AFNs to cross the endothelial layer, to bind on macrophage surface and to participate in the cholesterol efflux from the macrophage-foam cells; (ii) In vivo AFNs ability to reduce the atherosclerotic plaques in animal models. Targeting apoE at the plaque using novel biotechnologies will provide an efficient cholesterol efflux from the lipid loaded cells, having significant anti-atherosclerotic benefits. This strategy may open new horizons for developing “synthetic lipoproteins” based on recombinant proteins attached to spherical nanoparticles for diverse therapeutic purposes.

Rezumat: Apolipoproteina E (apoE) joaca un rol esential in protectia impotriva aterosclerozei. Acest rol se exercita doar de apoE furnizat la situsul lezional. Scopul principal al acestui proiect este de a obtine apolipoproteine in nano-sisteme bazate pe fulerene (ANF) directionate catre placa aterosclerotica de catre receptorul la transferina (TfR). Transferina (Tf) sau anticorpii monoclonali anti-TfR inclusi in ANF vor facilita transcitoza prin endoteliu si vor potenta legarea pe macrofagele spumoase. Ne propunem urmatoarele obiective: (1) Obtinerea unei serii de ANF utilizand apoE3 recombinant sau himera apoE3-HRP singure sau impreuna cu Tf sau anticorpi monoclonali anti-TfR; (2) Caracterizarea biochimica a ANF obtinute: marime, raport apolipoproteina/Tf sau antiTfR, capacitate de legare a lipidelor; (3). Caracterizarea functionala a ANF: (i) Teste in vitro ale capacitatii ANF de a traversa bariera endoteliala, de a se lega la suprafata macrofagelor si de a participa la efluxul de colesterol din celulele spumoase; (ii) Teste in vivo ale capacitatii ANF de a reduce placile aterosclerotice in modele animale. Directionarea apoE la nivelul placii utilizand biotehnologii noi va sustine un eflux eficient de colesterol din celulele incarcate cu lipide, avand beneficii semnificative in ateroscleroza. Aceasta strategie va dechide noi orizonturi pentru dezvoltarea “lipoproteinelor sintetice” bazate pe proteine recombinante atasate la nanoparticule sferice pentru diverse scopuri terapeutice.

Team of the project:

1	Violeta Anca Gafencu	Project manager
2	Elena Valeria Fuior	PhD Student
3	Violeta Georgeta Trusca	Postdoctoral fellow
4	Ioana Madalina Fenyo	Postdoctoral fellow
5	Madalina Dumitrescu	Postdoctoral fellow
6	Cristina Uratu	Postdoctoral fellow
7	Irina Florina Tudorache	PhD Student

The activities performed so far are done to attain the objective 1: “Setting up a series of AFNs using recombinant apoE3 or apoE3-HRP chimera alone or together with transferrin or with anti-transferrin receptor monoclonal antibodies” and the objective 2: “Biochemical characterization of AFN”

Results:

1. We obtained plasmids encoding apoE with:

(i) His6-Tag;

(ii) Halo-Tag;

(iii) biotin label;

(iv) HRP label.

Dissemination:

1 Apolipoprotein E expression in macrophages: from basic transcription to cell -specific gene modulation 2016, Prezentare Orala la 8th national congress with international participation and 34th annual scientific session of the Romanian society for cell biology, Oradea, Iunie 2016 Bulletin of Romanian Society for Cell Biology No.44, pag 38

2. Biotechnological approaches for labeling of apolipoprotein E secreted by mammalian cells, 2016, Prezentare Orala la 8th national congress with international participation and 34th annual scientific session of the Romanian society for cell biology, Oradea, Iunie 2016 Bullletin of Romanian Society for Cell Biology No. 44 pag 20